the silkworm

Interview with Dr. RK. Datta.

gkrajeshrajesh@gmail.com — Tue, 18 Aug 2009 19:43:00 +0000

Dr. RK. Datta is a prominent name in Tropical sericulture research. He belongs to that rare genre of technocrats who successfully combined scholarship, research and administration in their career. He was born in 1943 in Bengal, India. After obtaining Ph.D in Genetics, Dr. Datta built up his career as a teacher and a researcher. During his 31 years long career with Central Silk Board, India, as a researcher, Dr. Datta gained rich experience by working in various capacities and heading reputed national and international projects such as: National Sericulture Project of Government of India, JICA Project. FAO Project (Iran), Malaysian sericulture Project, Bangladesh sericulture Project, IFAD Project in DPR of Korea etc. He has also served as Member of Task Force, Dept. of Biotechnology, India, member of research Advisory Committee of CSR&TI, Mysore, Member of Editorial board of the Journals “Sericologia”, Lyon, France and Indian Journal of Sericulture.

As the Director of the Premier Sericulture Research Institute (CSRTI) at Mysore Dr. Datta steered the institution to higher levels of Science, Technology & training. He is widely regarded as the founder of a new school of silkworm breeding in India with a well directed application of principles of genetics and biotechnology. His work has earned him several laurels such as the prestigious Louis Pasteur Award of 1999, National Research Development Corporation awards in 1999 and 2004, Baldeodas Shah National Award (thrice) 1990-91; 1994-95; 1998-1999. A dedicated researcher, an inspiring teacher and a prolific writer, Dr. Datta has published more than 250 research papers in National and International journals, authored twelve books and supervised nearly ten PhD theses under his guidance. Dr. Datta is widely traveled. He has visited almost every part of the world which fostered interest in silk such as USA, Japan, France, China, CIS (Erstwhile USSR), Greece, UK, Italy, Belgium, Brazil Iran, Thailand, Malaysia, Bangladesh, Kenya and Cairo on various assignments.

At 65, Dr. Datta is still involved in active research. Currently he is the R & D consultant to the Bangalore Company “Sericare” who produces various products useful to the silk industry as also large scale production of silk proteins for use in animal tissue culture, nutraceuticals and silk films. During 2005, he could initiate a project on burn wound cover utilizing the bioengineered silk proteins. Results of this research offers solution to the much awaited demand for covering the burn and ulcers with non allergenic- epidermal growth promoting biomaterials.

Talking with Dr. Datta has been a pleasant and enlightening experience, given his flair for conversation and humour. Dr. Datta can be contacted at: rkdatta1943@hotmail.com

What is the importance of Indian sericulture, in the national and global contexts?

In India, importance of Sericulture at National level lies with its Immense potentiality to provide employment opportunity to rural people even more to women folk by avoiding their migration to alien urban areas (around 5 million people are engaged as full time or part time workers); also notable are its role in supporting to the need for recurrent income from sale of its products, its vital role in transferring wealth from rich to the poor; Employment support and continuance of handloom and decentralized power loom sectors both in rural or semi-urban areas of the country; Urban employment support like manufacturing seri-equipments, making silk handlooms, disinfectants, pest control products, growth hormones for mulberry and silkworm and finally and most importantly, it is catering to the demands of silk Saris, dress materials, silk carpets, blended fabrics etc in both rural and urban areas.

Its importance at the global context: Indian handloom materials like blended fabrics, dress materials, carpets which are unique in their quality and design are appreciated in the world market and demands are in increasing trend. India has trained large number of persons from the developing countries like many African and Asian personnel who later participated in promoting sericulture in their countries.

India is designated as world’s second largest silk producer. However we have not yet been capable of making any significant impact in the global silk market. What are the reasons?

Sericulture in India is practiced in tropical zone and thus success of bivoltine crops as seen in Seri-culturally developed country like Japan, China where it is concentrated only in temperate zone assuring higher silk yield stadium, is not found in India. Moreover, traditionally we grow silk to cater to the need of saris where preference is for handloom designs and of heavier weight of the cloth. Charka reeling which is highly cost effective and economic but makes uneven quality of weft (70%) is sufficient for making high quality handloom saris using filature reeled silk for warp (30%). Thus transformation of charka reeling to filature/ multi-end machined reeling remained a paradox over the decades. To capture the world market demand we require producing high quality warp and weft as well. Availability of cheap Chinese silk yarn and fabric remained as a bottleneck to expanding silk industry in India. Indian export failed to lead over that of Chinese silk fabrics due to price and quality differences.

During your tenure as the Director of CSRTI, Mysore, a large number of technologies were developed for tropical sericulture. Could you tell us three most important contributions during your tenure?

The three major achievements that I would like to high light are One- release of high yielding mulberry variety like V1 along with its cultivation practices which produces one and half times to that of the prevailing variety. Two- release of high yielding bivoltine hybrid, CSR2 X CSR4 and the full proof package of practices for successful rearing. And three- Development of a non-corrosive, non-irritating rearing room disinfectant, Sanitech replacing age-old use of formalin as well as unique bed disinfectant, Vijetha.

India is the only country that produces all the four commercial varieties of silk. How well have we exploited this opportunity to contribute to the country’s GDP?

Though India produces all the four commercial varieties of silk viz. Eri, Muga, Tropical and Temperate Tasars, exploitation of these varieties continued to be limited due to its existence in hilly forest terrenes of different states. Aboriginals residing in different forests though practice these wild silkworms, total production remained limited except in case of Eri culture. The latter is widely used as spun silk that blends well with mulberry yarn for making fabrics of different quality especially used for making male garments including suits. Of late tasar garments and suiting have gained momentum in export. However, overall contribution to country’s GDP is very negligible.

As a silkworm breeder and being at the helm of the most important tropical silkworm breeding station at a crucial period of Indian sericulture growth, could you tell us your experiences in developing various silkworm hybrids for the tropic?

In our main breeding Institutes of Central silk Board the thrust on silkworm breeding is a two pronged approach, i.e. evolution of both bivoltine and multivotine hybrids. Some of the new multivoltine hybrids (Multi- x bivoltine breeds) evolved are found highly promising having improved shell ratio and yarn neatness. In tropical climate, farmers prefer to rear multi x bivoltine hybrids over that of bivoltines due to low risk factor. Fairly large number of bivoltine hybrids including double hybrids evolved at CSRTI, Mysore is found very good for silk industry since raw silk percentage and neatness are very high in those hybrids compared to multi-bivoltine hybrids. Some of these hybrids are in field and India is able to produce 1200 MT of bivoltine silk now.

Since National Sericulture Project, Bivoltine Sericulture has been given major stress. Do you think the salvation of Indian sericulture is through Bivoltine sericulture alone?

No. As I have mentioned in response to your question earlier, Indian sericulture is dependent on both multivoltine and bivoltine hybrids. This is so due to massive demand for silk saris in the country, where multivoltine silk support is crucial. Moreover, in tropical climatic conditions one can’t rear bivoltine hybrids in all the seasons.

Why in spite of technological advancements, foreign technology support and huge investments for past 15 years, diffusion of Bivoltine hybrids is just 5% in India?

It is unfortunate, despite evolution of excellent bivoltine hybrids only 9% of the total silk is the bivoltine (1200MT) one and the rest is multivoltine. However, some quantity of bivoltine hybrids also goes for preparing multi x bivoltine hybrids. Reasons are not difficult to understand- (i) bivoltine cocoons many a times do not fetch the expected higher price, since demand for multi-bivoltine cocoons is higher from the charka and cottage basin reelers [bivoltine cocoons are transacted in Ramnagaram (Karnataka State) market only]; (ii) processing cost of bivoltine silk is higher (iii) Chinese bivoltine silk are comparatively cheaper compared to bivoltine silk produced in India (iv) Farmers experience difficulty in raising bivoltine cocoons in tropical climate.

Given the potentials of silkworm as a model genetic system and a bio reactor, how well did India exploit it either industrially or academically? What are the reasons for the sluggish progress?

Significant progress has been made in Indian research on the molecular genetics and biotechnological researches on silkworm and mulberry including the use of silkworm bye-products. Research Publications of last ten years confirm that. Now transgenic silkworms were utilized for developing silkworm strains controlling the virulent silkworm disease like NPV using the latest technique of RNAi. Private Company researches are initiated on the use of silk proteins like fibroin, sericin for use of biomedical devices, nutraceuticals, mulberry tea, health drink etc. Department of Biotechnology (DBT), Govt. of India has now decided to establish a full fetched Silk-biotechnology Research Institute in Bangalore.

Sericulture is one of the most technology rich areas in Indian agriculture. Do you think matching technological progress was made in the post cocoon sector? What are the reasons?

So far as my knowledge goes silk technological researches undertaken at Central Silk Technological Research Institute (CSTRI), Bangalore have done impressive work in last two decades. Problem lies with the implementation of those technologies in the silk industry which is uniquely different from that of Japan or China. Silk Industry in India remained as an unorganized decentralized sector. Only a few progressive reelers or silk factories are coming forward to utilize the expertise available with CSB. However, CSTRI has also done lot of work for the improvement of decentralized sectors too, like existing charka reelers, multiend reeling, handlooms, Jacquard machines, cheaper drying equipments for cocoons, dyeing and printing of handloom fabrics. Some of the Bangalore silk fabrics manufacturing companies are able to compete with the renowned international companies for the sale of silk fabrics at a very high cost in USA.

Do you think that the allegation that Peoples Republic of China is dumping silk in India is true? Isn’t it a natural outcome of liberalization of Indian economy, and a part of the globalization phenomenon? Why couldn’t we utilize this opportunity to strengthen the domestic silk weaving sector in an export oriented manner?

I don’t call it an intentional dumping of silk. National demand of silk is very high (23000MT or above) compared to that produced in India (17000MT). Chinese silk being cheaper, Indian companies purchase the silk in order to cater to the need of export market. In fact, good silk fabric manufacturing companies have come up in India using imported silk yarns for exporting Indian silk goods in foreign countries. Unfortunately, powered handloom and small power loom sectors are also using the Chinese silk to meet their need for warp and creating pressure on development of domestic silk industry. Good news now a few entrepreneurs have now installed 400 end multiend reeling machine in the States of Karnataka, Tamil Nadu and Andhra Pradesh with the support of Govt. subsidy (50:50) for machines procured from China. One thus wishes more number of such entrepreneurs enters in the reeling business with the increase in production of bivoltine cocoons.

We see that sericulture gets gradually eliminated as countries improve their GDP. What is the fate of Indian sericulture in another 20 years time?

It is a fact and no one can deny that. Sericulture being highly labour intensive, it is difficult for making silk products at a cheaper cost. Owing to the huge internal demand of silk in the country we are able to sustain this enterprise. As long as elite classes pay for silk at a higher price, sericulture will continue to survive in the villages. But most alarming situation has arisen now due to sudden hike in labour cost that does not commensurate with the increase of silk price fetched in the market. Thus production drop in different states can’t be ruled out. However, Indian sericulture will survive as long as sale of Chinese silk remain moderately competitive or high in the market.

Is China the Villain? Silk ‘dumping’ and the plight of Indian silk reeler

gkrajeshrajesh@gmail.com — Fri, 31 Jul 2009 11:04:00 +0000

In spite of its key role in the process of silk production and it’s high and growing potential to employ poor workers particularly women, the reeling activity is a relatively neglected area from the research point of view. The post WTO developments especially the relaxation of import restrictions in the post liberalization regime also has apparently taken its toll on the silk reeling sector. Not many studies were conducted on this issue. The objective of this paper is two fold 1. To throw up a few important issues faced by the silk reeling sector in India and 2. To suggest a broad direction of research in the Indian silk sector in the wake of globalization.

Silk reeling sector in India

There are around 24 thousand reelers (as per the registered number of reeling units up to March 97) in the state (Vasumathi, 2000). They all own small reeling units and the maximum size of a reeling unit does not exceed 20 basins. Manual silk reeling provides employment to around 50,000 persons in semi-urban locations; most of these live in conditions of absolute poverty and exist on subsistence incomes. Yet the economic and working conditions of reelers and the reeling activity have received little research attention when compared with other aspects of sericulture (Economic development associates, 1990)

Liberalisation and Rawsilk imports

Since liberalization of imports in 2001, import of raw silk has been steadily increasing with low prices. Raw silk imports have increased from 4713MT in 2000-01 to 9258 MT in 2003-04 (Ministry of Textiles, Government of India, 2005). Increase in imports from 2001-02 to 2002-03 reportedly impacted the domestic price of raw silk. Price of imported silk in India fell drastically from US $ 24.50 per kg in May 2001 to US $ 13.50 per kg. in June 2003. This apparently disrupted the domestic silk market and prices of cocoons and raw silk fell by about 40% due to large volume of import at relatively low prices. As a consequence farmers and the reelers, who were the most affected segment, approached the Directorate General of Anti Dumping and Allied Duties (DGAD) requesting to impose levy of anti-dumping duty against China. As dumping of raw silk by China was established, the GOI imposed antidumping duty against the import of mulberry raw silk (not thrown) of international grade 2A and below, originated in or exported from People’s Republic of China (DGAD, 2003). According to government sources, in spite of antidumping duty on all imports of mulberry raw silk of grade 2A and below from China, the imports of raw silk were on the increase and the antidumping duty was being evaded by China by exporting to India raw silk of Grade-2A and above and twisted silk yarn which is beyond the purview of the antidumping notification (Ministry of Textiles, GOI, 2005).

The case concerns the silkworm rearers and silk reelers on the one side and handloom weavers and power loom weavers on the other side. The rearers and reelers are adversely affected by (cheap) imports of rawsilk by experiencing fall in price of rawsilk and cocoon. Though the weavers are supposed to be benefited by the cheap imports of rawsilk, it can’t be expected that the imports will continue to be cheap for ever. There are reports of closure of many reeling units (Vasumathi, 2000), the exact figures of which are not available. This indicates that the reeling industry is incapable of buffering against the shocks of price fall, presumably due to the small firm size. The farm sector is also reportedly affected by the downward price shifts of cocoon which is reflected on the down fall in acreage over the years (CSB, 2006). The area under mulberry plantation had increased from 170000 hectare during 1980-81to 343000 hectare during 1992-93. Since then it had fallen gradually to 172000 hectare during 2004-05. (CSB, 2006)

The phenomenal decline in mulberry area did not result in drastic downfall in cocoon production only because of productivity gains, thanks to the huge R&D expenditure borne by the government in the farm sector. The cocoon production during 1997-98 was 127495 tons. During 2004-05 it fell to 120027 tons. Raw silk production statistics also reveal the stagnation of the industry over the period, save a slight increase from 14048 tons to 14620 tons (Ministry of textiles, 2005). Thus sericulture farmers and silk reelers organized against cheap rawsilk imports to the country and demanded anti dumping sanctions.

Cheap imports benefit weaving sector

The silk weaving sector favored cheap imports of Chinese rawsilk. The organized power loom weavers represented to the DGAD in favour of Chinese rawsilk imports. To quote from their representation: ‘The indigenous prices have come down on account of increase in production and quality as a result of R & D. As against a total of 16000 tons of silk produced in the country, the current domestic demand is 23000 tons leaving a huge gap in demand and supply to the extent of 7000 metric tons. The demand-supply gap in mulberry raw silk exists basically for warp-grade silk for power looms. Warp-grade silk preferred by powerlooms can usually be had only from bivoltine races and is being met by imported Chinese bivoltine silk. There are more than 30000 powerlooms and 180000 handlooms in the country using pure silk yarn for manufacturing fabrics. In the production of pure silk fabrics, 6 handlooms are equal to 1 powerloom. Thus the production of both the sectors is almost equal. Both the sectors employ more than one million workers. If Chinese imports are restricted the powerlooms will stop production and the workers will be thrown out of employment’ (DGAD, 2003). Thus there started a hot debate between parties in favour and against cheap imports of Chinese silk.

Who purchase Chinese silk and why?

The argument that ‘the imported Chinese silk is consumed only by the power loom sector’ is to be taken with a pinch of salt. As per the official statistics over the six year period from 1999-2000 to 2004-05, the number of powerlooms in the country remained stagnant at 29340 where as the number of handlooms increased from 227701 to 258000, which is a 13% increase (Ministry of Textiles, 2006). During this period the domestic demand-supply gap of rawsilk increased from 5852 tons to 10180 tons which is 74% increase (COMTRADE, 2007). It takes only logic to understand that the excess demand has been being created by the handloom sector and that the imported Chinese silk were being absorbed by the handloom sector as well. Because of the price differential the natural choice of the hand looms would be imported Chinese silk. It is also important to note that during this period the number of reeling units (filature, charka and multi end) in the country has fallen from 60838 to 54846 (Ministry of Textiles, 2006). This means that 10% of the reeling units have closed down, as an after effect of the fall in price of rawsilk. It is clear that the increase in number of hand looms has not helped the domestic reeling industry by creating matching demand for domestic rawsilk. Thus the answer to the question ‘why Chinese silk?’ doesn’t seem to be ‘material quality’ but ‘price’ instead.

Argument of the (organized) power loom weavers that ‘the indigenous prices have come down on account of increase in production’ (DGAD, 2003) is not true. During the above said period the increase in domestic rawsilk production was from 15214 ton to 15445 ton, the percentage increase being only 1.5% (COMTRADE, 2007). This meager increase in production is incapable of creating a fall in rawsilk price, given the 74% increase in demand-supply gap over the same period. When the imported quantity is taken as percentage of domestic production it can be seen that over the six years period from 1999-00 to 2004-05 it has increased from 39% to 68%. The absolute quantity of ‘cheap imports’ has increased by 75% during this period (ibid). Thus the role played by the imported silk on the price fall of domestic raw silk is evident.

It is reported that the cocoon cost constitute about 80% of the silk yarn price (DGAD, 2006). Other studies show that the cocoon price constitutes nearly 90% of the yarn price (Vasumathi, 2000). Owing to this very little value addition takes place. The pricing of cocoons in turn is guided by government norms and the marketing of cocoons is done through regulated markets. Thus Reelers do not enjoy sufficient economic profits to purchase sophisticated reeling machinery and to adopt advanced processing technology or to establish more number of reeling units. Official sources report closure of 5992 reeling basins during the six year period from 1999-00 to 2004-05 (Ministry of Textiles, 2006).

Thus it is evidenced that the Indian reeling industry operates with wafer thin margins and might easily get whiffed-off in adverse situations. The sector is financially weak and has no bargaining power. Credit flow into the sector being extremely poor, purchase of cocoons and selling of raw silk in opportune situations is not possible. The government regulated cocoon market on the one hand is offering a tough competition for the reelers due to supply side domination. On the other hand, failure of the Silk Exchange in facilitating raw silk sale has placed them at the receiving end in the environs of a buyers market. The reeler is thus sandwiched between the domination of supply side on the one side and weaver or trader on the other. On top of this, the after effects of trade liberalization have added to the suffering of the reeling sector.

What is Dumping?

The term ‘dumping’ is used to denote ‘export of commodities at prices below the cost of production’. The most common definition of dumping at the WTO is ‘the sale of exports at prices below the prevailing prices in the domestic market’. Dumping is formally prohibited by Article VI of the GATT. Trade officials presume dumping as a good thing for the importing country (they are getting cheap merchandise) unless the country complains (usually because the cheap imports are threatening domestic producers). [For a theoretical explanation of ‘dumping’ please read the previous story in this blog].

So it is up to countries to put in place the national legislation they need to protect themselves from dumping, and the onus is on the country receiving the dumped production to prove harm to its domestic producers before anti-dumping duties can be imposed (Institute for Agriculture and Trade Policy, 2004). ‘Contingent protection (CTP)’ may be used by the governments to protect their domestic producers from unfair competition. Contingent protection measures fall under three categories – antidumping, countervailing and safeguard measures. Of these antidumping remains the most commonly used contingent protection measure. It proliferated in the 1990s and is now used extensively by developed and developing countries alike (Agarwal, 2003). The use of CTP implies a violation of one of the GATT fundamental principle, namely the reciprocity principle codified by article 28. There are theoretical explanations justifying the presence of CTP within WTO trade liberalizing agreements which show that the use of CTP is a necessary condition for optimal trade liberalization (Kohler, 2001). At the same time a growing body of literature criticizes the dramatic increase of anti dumping measures over the last two decades (Miranda, Torres and Ruiz, 1998; Prusa, 2001), the proponents of which believe that the rise in anti dumping (AD) activity can’t be solely explained by an increase in unfair trade. According to a recent study by Aggarwal (2002), the surge in the use of anti dumping measures by India cannot be justified on economic grounds.

Is China dumping silk in India?

Indian silk industry is a bundle of contradictions. Second largest producer of silk, the largest silk consumer and the largest importer of rawsilk. Unable to exploit export potential by equipping processing industry to value add cheap imported rawsilk. In spite of huge R&D investments and extension efforts for 15 years the (desired) technology diffusion in the farm sector is below 5%. The legislations and regulations favoring the farming sector have nearly suffocated the reeling industry. The processing industry is marked by low end technology use, lack of investment, poor quality produce and high cost of production.

Statistical evidence indicates that the agro industry has received blows due to cheap imports of rawsilk. However whether these cheap imports could be designated ‘dumping’ and whether anti dumping measures can save the situation are questions not easy to find answer but important enough to be probed in greater depths. In the mainstream economic theory, ‘predatory dumping’ requires fulfillment of a number of stringent conditions. For example the firm who has the predatory motive should have a dominant position in home as well as in the global market. Again, predator must be in a position to check entry of other firms to that market. Thus one possibility of the exporter using predatory power is when it has higher share in the total import of the product as well as in the total domestic consumption of the product. The share of the named country in total domestic consumption of the product or the ‘import penetration ratio’ (Dumped Imports as a Percentage of Domestic Consumption) should be calculated to verify whether there is predatory dumping.

There are not many well directed and theoretically sound economic studies in this area. The fact that such an interesting and important issue escaped scholastic attention is surprising. Considering the efforts made by the Government of India to modernize its silk industry vis-à-vis its dismal performance over the years raises the question whether any anti dumping and countervailing measures could save it from cheap imports and for how long such measures can prevail in a fast globalizing world? The situation demands a realistic and impartial investigation to arrive at a broad direction of policy in order to utilize the strengths of the domestic industry as well as to exploit the cheap raw material imports from China constructively for modernizing the domestic silk weaving industry in an export oriented perspective. The following fundamental studies are suggested as a ground work for better understanding the dynamics of the silk imports from China.

Assess and quantify the material injury suffered by the Indian reeling industry due to cheap imports of rawsilk.
Study the impact of cheap rawsilk imports on cocoon price and its consequent farm level implications.
Study how China is able to produce silk at relatively low cost
Establish whether the anti dumping measures adopted by India against Chinese rawsilk import are justifiable on economic grounds.
Study the extent of absorption of imported rawsilk by hand looms in the country over the years and to analyze the dynamics of imported silk.
Analyze the performance of sericulture farm sector and reeling sector before and after the imposition of anti dumping duties on Chinese silk.
Understand and document the factors responsible for the reportedly low end technology use by the reeling sector.
Evaluate whether the country is prepared for producing sufficient quantities of high quality silk with in the time span offered by AD and CV measures against silk imports from China by analyzing a) the extent of diffusion of the bivoltine hybrid, b) the price differential enjoyed by the bivoltine hybrid rearers and bivoltine silk reelers over those subscribing to traditional and cross breed silk worm/ silk c) whether the domestic reeling sector is prepared absorb the high quality cocoons produced by the farm sector and process it to produce high quality of silk usable in power looms

Reference:

Aggarwal A (2002): ‘‘Anti Dumping Law and Practice: An Indian Perspective’’, Indian Council Research on International Economic Relations (ICRIER) Working Paper, No. 85

Aggarwal A (2003): The WTO Antidumping Code: Issues for Review in Post-Doha Negotiations, ICRIER working paper No. 99

Baruah Nandana (2005): Anti Dumping Duty As A Measure Of Contingent Protection: An Analysis Of Indian Experience. Center for Development Studies Working paper No. 377

COMTRADE (2007): United Nations, Comtrade Data Base (New York: United Nations Statistical Office, 2007).

CSB (2006): annual report of Central Silk Board 2005.

Curry Ronald (1997): Global silk industry: today and tomorrow. Indian silk vol 35., No. 12, 1997

Czako J, Human J and J Mirinda (2003): A Handbook of Anti dumping Investigations, Cambridge University Press, UK

DGAD (2003): Notification No: 14/28/2002-DGAD DATE: 03/07/2003 Anti dumping Investigations Concerning Imports of Mulberry Raw Silk (not thrown) originating in or Exported from China PR.-Final Findings.

Economic development associates (1990): Manual silk reeling in south India, a preliminary study of technology transfer. Report by Economic Development associates

Feinberg R M. and B T Hirsch (1989): “Industry Rent Seeking and the Filing of ‘Unfair Trade’ complaints,” International Journal of Industrial Organization, 7 (3) 325-340

Institute for Agriculture and Trade Policy (2004): Glossary for the WTO agreement on agriculture

Kohler Philippe (2001): The role of contingent protection in WTO agreements. Working version, GEM, Institut d’Etudes Politiques de Paris – 2, square de Luynes – 75007 Paris.

Ministry of Textiles, GOI (2005): Sericulture Industry a report.

Miranda, Jorge, Raul A. Torres and Mario Ruiz. (1998): “The International Use of Antidumping: 1987 – 1997.” Journal of World Trade 32(5), pp. 5-71.

Naik Gopal and Babu KH, (1993): Demand and Supply Prospects for High Quality Silk, Oxford & IBH Publishing Co. Pvt. Ltd, New Delhi.

Prusa T (2001): ‘‘On the spread and Impact of Anti dumping’’ Canadian Journal of Economics 34 (3) 591-611.

Prusa T and S Skeath (2001): ‘‘The Economic and Strategic Motives for Antidumping Filings’’, NBER Working Paper, No. 8424.

Prusa, Thomas J. (2001): “On the Spread and Impact of Antidumping.” Canadian Journal

Tikku .MK. (1999): Tangled threads: Silk growers and imports EPW, March 6-13., 1999

Vasumathi. B. V (2000): An Analytical Study of the Silk Reeling Operations in Karnataka, PhD thesis, Department Of Management Studies Indian Institute Of Science, Bangalore, India.

RESEARCH UPDATES-04

gkrajeshrajesh@gmail.com — Fri, 01 May 2009 07:14:00 +0000

Silk as a fiber of remarkable physical properties has attracted human attention since its origins. However it has not stopped enticing human interest and imagination over 5000 years. The more it was investigated the more mysterious its properties became. The recent developments in silk protein research has revealed its possible applications as/in new generation of biomaterials, scaffold materials for tissue engineering, designer biomaterial for medical, commercial, and military applications, nano technology etc. The picture is still emerging. Silk will have much more in store for the ever inquisitive human mind and hopefully will tie it down more towards bio materials, in opposition to its desire to break free towards “cheaply manufacturable” synthetic materials. This however is heartening news for the silk enthusiast. Here we provide citations of 22 recent research papers on this topic. No effort has been made to arrange the papers under any particular categorization. Papers on both mulberry silk and spider silk are included.

1. Engineered disulfides improve mechanical properties of recombinant spider silk.
S Grip, J Johansson, and M Hedhammar
Protein Sci, March 16, 2009; 18(5): 1012-1022.

Nature's high-performance polymer, spider silk, is composed of specific proteins, spidroins, which form solid fibers. So far, fibers made from recombinant spidroins have failed in replicating the extraordinary mechanical properties of the native material. A recombinant miniature spidroin consisting of four poly-Ala/Gly-rich tandem repeats and a nonrepetitive C-terminal domain (4RepCT) can be isolated in physiological buffers and undergoes self assembly into macrofibers. Herein, we have made a first attempt to improve the mechanical properties of 4RepCT fibers by selective introduction of AA --> CC mutations and by letting the fibers form under physiologically relevant redox conditions. Introduction of AA --> CC mutations in the first poly-Ala block in the miniature spidroin increases the stiffness and tensile strength without changes in ability to form fibers, or in fiber morphology. These improved mechanical properties correlate with degree of disulfide formation. AA --> CC mutations in the forth poly-Ala block, however, lead to premature aggregation of the protein, possibly due to disulfide bonding with a conserved Cys in the C-terminal domain. Replacement of this Cys with a Ser, lowers thermal stability but does not interfere with dimerization, fiber morphology or tensile strength. These results show that mutagenesis of 4RepCT can reveal spidroin structure-activity relationships and generate recombinant fibers with improved mechanical properties. http://highwire.stanford.edu/cgi/medline/pmid;19388023

2. Advancing Towards a Tissue-Engineered Tympanic Membrane: Silk Fibroin as a Substratum for Growing Human Eardrum Keratinocytes

Reza Ghassemifar, Sharon Redmond, . Zainuddin, and Traian V Chirila

Journal of Biomaterials Applications 2009, doi: 10.1177/0885328209104289

Human tympanic membrane cells (hTMCs), harvested from tympanicmembrane (TM) explants, were grown in culture and then seededon membranes prepared from silkworm (Bombyx mori) silk fibroin(BMSF) and on tissue-culture plastic membranes (PET). Fibroinwas isolated from silk cast into membranes with a thicknessof 10–15 µm. The hTMCs were cultured on both materialsfor 15 days in a serum-containing culture medium. The cellsgrown on both substrata were subjected to nuclear staining (DAPI)and counted. Further, the cultures were immunostained for anumber of protein markers related to the epithelial/keratinocytephenotype and cell adhesion complexes. The BMSF membranes supportedlevels of hTMC growth higher than that observed on the PET membranes.The immunofluorochemical analysis indicated unequivocally thatBMSF is a more suitable substratum than PET with respect tothe growth patterns, proliferation, and cell–cell contactand adhesion. BMSF appear as a promising substratum in the tissue-engineeredconstructs for the replacement of TM in case of nonhealing perforations. http://jba.sagepub.com/cgi/content/abstract/0885328209104289v1

3. Greatly increased toughness of infiltrated spider silk.

Lee SM, Pippel E, Gösele U, Dresbach C, Qin Y, Chandran CV, Bräuniger T, Hause G, Knez M.

Science. 2009 Apr 24;324(5926):488-92

In nature, tiny amounts of inorganic impurities, such as metals, are incorporated in the protein structures of some biomaterials and lead to unusual mechanical properties of those materials. A desire to produce these biomimicking new materials has stimulated materials scientists, and diverse approaches have been attempted. In contrast, research to improve the mechanical properties of biomaterials themselves by direct metal incorporation into inner protein structures has rarely been tried because of the difficulty of developing a method that can infiltrate metals into biomaterials, resulting in a metal-incorporated protein matrix. We demonstrated that metals can be intentionally infiltrated into inner protein structures of biomaterials through multiple pulsed vapor-phase infiltration performed with equipment conventionally used for atomic layer deposition (ALD). We infiltrated zinc (Zn), titanium (Ti), or aluminum (Al), combined with water from corresponding ALD precursors, into spider dragline silks and observed greatly improved toughness of the resulting silks. The presence of the infiltrated metals such as Al or Ti was verified by energy-dispersive x-ray (EDX) and nuclear magnetic resonance spectra measured inside the treated silks. This result of enhanced toughness of spider silk could potentially serve as a model for a more general approach to enhance the strength and toughness of other biomaterials. http://www.ncbi.nlm.nih.gov/pubmed/19390040?ordinalpos=1&itool=Email.EmailReport.Pubmed_ReportSelector.Pubmed_RVDocSum

4. Spider silk fibers spun from soluble recombinant silk produced in mammalian cells.

Lazaris A, Arcidiacono S, Huang Y, Zhou JF, Duguay F, Chretien N, Welsh EA, Soares JW, Karatzas CN.

Science. 2002 Jan 18; 295(5554):472-6

Spider silks are protein-based "biopolymer" filaments or threads secreted by specialized epithelial cells as concentrated soluble precursors of highly repetitive primary sequences. Spider dragline silk is a flexible, lightweight fiber of extraordinary strength and toughness comparable to that of synthetic high-performance fibers. We sought to "biomimic" the process of spider silk production by expressing in mammalian cells the dragline silk genes (ADF-3/MaSpII and MaSpI) of two spider species. We produced soluble recombinant (rc)-dragline silk proteins with molecular masses of 60 to 140 kilodaltons. We demonstrated the wet spinning of silk monofilaments spun from a concentrated aqueous solution of soluble rc-spider silk protein (ADF-3; 60 kilodaltons) under modest shear and coagulation conditions. The spun fibers were water insoluble with a fine diameter (10 to 40 micrometers) and exhibited toughness and modulus values comparable to those of native dragline silks but with lower tenacity. Dope solutions with rc-silk protein concentrations >20% and postspinning draw were necessary to achieve improved mechanical properties of the spun fibers. Fiber properties correlated with finer fiber diameter and increased birefringence. http://www.ncbi.nlm.nih.gov/pubmed/11799236?ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_DiscoveryPanel.Pubmed_Discovery_RA&linkpos=1&log$=relatedarticles&logdbfrom=pubmed

5. The elaborate structure of spider silk: structure and function of a natural high performance fiber.

Römer L, Scheibel T.

Prion. 2008 Oct;2(4):154-61. Epub 2008 Oct 20

Universität Bayreuth, Fakultät für angew. Naturwissenschaften, Lehrstuhl für Biomaterialien, Bayreuth, Germany.

Biomaterials, having evolved over millions of years, often exceed man-made materials in their properties. Spider silk is one outstanding fibrous biomaterial which consists almost entirely of large proteins. Silk fibers have tensile strengths comparable to steel and some silks are nearly as elastic as rubber on a weight to weight basis. In combining these two properties, silks reveal a toughness that is two to three times that of synthetic fibers like Nylon or Kevlar. Spider silk is also antimicrobial, hypoallergenic and completely biodegradable. This article focuses on the structure-function relationship of the characterized highly repetitive spider silk spidroins and their conformational conversion from solution into fibers. Such knowedge is of crucial importance to understanding the intrinsic properties of spider silk and to get insight into the sophisticated assembly processes of silk proteins. This review further outlines recent progress in recombinant production of spider silk proteins and their assembly into distinct polymer materials as a basis for novel products. http://www.ncbi.nlm.nih.gov/pubmed/19221522?ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_DiscoveryPanel.Pubmed_Discovery_RA&linkpos=5&log$=relatedreviews&logdbfrom=pubmed

6. The mechanical design of spider silks: from fibroin sequence to mechanical function.

Gosline JM, Guerette PA, Ortlepp CS, Savage KN.

J Exp Biol. 1999 Dec;202(Pt 23):3295-303

Spiders produce a variety of silks, and the cloning of genes for silk fibroins reveals a clear link between protein sequence and structure-property relationships. The fibroins produced in the spider's major ampullate (MA) gland, which forms the dragline and web frame, contain multiple repeats of motifs that include an 8-10 residue long poly-alanine block and a 24-35 residue long glycine-rich block. When fibroins are spun into fibres, the poly-alanine blocks form (&bgr;)-sheet crystals that crosslink the fibroins into a polymer network with great stiffness, strength and toughness. As illustrated by a comparison of MA silks from Araneus diadematus and Nephila clavipes, variation in fibroin sequence and properties between spider species provides the opportunity to investigate the design of these remarkable biomaterials.

Full paper: http://jeb.biologists.org/cgi/reprint/202/23/3295

7. Comparative architecture of silks, fibrous proteins and their encoding genes in insects and spiders

Craig CL, Riekel C.

Comp Biochem Physiol B Biochem Mol Biol. 2002 Dec;133(4):493-507

The known silk fibroins and fibrous glues are thought to be encoded by members of the same gene family. All silk fibroins sequenced to date contain regions of long-range order (crystalline regions) and/or short-range order (non-crystalline regions). All of the sequenced fibroin silks (Flag or silk from flagelliform gland in spiders; Fhc or heavy chain fibroin silks produced by Lepidoptera larvae) are made up of hierarchically organized, repetitive arrays of amino acids. Fhc fibroin genes are characterized by a similar molecular genetic architecture of two exons and one intron, but the organization and size of these units differs. The Flag, Ser (sericin gene) and BR (Balbiani ring genes; both fibrous proteins) genes are made up of multiple exons and introns. Sequences coding for crystalline and non-crystalline protein domains are integrated in the repetitive regions of Fhc and MA exons, but not in the protein glues Ser1 and BR-1. Genetic 'hot-spots' promote recombination errors in Fhc, MA, and Flag. Codon bias, structural constraint, point mutations, and shortened coding arrays may be alternative means of stabilizing precursor mRNA transcripts. Differential regulation of gene expression and selective splicing of the mRNA transcript may allow rapid adaptation of silk functional properties to different physical environments. http://www.ncbi.nlm.nih.gov/pubmed/12470814?ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_DiscoveryPanel.Pubmed_Discovery_RA&linkpos=5&log$=relatedreviews&logdbfrom=pubmed

8. Biotechnological production of spider-silk proteins enables new applications.

Vendrely C, Scheibel T.

Macromol Biosci. 2007 Apr 10;7(4):401-9

The outstanding mechanical properties of spider silks have motivated many researchers to establish biotechnological production techniques which are necessary to provide sufficient amounts of silk proteins for industrial applications. Based on recent developments in genetic engineering, two strategies for the recombinant production of spider-silk proteins have been established which are discussed in detail. Further, protein-design strategies are described, enabling the combination of silk properties with additional biological, chemical, or technical features. We highlight the potential of engineered and recombinantly-produced spider-silk proteins to provide the basis for a new generation of biomaterials. http://www.ncbi.nlm.nih.gov/pubmed/17429812?ordinalpos=4&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_DefaultReportPanel.Pubmed_RVDocSum

9. A protocol for the production of recombinant spider silk-like proteins for artificial fiber spinning.

Teulé F, Cooper AR, Furin WA, Bittencourt D, Rech EL, Brooks A, Lewis RV.

Nat Protoc. 2009;4(3):341-55

The extreme strength and elasticity of spider silks originate from the modular nature of their repetitive proteins. To exploit such materials and mimic spider silks, comprehensive strategies to produce and spin recombinant fibrous proteins are necessary. This protocol describes silk gene design and cloning, protein expression in bacteria, recombinant protein purification and fiber formation. With an improved gene construction and cloning scheme, this technique is adaptable for the production of any repetitive fibrous proteins, and ensures the exact reproduction of native repeat sequences, analogs or chimeric versions. The proteins are solubilized in 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) at 25-30% (wt/vol) for extrusion into fibers. This protocol, routinely used to spin single micrometer-size fibers from several recombinant silk-like proteins from different spider species, is a powerful tool to generate protein libraries with corresponding fibers for structure-function relationship investigations in protein-based biomaterials. This protocol may be completed in 40 d. http://www.ncbi.nlm.nih.gov/pubmed/19229199?ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_DiscoveryPanel.Pubmed_Discovery_RA&linkpos=2&log$=relatedarticles&logdbfrom=pubmed

10. Probing the elastic nature of spider silk in pursuit of the next designer fiber.

Brooks AE, Lewis RV.

Biomed Sci Instrum. 2004;40:232-7

Spider silk, one of nature's greatest accomplishments, has a combination of strength and elasticity that is unrivaled. Spiders produce up to 7 different silks; each one with a unique combination of tensile strength and elasticity that allows the spiders' web to hold prey while being resilient enough not to break upon impact. In an attempt to determine the sequences responsible for these enviable mechanical properties, several different amino acid motifs have been defined. Much of the recent work is now concentrated on correlating amino acid motifs with a specific mechanical property. The current hypothesis is that the strength property of spider silk is conferred by a poly-alanine or alanine rich (An or GAn) motif whereas the elastic nature of spider silk is conferred by the amino acid motif, GPGXX, where X is Q, S, A, G, or Y. Despite the fact that these different motifs are now known, the combination of strength and elasticity are yet to be duplicated ex vivo. In an attempt to verify that the GPGXX motif imparts elasticity to the spider silk, the number of repeats and/or the amino acid composition of the Argiope aurantia "elastic motif" were varied and expressed in various strains of E. coli to change the elastic properties of the resulting film and/or fiber. Concurrent with work on the elasticity motif is ongoing work on the strength module. Understanding these two different motifs will aide efforts to produce a designer biomaterial for medical, commercial, and military applications. http://www.ncbi.nlm.nih.gov/pubmed/15133963?ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_DiscoveryPanel.Pubmed_Discovery_RA&linkpos=4&log$=relatedarticles&logdbfrom=pubmed

11. Design, expression and solid-state NMR characterization of silk-like materials constructed from sequences of spider silk, Samia cynthia ricini and Bombyx mori silk fibroins.

Yang M, Asakura T.

J Biochem. 2005 Jun;137(6):721-9

Silk has a long history of use in medicine as sutures. To address the requirements of a mechanically robust and biocompatible material, basic research to clarify the role of repeated sequences in silk fibroin in its structures and properties seems important as well as the development of a processing technique suitable for the preparation of fibers with excellent mechanical properties. In this study, three silk-like protein analogs were constructed from two regions selected from among the crystalline region of Bombyx mori silk fibroin, (GAGSGA)(2), the crystalline region of Samia cynthia ricini silk fibroin, (Ala)(12), the crystalline region of spider dragline silk fibroin, (Ala)(6), and the Gly-rich region of spider silk fibroin, (GGA)(4). The silk-like protein analog constructed from the crystalline regions of the spider dragline silk and B. mori silk fibroins, (A(6)SCS)(8), that constructed from the crystalline regions of the S. c.ricini and B. mori silk fibroins, (A(12)SGS)(4), that constructed from and the crystalline region of S. c.ricini silk fibroin and the glycine-rich region of spider dragline silk fibroin, (A(12)SGS)(4),were expressed their molecular weights being about 36.0 kDa, 17.0 kDa and 17.5 kDa, respectively in E. coli by means of genetic engineering technologies. (A(12)SCS)(4) and (A(12)SGS)(4 )undergo a structural transition from alpha-helix to beta-sheet on a change in the solvent treatment from trifluoroacetic acid (TFA) to formic acid (FA). However, (A(6)SCS)(8) takes on the beta-sheet structure predominantly on TFA treatment and FA treatment. Structural analysis was performed on model peptides selected from spider dragline and S. c.ricini silks by means of (13)C CP/MAS NMR. http://www.ncbi.nlm.nih.gov/pubmed/16002994?ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_DiscoveryPanel.Pubmed_Discovery_RA&linkpos=5&log$=relatedarticles&logdbfrom=pubmed

12. Structures of Bombyx mori and Samia cynthia ricini silk fibroins studied with solid-state NMR.

Yao J, Nakazawa Y, Asakura T.

Biomacromolecules. 2004 May-Jun;5(3):680-8

There are many kinds of silks spun by silkworms and spiders, which are suitable to study the structure-property relationship for molecular design of fibers with high strength and high elasticity. In this review, we mainly focus on the structural determination of two well-known silk fibroin proteins that are from the domesticated silkworm, Bombyx mori, and the wild silkworm, Samia cynthia ricini, respectively. The structures of B. mori silk fibroin before and after spinning were determined by using an appropriate model peptide, (AG)(15), with several solid-state NMR methods; (13)C two-dimensional spin-diffusion solid-state NMR and rotational echo double resonance (REDOR) NMR techniques along with the quantitative use of the conformation-dependent (13)C CP/MAS chemical shifts. The structure of S. c. ricini silk fibroin before spinning was also determined by using a model peptide, GGAGGGYGGDGG(A)(12)GGAGDGYGAG, which is a typical repeated sequence of the silk fibroin, with the solid-state NMR methods. The transition from the structure of B. mori silk fibroin before spinning to the structure after spinning was studied with molecular dynamics calculation by taking into account several external forces applied to the silk fibroin in the silkworm. http://www.ncbi.nlm.nih.gov/pubmed/15132647?ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_DiscoveryPanel.Pubmed_Discovery_RA&linkpos=1&log$=relatedarticles&logdbfrom=pubmed

13. Synthesis and characterization of chimeric silkworm silk.

Asakura T, Nitta K, Yang M, Yao J, Nakazawa Y, Kaplan DL.

Biomacromolecules. 2003 May-Jun;4(3):815-20

A synthetic gene encoding a chimeric silklike protein was constructed that combined a polyalanine encoding region (Ala)(18), a sequence slightly longer than the (Ala)(12-13) found in the silk fibroin from the wild silkworm Samia cynthia ricini, and a sequence encoding GVGAGYGAGAGYGVGAGYGAGVGYGAGAGY, found in the silk fibroin from the silkworm Bombyx mori. A tetramer of the chimeric repeat sequence encoding a approximately 29 kDa protein was expressed as a fusion protein in Escherichia coli. In comparison to S. c. ricini silk, the chimeric protein demonstrated improved solubility because it could be dissolved in 8 M urea. The purified protein assumed an alpha-helical structure based on solid-state (13)C CP/MAS NMR and was less prone to conformational transition to a beta-sheet, unlike native silk proteins from S. c. ricini. Model peptides representing the crystalline region of S. c. ricini silk fibroin, (Ala)(12) and (Ala)(18), formed beta-sheet structures. Therefore, the solubility and structural transitions of the chimeric protein were significantly altered through the formation of this chimeric silk. This experimental strategy to the study of silk structure and function can be used to develop an improved understanding of the contributions of protein domains in repetitive silkworm and spider silk sequences to structure development and structural transitions. http://www.ncbi.nlm.nih.gov/pubmed/12741803?ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_DiscoveryPanel.Pubmed_Discovery_RA&linkpos=3&log$=relatedarticles&logdbfrom=pubmed

14. Silk fibroin: structural implications of a remarkable amino acid sequence

Zhou CZ, Confalonieri F, Jacquet M, Perasso R, Li ZG, Janin J.

Proteins. 2001 Aug 1;44(2):119-22

The amino acid sequence of the heavy chain of Bombyx mori silk fibroin was derived from the gene sequence. The 5,263-residue (391-kDa) polypeptide chain comprises 12 low-complexity "crystalline" domains made up of Gly-X repeats and covering 94% of the sequence; X is Ala in 65%, Ser in 23%, and Tyr in 9% of the repeats. The remainder includes a nonrepetitive 151-residue header sequence, 11 nearly identical copies of a 43-residue spacer sequence, and a 58-residue C-terminal sequence. The header sequence is homologous to the N-terminal sequence of other fibroins with a completely different crystalline region. In Bombyx mori, each crystalline domain is made up of subdomains of approximately 70 residues, which in most cases begin with repeats of the GAGAGS hexapeptide and terminate with the GAAS tetrapeptide. Within the subdomains, the Gly-X alternance is strict, which strongly supports the classic Pauling-Corey model, in which beta-sheets pack on each other in alternating layers of Gly/Gly and X/X contacts. When fitting the actual sequence to that model, we propose that each subdomain forms a beta-strand and each crystalline domain a two-layered beta-sandwich, and we suggest that the beta-sheets may be parallel, rather than antiparallel, as has been assumed up to now. Copyright 2001 Wiley-Liss, Inc. http://www.ncbi.nlm.nih.gov/pubmed/11391774?ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_DiscoveryPanel.Pubmed_Discovery_RA&linkpos=4&log$=relatedreviews&logdbfrom=pubmed

15. Structural study of irregular amino acid sequences in the heavy chain of Bombyx mori silk fibroin.

Ha SW, Gracz HS, Tonelli AE, Hudson SM.

Biomacromolecules. 2005 Sep-Oct;6(5):2563-9

Recently, genetic studies have revealed the entire amino acid sequence of Bombyx mori silk fibroin. It is known from X-ray diffraction studies that the beta-sheet crystalline structure (silk II) of fibroin is composed of hexaamino acid sequences of GAGAGS. However, in the heavy chain of B. mori silk fibroin, there are also present 11 irregular sequences, with about 31 amino acid residues (irregular GT approximately GT sequences). The structure and role of these irregular sequences have remained unknown. One of the most frequently appearing irregular sequences was synthesized and its 3-D solution structure was studied by high-resolution 2-D NMR techniques. The 3-D structure determined for this peptide shows that it makes a loop structure (distorted omega shape), which implies that the preceding backbone direction is changed by 180 degrees, i.e., reversed, by this sequence. This may facilitate the beta-sheet formation between the crystal-forming building blocks, GAGAGS/GY approximately GY sequences, in the fibroin heavy chain. http://www.ncbi.nlm.nih.gov/pubmed/16153093?ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_DiscoveryPanel.Pubmed_Discovery_RA&linkpos=3&log$=relatedarticles&logdbfrom=pubmed

16. New silk protein: modification of silk protein by gene engineering for production of biomaterials

Mori H, Tsukada M.

J Biotechnol. 2000 Aug;74(2):95-103

The interest in silk fibroin morphology and structure have increased due to its attractiveness for bio-related applications. Silk fibers have been used as sutures for a long time in the surgical field, due to the biocompatibility of silk fibroin fibers with human living tissue. In addition, it has been demonstrated that silk can be used as a substrate for enzyme immobilization in biosensors. A more complete understanding of silk structure would provide the possibility to further exploit silk fibroin for a wide range of new uses, such as the production of oxygen-permeable membranes and biocompatible materials. Silk fibroin-based membranes could be utilized as soft tissue compatible polymers. Baculovirus-mediated transgenesis of the silkworm allows specific alterations in a target sequence. Homologous recombination of a foreign gene downstream from a powerful promoter, such as the fibroin promoter, would allow the constitutive production of a useful protein in the silkworm and the modification of the character of silk protein. A chimeric protein consisted of fibroin and green fluorescent protein was expressed under the control of fibroin in the posterior silk gland and the gene product was spun into the cocoon layer. This technique, gene targeting, will lead to the modification and enhancement of physicochemical properties of silk protein. http://www.ncbi.nlm.nih.gov/pubmed/11763506?ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_DiscoveryPanel.Pubmed_Discovery_RA&linkpos=5&log$=relatedreviews&logdbfrom=pubmed

17. Silk fibroin protein from mulberry and non-mulberry silkworms: cytotoxicity, biocompatibility and kinetics of L929 murine fibroblast adhesion

Acharya C, Ghosh SK, Kundu SC.

J Mater Sci Mater Med. 2008 Aug;19(8):2827-36. Epub 2008 Mar 6

Silks fibers and films fabricated from fibroin protein of domesticated mulberry silkworm cocoon have been traditionally utilized as sutures in surgery and recently as biomaterial films respectively. Here, we explore the possibility of application of silk fibroin protein from non-mulberry silkworm cocoon as a potential biomaterial aid. In terms of direct inflammatory potential, fibroin proteins from Antheraea mylitta and Bombyx mori are immunologically inert and invoke minimal immune response. Stimulation of murine peritoneal macrophages and RAW 264.7 murine macrophages by these fibroin proteins both in solution and in the form of films assayed in terms of nitric oxide and TNFalpha production showed comparable stimulation as in collagen. Kinetics of adhesion of L929 murine fibroblasts, for biocompatibility evaluation, monitored every 4 h from seeding and studied over a period of 24 h, reveal A. mylitta fibroin film to be a better substrate in terms of rapid and easier cellularization. Cell viability studies by MTT assay and flow cytometric analyses indicate the ability of fibroin matrices to support cell growth and proliferation comparable to collagen for long-term culture. This matrix may have potential to serve in those injuries where rapid cellularization is essential. http://www.ncbi.nlm.nih.gov/pubmed/18322779?ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_DiscoveryPanel.Pubmed_Discovery_RA&linkpos=5&log$=relatedarticles&logdbfrom=pubmed

18. Growth of human cells on a non-woven silk fibroin net: a potential for use in tissue engineering.

Unger RE, Wolf M, Peters K, Motta A, Migliaresi C, James Kirkpatrick C.

Biomaterials. 2004 Mar;25(6):1069-75

We have examined a novel biomaterial consisting of a non-woven fibroin net produced from silk (Bombyx mori) cocoons for its ability to support the growth of human cells. Various human cells of different tissue and cell types (endothelial, epithelial, fibroblast, glial, keratinocyte, osteoblast) were examined for adherence and growth on the nets by confocal laser microscopy after staining of the cells with calcein-AM and by electron microscopy. All the cells readily adhered and spread over the individual fibers of the nets. Most of the cells were able to grow and survive on the nets for at least 7 weeks and growth not only covered the individual fibers of the net but generally bridged the gaps between individual fibers forming tissue-like structures. Scanning electron microscopic examination of the nets demonstrated a tight association of individual cells with the fibers and nets examined after removal of cells showed no evidence that the growth of cells in any way changed the structure of the fibers. Thus, silk fibroin nets are highly human cell-compatible and should be a useful new scaffolding biomaterial applicable for a wide range of target tissues in addition to supporting endothelial cells required for the vascularization of the newly formed tissue. http://www.ncbi.nlm.nih.gov/pubmed/14615172?ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_DiscoveryPanel.Pubmed_Discovery_RA&linkpos=4&log$=relatedarticles&logdbfrom=pubmed

19. Molecular mechanisms of spider silk.

Hu X, Vasanthavada K, Kohler K, McNary S, Moore AM, Vierra CA.

Cell Mol Life Sci. 2006 Sep;63(17):1986-99

Spiders spin high-performance silks through the expression and assembly of tissue-restricted fibroin proteins. Spider silks are composite protein biopolymers that have complex microstructures. Retrieval of cDNAs and genomic DNAs encoding silk fibroins has revealed an association between the protein sequences and structure-property relationships. However, before spider silks can be subject to genetic engineering for commercial applications, the complete protein sequences and their functions, as well as the details of the spinning mechanism, will require additional progress and collaborative efforts in the areas of biochemistry, molecular biology and material science. Novel approaches to reveal additional molecular constituents embedded in the spider fibers, as well as cloning strategies to manipulate the genes for expression, will continue to be important aspects of spider biology research. Here we summarize the molecular characteristics of the different spider fibroins, the mechanical properties and assembly process of spidroins and the advances in protein expression systems used for recombinant silk production. We also highlight different technical approaches being used to elucidate the molecular constituents of silk fibers. http://www.find-health-articles.com/rec_pub_16819558-molecular-mechanisms-spider-silk.htm

20. Silk-based biomaterials.

Altman GH, Diaz F, Jakuba C, Calabro T, Horan RL, Chen J, Lu H, Richmond J, Kaplan DL.

Biomaterials. 2003 Feb;24(3):401-16

Silk from the silkworm, Bombyx mori, has been used as biomedical suture material for centuries. The unique mechanical properties of these fibers provided important clinical repair options for many applications. During the past 20 years, some biocompatibility problems have been reported for silkworm silk; however, contamination from residual sericin (glue-like proteins) was the likely cause. More recent studies with well-defined silkworm silk fibers and films suggest that the core silk fibroin fibers exhibit comparable biocompatibility in vitro and in vivo with other commonly used biomaterials such as polylactic acid and collagen. Furthermore, the unique mechanical properties of the silk fibers, the diversity of side chain chemistries for 'decoration' with growth and adhesion factors, and the ability to genetically tailor the protein provide additional rationale for the exploration of this family of fibrous proteins for biomaterial applications. For example, in designing scaffolds for tissue engineering these properties are particularly relevant and recent results with bone and ligament formation in vitro support the potential role for this biomaterial in future applications. To date, studies with silks to address biomaterial and matrix scaffold needs have focused on silkworm silk. With the diversity of silk-like fibrous proteins from spiders and insects, a range of native or bioengineered variants can be expected for application to a diverse set of clinical needs. http://www.ncbi.nlm.nih.gov/pubmed/12423595?ordinalpos=15&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_DefaultReportPanel.Pubmed_RVDocSum

21. Skeletal tissue engineering using silk biomaterials

MacIntosh AC, Kearns VR, Crawford A, Hatton PV.

J Tissue Eng Regen Med. 2008 Mar-Apr;2(2-3):71-80

Silks have been proposed as potential scaffold materials for tissue engineering, mainly because of their physical properties. They are stable at physiological temperatures, flexible and resist tensile and compressive forces. Bombyx mori (silkworm) cocoon silk has been used as a suture material for over a century, and has proved to be biocompatible once the immunogenic sericin coating is removed. Spider silks have a similar structure to silkworm silk but do not have a sericin coating. This paper provides a general overview on the use of silk protein in biomaterials, with a focus on skeletal tissue engineering. http://www.ncbi.nlm.nih.gov/pubmed/18383453?ordinalpos=7&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_DefaultReportPanel.Pubmed_RVDocSum

22. Spider silks and their applications

Kluge JA, Rabotyagova O, Leisk GG, Kaplan DL.

Trends Biotechnol. 2008 May;26(5):244-51. Epub 2008 Mar 25

Spider silks are characterized by remarkable diversity in their chemistry, structure and functions, ranging from orb web construction to adhesives and cocoons. These unique materials have prompted efforts to explore potential applications of spider silk equivalent to those of silkworm silks, which have undergone 5,000 years of domestication and have a variety of uses, from textiles to biomedical materials. Recent progress in genetic engineering of spider silks and the development of new chimeric spider silks with enhanced functions and specific characteristics have advanced spider silk technologies. Further progress in yields of expressed spider-silk proteins, in the control of self-assembly processes and in the selective exploration of material applications is anticipated in the future. The unique features of spider silks, the progress and challenges in the cloning and expression of these silks, environmentally triggered silk assembly and disassembly and the formation of fibers, films and novel chimeric composite materials from genetically engineered spider silks will be reviewed. http://www.ncbi.nlm.nih.gov/pubmed/18367277?ordinalpos=5&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_DefaultReportPanel.Pubmed_RVDocSum

TEMPERATURE STRESS INDUCED TRANSCRIPTION AND ITS POSSIBLE APPLICATION IN SILKWORM BREED IMPROVEMENT.

gkrajeshrajesh@gmail.com — Wed, 22 Apr 2009 14:16:00 +0000

The mulberry silkworm (Bombyx mori L.) is one of the most thermal-sensitive organisms. Intensive and careful domestication over centuries has apparently deprived this taxon of opportunities to acquire thermo tolerance. This vulnerability is more pronounced in bivoltine races compared to polyvoltine ones. Thus, among many factors attributed to poor performance of the bivoltine strains under tropical conditions, the major one is lack of thermo tolerance. Many quantitative characters decline sharply at higher temperatures. Therefore, one of the key considerations in developing bivoltine hybrids for tropics could be need for thermo tolerant bivoltine races. This could be achieved through hybridization of polyvoltine with bivoltine races which is a prolonged and tedious procedure mainly due to the delay in fixation of economic characters. In India earlier efforts in this direction using Pure Mysore, a comparatively robust but poor silk yielding Indian race did not yield expected outcome which pointed out that- economic cocoon traits were negatively correlated with high temperate resistance (Tazima & Ohnuma, 1995). The recent advances in silkworm breeding and those in stress induced protein synthesis have opened up new avenues to evolve robust productive silkworm hybrids.

The Phenomenon of Heat Shock:

Heat shock is nothing but a thermal injury caused by sudden increase in temperature in biological molecules like DNA, RNA, lipids, etc., of the cell which are vulnerable to heat stress. This leads to a number of abnormalities at cellular level. Normal pattern of protein synthesis halts. Transfer RNA and ribosomal RNA loose conformational integrity leading to degradation. DNA looses ability to function properly. There is aggregation of intermediate filamentous proteins at the nucleus instead of forming the cytoskeleton. At the same time pH of body fluid also drops. Increase of temperature leads to increase in kinetic energy of macromolecules, decrease of ionic bonds, hydrogen bonds, Van - der - Waals bonds etc., and increase its hydrophobic interactions; leading to loss of its shape. Denatured proteins also get adhered to DNA and restrict enzymatic access to DNA causing large-scale DNA damage. The heat shock thus ultimately leads to death of cells.

Another most important effect of temperature (or stress of any kind) is on cellular proteins by unfolding them. Cellular proteins are typically folded in their native conformations while functioning in cells. They unfold at the following contexts: 1) during de novo synthesis of polypeptides and assembly of multimetric proteins; 2) during intra cellular transport and organellar import when a protein must unfold or remain unfolded to cross the boundary of a cellular compartment; 3) during or after exposure to a protein denaturing stress. At these times unfolded proteins may be susceptible to inappropriate interactions with one another or with other cellular components. More over once unfolded, a protein can prospectively interact with folded proteins and induce them to unfold. Such interactions can result in aggregates of unfolded protein that at best diminish the pool of functional proteins and at worst are cytotoxic (Feder, 1996).

However, a brief exposure of cells to sub-lethal high temperature was found to render protection to the organism from subsequent and more severe temperature. In a study with heat shocked Drosophila subobscura Digley and Smith (1968) reported continued survival and acclimatization of the experimental insects at higher temperatures.

Heat Shock Proteins:

It was Ritossa (1962) who reported that the metabolic inhibitor dinitrophenol and heat induced a characteristic pattern of puffing in the chromosomes of Drosophila. It was observed by the author that when Drosophila larvae were shifted from 27°C to 37°C temperature similar puffs appeared in the polytene chromosomes. This discovery eventually led to the identification of the heat-shock proteins (Hsp) or stress proteins whose expression these puffs represented. By the mid-1980's, investigators recognized that many Hsps function as molecular chaperones. The word chaperone (pronounced as 'sha-pə-"rōn) means ‘an older person who accompanies young people at a social gathering to ensure proper behavior; broadly: one delegated to ensure proper behavior’. Molecular chaperones work more or less in a similar fashion. They are a class of proteins that enable the cell to cope with the problem of protein unfolding subsequent to a stress. Chaperones can recognize and bind to the exposed side groups that charecterise unfolded proteins. In so doing molecular chaperones prevent the bound side groups from engaging in inappropriate interactions with other cellular components, as well as stabilize the bound proteins in an unfolded state. Alternatively, chaperones can target bound proteins for degradation or removal from the cell. The constitutively expressed heat shock proteins (heat shock cognates) perform these roles for nascent polypeptides or proteins that unfold during normal cellular processes, while the inducible hsps function in response to the protein denaturation due to stress.

A summary of the mechanism for expression of heat shock proteins within a cell is given by Kregel (2002) as follows. ‘Heat shock factors (HSFs), present in the cytosol, are bound by heat shock proteins (HSPs) and maintained in an inactive state. A broad array of physiological stimuli (“stressors”) are thought to activate HSFs, causing them to separate from HSPs. HSFs are phosphorylated by protein kinases and form trimers in the cytosol. These HSF trimer complexes enter the nucleus and bind to heat shock elements (HSE) in the promoter region of the Hsp gene. Hsp mRNA is then transcribed and leaves the nucleus for the cytosol, where new Hsp is synthesized’.

Subsequently, every form of stress is known to induce these proteins in all tested organisms from bacteria to man. Inducing stresses include ethanol, heavy metals, hypoxia, hyperoxia, changes in pH, free radicals, various poisons and toxins, ischemia, osmotic shock, ionizing radiation and many others (Feder, 1996). Thus the term “heat shock protein” is a bit of a misnomer, and it is more accurate to refer to these proteins as “stress proteins”. Yet the term heat shock proteins is so deeply entrenched in the literature and so accurate a descriptor of the response at high temperature that, it is likely to persist for years to come (Denlinger and Yocum -1998).

Heat Shock Proteins (HSP) – Types and functions:

Heat-shock proteins are classified into families on the basis of sequence homology and typical molecular weight as Hsp 110, Hsp 100, Hsp 90, Hsp 70, Hsp 40, Hsp 10 and small heat- shock protein families. In eukaryotes many families comprise multiple members that differ in inducibility, intra cellular localisation and function which are presented briefly in the following table:-
[click to enlarge the table]

(After Moseley, 1997)

The threshold temperature for Hsp induction is correlated with the typical temperature at which species live. Thermophilic species have a higher threshold than the psychrophilic species. Many species exhibit characteristic and distinctive patterns of Hsp expression (or non expression) during the various stages of development, including gametogenesis, embryogenesis and metamorphosis. Extensive studies have been conducted on the heat- shock response by a large number of workers across the world in a variety of insect species such as Drosophila sp. (Tissiers et.al., 1974; Lindquist, 1980, Gilchrist & Huey 1999; Karunanidhi et al, 1999), the locust Locusta migratoria (Whyard et al., 1986) Anopheles stephensi (Nath and Lakhotia, 1989), the tobacco hornworm - Manduca sexta (Fittinghoff and Riddiford 1990), the fleshfly-Sarcophaga crassipalpis, (Joplin and Denlinger 1990), Lymantria dispar (Denlinger et al., 1992). Studies examining stress protein expression in the wild or in response to laboratory stimulations or of natural stress regimes are still few. Nonetheless, even these few studies are sufficient to demonstrate that patterns of stress protein expression can be correlated with specie’s natural thermal environments; that is, cells and species from warm environments undergo a stress response at warmer temperatures than counterparts from cool environments (Lindquist, 1986; Huey& Bennet, 1990; Sarge et al., 1995; Somero, 1995). Numerous studies have now demonstrated that heat shock proteins are responsible for a large component of inducible thermotolerance (Morimoto et al., 1994). Studies in which hsp genes were either removed or their expression inhibited demonstrate a reduction in inducible thermotolerance (Sanchez and Lindquist, 1990; Craig and Jacobsen, 1984; Johnston and Kucey, 1988). Likewise the insertion of extra copies of the hsp70 gene increased thermotolerance in both cells (Li et al, 1991; Solomon et al., 1991; Li and Duncan, 1995) and whole organisms (Welte et al., 1993; Feder et al., 1996).

The quest for heat tolerant silkworm breeds.

Among many factors attributed to poor performance of the bivoltine silkworm strains under tropical conditions the major aspect is that many quantitative characters decline sharply when temperature is higher than 28°C. The risk of hybridization of polyvoltine to bivoltine could not be taken due to the delay in fixation of economic characters. The long and hard struggle to evolve robust-productive silkworm hybrids has not so far met with satisfactory results. Veteran silkworm geneticist Y. Tazima made one of the first attempts in this direction. He started a breeding experiment by using Pure Mysore and C134a as parent breeds, but did not yield expected outcome. Prof. Tazima concluded with an apprehension whether the heavy cocoon trait was negatively correlated with that of high temperature resistance (Tazima and Ohnuma, 1995). The only way out was to evolve tropical bivoltine breeds. In this direction a breeding technique was evolved at CSR&TI, Mysore, to select the robustness genes along with its modifiers in high temperature conditions. Consequently in 1988 a temperature tolerant bivoltine hybrid namely CSR18 X CSR19 was developed and authorized for commercial exploitation (Sureshkumar, et.al., 2002). Though the introduction of CSR18xCSR19 in the field for commercial rearing during summer months was seen as a big success, the productivity level and returns realized did not match to that of other productive CSR hybrids. Therefore, the acceptance level of this hybrid among farmers was not up to the expected level.

As conventional breeding experiments encounter bottlenecks the silkworm breeder is bound to look for other possible options as molecular marker assisted breeding and transgenesis breeding. In spite of remarkable progress achieved in the disciplines of silkworm genomics, silkworm genetics, silkworm biotechnology and silkworm breeding no much result have been forthcoming in evolving temperature tolerant bivoltine silkworm breeds. Probably the reason for this lies in the fact that such a breed is largely the requirement of countries in the tropical sericulture belt as India, where interdisciplinary research is yet to materialize in its true sense.

Heat shock proteins – a missing link in silkworm breeding for robustness

Though there are reports on the activity of heat shock proteins in silkworm, the body of literature available on the molecular mechanism of temperature sensitivity and heat shock response in silkworm is rather thin as compared to the enormous work done on other insects. Evegnev et al., (1987) studied heat shock response in Bombyx mori cells and found that temperature elevation induced active transcription of heat shock mRNAs in infected cells. But at the level of translation heat shock treatment failed to induce Hsp synthesis and was not able to inhibit production of polyhedrin in such cells. Joy and Gopinathan in 1995 reported the appearance of 93, 70, 46 and 28 kDa protein bands consequent to high temperature exposure in Bombyx mori. in both bivoltine and multivoltine strains, but with varying kinetics. The isolated hemocytes of multivoltine race exhibited the induction of 70 kDa protein. Lee et.al., in 2003 cloned a genomic DNA fragment containing a promoter region for the gene encoding an HSC70-4 homologue, the structure of which was deduced from the partial cDNA sequences that were registered in a Bombyx mori EST data base. The deduced amino acid sequence with 649 residues was 89% and 96% identical to those from Drosphilla melanogaster hsc-4 and Muduca sexta HSC-70-4 respectively. The expression analysis by reverse transcription PCR demonstrated that mRNA transcription occurred in all tissues examined and was not stimulated by heat shock. Thus HSC70-4, the molecular chaperon is ubiquitously expressed in every tissue of Bombyx mori. In a recent study Vasudha et.al., (2006) observed differential expression of hsps in silkworm strains. 90 kDa in the first, second and third instars, 84 kDa in the fourth instar and 84, 62, 60, 47 and 33 kDa heat shock proteins in fifth instar was observed in response to heat shock. Use of Heat shock proteins as molecular markers for evaluation and evolution of thermotolerant silkworm strains has been suggested by them.

Considering the enormous investigations conducted on HSPs in a plethora of organisms ranging from bacteria to man, it is felt that there is an acute shortage of literature on the heat shock response of the silkworm Bombyx mori. However, the literature available on the heat-shock protein synthesis in other lepidopteran insects suggest that there is much scope for similar studies in silkworm HSPs in detail with the set target of harnessing the genes responsible for rendering thermo-tolerance in hardy polyvoltine that can be transferred in bivoltine breeds since high temperature resistance is recognized as heritable character in silkworm (Kato et al., 1989). In order to achieve greater success in this regard, there is dire necessity for (1) Understanding the molecular mechanism of temperature tolerance in silkworm; (2) Identification of the various families of HSPs synthesized and the threshold temperature, which induce their expression; (3) Understanding the differential expression pattern of various HSPs in bivoltine and polyvoltine races; and (4) To locate the genes responsible for the heat inducible HSPs and subsequent steps to introgress the same into the silkworm genome either by conventional breeding or by use of molecular techniques. The recent success stories in silkworm transgenesis (Tomita et al., 2003) shed rays of hope in this direction.

References:

Craig, E.A., and K. Jacobsen. 1984. Mutations of the heat- inducible 70 kilodalton genes of yeast confer temperature-sensitive growth. Cell 38:841-849.

Denlinger DL and Yoccum GD (1998). Physiology of Heat Sensitivity in Insects. In: Lethal Temperatures in Integrated Pest Management. Eds: GJ Hallman and DL Denlinger. Westview Press, Boulder Co., pp:7

Denlinger DL, Lee RE, Yocum GD and Kukal O (1992). Role of chilling in the acquisition of cold tolerance and the capacitation to express stress proteins in diapausing pharate larvae of the gypsy moth Lymantria dispar. Arch. Insect Biochem. Physiol., 21:271-80.

Dingley F, Maynard Smith J (1968). Temperature acclimatization in the absence of protein synthesis of Drosophila subobscura. J. Insect Physiol., 14:1185-94.

Evgenev MB, Braude-Zolotareva TY, Titarenko EA, Levin AV, Denisenko ON, Ulmasov K and Karaev K (1987). Heat Shock response in Bombyx mori cells infected by Nuclear Polyhedrosis Virus. Mol. Gen. Genetics, 215:322-325.

Feder. M. E. (1996) Ecological and evolutionary physiology of stress proteins and the stress response: the Drosophila melanogaster model. In Animals and Temperature: Phenotypic and Evolutionary Adaptation to Temperature Eds. IA. Johnston and A.F. Bennett, Cambridge University Press. pp.79-102

Feder, M.E., N.V. Cartaño, L. Milos, R.A. Krebs, and S.L. Lindquist. 1996. Effect of engineering hsp70 copy number on hsp70 expression and tolerance of ecologically relevant heat shock in larvae and pupae of Drosophila melanogaster. J. Exp. Biol. 199:1837-1844.

Fernando P and Heikkila JJ (2000). Functional charecterisation of Xenopus small heat shock protein, Hsp 30 c: the carboxyl end is required for stability and chaperone activity. Cell Stress and Chaperons, 5:148-159.

Fittinghoff CM and Riddiford LM (1990). Heat Sensitivity and Protein synthesis during heat shock in the tobacco horn worm, Manduca Sexta. J. Comp. Physiol., B160: 349-356.

Gilchrist GW, Raymond B. Huey (1999). The direct response of Drosophila melanogaster to selection on knock-down temperature. Heredity, 83:15-29.

Huey, R.B. & Bennet, A. F. (1990) Physiological adjustments to fluctuating thermal environments: an ecological and evolutionary perspective. In Stress Proteins in Biology and Medicine, ed. R.I. Morimoto, A. Tissieres &C. Georgopoulus, pp.37-59. Cold Spring Harbor, NY, Cold Spring Harbor Laboratory Press.

Johnston, R.N., and B.L. Kucey. 1988. Competitive inhibition of hsp70 gene expression causes thermosensitivity. Science 242:1552-1554.

Joplin KH, Yocum GD and Denlinger DL (1990) Cold shock elicits expression of heat shock proteins in the flesh fly Sarcophaga crassipalpis. J. Insect Physiol., 36:825-834.

Joy O, Gopinathan KP (1995). Heat-shock response in mulberry silkworm races with different thermo tolerances. J. Biosci., 20:499-513.

Karunanidhi S, Barclay JW, Robertson RM, Brown IR and Atwood HL (1999). Neuro protection at Drosophilla synapsis conferred by prior heat shock. The Journal of Neuro Science, 19:4360-4369.

Kato M, Nagayasu K, Ninagi O, Hara W and Watanabe A (1989). Study on resistance of the Silkworm Bombyx mori to high temperature. Proceedings of the 6th International Congress of SABRAO (II), pp:953

Kregel kevin C. (2002) Molecular Biology of Thermoregulation Invited Review: Heat shock proteins: modifying factors in physiological stress responses and acquired thermotolerance. J Appl Physiol 92: 2177–2186

Lee JM, Kusakave TE, Kawaguchi Y, Yasunaga-Aoki C, Nho S, Nakajima Y and Koga K (2003). Molecular characterisation of heat shock cognate 70-4 promoter from the silkworm Bombyx mori. J. Insect Biotech. Sericology, 72: 33-39.

Li, G.C., L.G. Li, Y.K. Liu, J.Y. Mak, L.L. Chen, and W.M. Lee. 1991. Thermal response of rat fibroblasts stably transfected with the human 70-kDa heat shock protein-encoding gene. Proc. Natl. Acad. Sci. 88:1681-1685.

Li, D., and R.F. Duncan. 1995. Transient acquired thermotolerance in Drosophila, correlated with rapid degradation of hsp70 during recovery. Eur. J. Biochem. 231:454-465.

Lindquist S (1980). Variying patterns of protein synthesis in Drosophilla during heat shock; implications for regulation. Developmental Biology, 77:463-479.

Lindquist, S. (1986) The heat shock response. Annual Review of Biochemistry 55, 1151-91

Morimoto, R.I., A. Tissières, C. Georgopoulos. 1994. The biology of heat shock proteins and molecular chaperones. Cold Spring Harbor Lab. Press. 610pp.

Moseley PL (1997). Heat shock proteins and heat adaptation of the whole organism. J. Appl. Physiol., 83:1413

Nath BB, Lakhotia SC. (1989). Heat shock response in ovarian nurse cells of Anopheles stephensi. J. Biosci., 14:143-52.

Ritossa FA (1962) A new puffing pattern induced by temperature shock and DNP in Drosophila. Experientia, 18: 571

Sanchez, Y. and S.L. Lindquist. 1990. HSP104 required for induced thermotolerance. Science 248:1112-1115.

Sarge, K. D., Bray, A.E. & Goodson, M. L. (1995) Altered Stress response in insects. Nature 374, 126

Solomon, J.M., J.M. Rossi, K. Golic, T. McGarry, and S. Lindquist. 1991. Changes in hsp70 alter thermotolerance and heat shock regulation in Drosophila. New Biol. 3:1106-1120.

Somero, G. N (1995) Proteins and temperature. Annual Review of Physiology 57, 43-68.

Suresh Kumar N, Basavaraja HK, Kishore Kumar CM, Malreddy N and Datta RK (2002). On the Breeding of “CSR18 x CSR19” – A Robust Bivoltine Hybrid of Silkworm Bombyx mori L. for the Tropics. Int. J. Indust. Entomol., 5: 153

Tazima Y and Ohnuma A (1995). Preliminary Experiments on the Breeding Procedure for Synthesising a High Temperature Resistant Commercial Strain of the Silkworm, Bombyx mori L. Silk. Sci. Res. Inst. Jpn., 43: 1

Tissiers A,Mitchell HK and Tracy UM (1974) Protein synthesis in salivary glands of Drosophila melanogaster : relation to chromosome puffs. J. Mol. Biol., 84:389-398.

Tomita M et al. (2003). Transgenic silkworms produce recombinant human type III procollagen in cocoons. Nature

Biotech 21: 52-56.

Vasudha B. Chavadi, Aparna H. Sosalegowda and Manjunatha H Boregowda (2006) Impact of heat shock on heat shock proteins expression, biological and commercial traits of Bombyx mori : Insect Science .,13 : 243

Welte, M.A., J.M. Tetrault, R.P. Dellavalle, and S.L. Lindquist. 1993. A new method for manipulating transgenes: engineering heat tolerance in a complex, multicellular organism. Current Biol. 3:842-853.

Whyard S, Wyatt GR and Walker VK (1986). The heat-shock response in Locusta migratoria. J. Comp. Physiol., 156B:813-817.

Links:

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2.http://sites.google.com/site/gkrajeshrajesh/Home/msc-dissertation--proteins

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Silkworm breeding-certain fundamental thoughts

gkrajeshrajesh@gmail.com — Sun, 17 Aug 2008 12:25:00 +0000

Breeding is defined as the over all improvement of a domesticated animal or plant for maximum exploitation of its genetic resources with reference or in relation to the climatic zone and geographic area where it is reared.
Breeding is an age old practice. It started when man first started domestication of plant and animals and when particular animals and plants were selected for various reasons including productivity, quality of produce, easiness of domestication, healthiness etc.
Silkworm is one of the most genetically exploited animals. Silkworms were first domesticated during the ‘Han Dynasty’ in China about 5000 years ago. Since then the silk production capacity of the species has increased nearly ten folds. Silkworm is one of the few organisms were in the principles of genetics and breeding were applied to harvest maximum output. It is next only to maize in exploiting the principles of ‘heterosis’ and ‘cross breeding’
Silkworm breeding is aimed at the overall improvement of silkworm in a human-commercial point of view. The major objectives of silkworm breeding are: Improving fecundity, improving healthiness of larvae, improving quantity of cocoon and silk production, improving quality of cocoon and silk production, for specific purposes based on cocoon and silk production, for disease resistance etc. Let us discuss each of this in short.
Fecundity: it refers to the egg laying capacity of a breed. It is a very important factor since commercial sericulture is strongly dependent on silkworm egg.
Healthiness of larvae: healthy larvae lead to healthy cocoon crop. Healthiness is dependent on factors such as better pupation rate, less number of dead larvae in the mountage, shorter larval duration (shorter the larval duration lesser the chances of infection) and bluish tinged fifth instar larvae (it is observed that bluish colored fifth instar larvae are healthier than the reddish brown ones).
Quantity of cocoon and silk: Quantity of cocoon produced is directly related to the pupation rate and larval weight. Healthier the larva more will be the pupation rate and cocoon weight.
Quality of cocoon and silk: This depends upon a number of factors including genetic factors.
Specific purposes: Apart from commercial purpose advanced countries are giving attention to specific breed development for specific purposes like sericin production, sex limited breeds, thin/ thick filament production etc.
Disease resistance breeding: The major reason for crop losses is pathogen infection. Efforts are in vogue to evolve breeds which are tolerant or resistant to various pathogens.
‘Heterosis’ and its relevance in silkworm breeding
Heterosis is defined as the ‘extra vigour’ or superiority shown by the F1 progeny over either of its parents or the mid parental value. It is given by the formula

The phenomenon of heterosis was first observed and explained by Shull in 1909 in silkworm. The progenies obtained by crossing two inbred lines showed better performance and extra vigour.
The factors determining heterosis are: genetic relationship, Compatibility and Origin.
The theoretical explanation of the phenomenon of heterosis gives four theories as given below.
1) Genes control characters: Genes are the functional units of chromosomes. Certain characters are determined by single genes while others are determined by more than one gene. The output of single gene controlled characters is less. The resultant effect of multiple genes can be improved by their optimum combination.
2) Theory of dominance: This theory suggests that out of the various allele combinations the homozygous dominant is stronger. That is out of ‘AA’, ‘aA’ and ‘aa’ AA will have stronger effect.
3) Theory of over dominance: This theory says that the heterozygous dominant allele will have a stronger effect. That is to say the crossing of a homozygous recessive parent with a homozygous dominant parent out of the various combinations the heterozygous dominant combination will be stronger.
4) Epistasis: Epistasis is the interaction between genes. Epistasis takes place when the action of one gene is modified by one or several other genes, which are sometimes called modifier genes. The gene whose phenotype is expressed is said to be epistatic, while the phenotype altered or suppressed is said to be hypostatic.
Though the above theories are different they are not mutually exclusive.
The phenomenon of heterosis if fully exploited in silkworm and maize breeding. In the case of all other animal breeding, pure line selection method is made use of. Hence those breeding programs are deprived of the beneficial effects of heterosis. It was Toyama (1909) who discovered heterosis in silkworm. Till then pure line selection was the sole breeding method in silkworm. Since the finding of heterosis, Japanese scientists conducted trials by crossing pure lines in order to harvest the luxuriousness, robustness and superiority in production. During the late 20th century Japanese cocoon and silk production increased manifold, by the terminal cross breeding strategy.
Hybrid vigour and environment
Hybrid vigour refers to positive heterosis. It is the extra vigour or improvement in performance shown by the F1 progeny over either of its parents or mid parent value. Hybrid vigour is the manifestation of interaction between the genes or alleles of two purelines. However the phenotypic expression is always dependent on the interaction between genotype and environment. Even if the organism posses a very good genetic make up the phenotype is the result of its interaction with the environment. Thus, for optimum expression of the genotype congenial environment is necessary.
The phenotypic expression is also dependent upon the genetic plasticity or buffering capacity of the organism. The extra vigour shown by the out cross between two inbred lines of silkworm could be due to the extra buffering capacity of the resultant organism.

Kerala’s SERISREE project for poverty eradication through sericulture promotion

gkrajeshrajesh@gmail.com — Sat, 17 May 2008 19:41:00 +0000

The Kerala State Sericulture Co operative Federation (SERIFED), Kerala, India has launched a poverty eradication project in the state, through intensive promotion of sericulture among the people Below Poverty Line. The federation receives financial support from the Swarnjayanti Gram Swarozgar Yojana (SGSY) of the Government of India. Under the SGSY, assistance is given to the poor families living below the poverty line in rural areas for taking up self employment. The persons taking up Self-Employment are called swarozgaris. They may take up the activity either individually or in Groups, called the Self-Help Groups. For successful Self-Employment, it is necessary to take up the right activity. For this purpose, 4 to 5 activities are selected in each Block with the help of officials, non-officials and the Bankers. These are called ‘Key Activities’, and should be such that they give the Swarozgaris an income of Rs. 2000 per month, net of Bank loan repayment.

The SERISREE project is formulated by SERIFED by following the example of the KUDUMBASREE project run by the state government. The major objectives of the project are to eradicate poverty through promotion of gainful employment and additional income generating opportunities through sericulture, to ensure economic empowerment of rural women and small & marginal farmers, to ensure sustainable development of sericulture industry, to exploit available natural resources to the best advantage of sericulture sector, to promote lease farming in the State as there is not sufficient cultivable land under the ownership of people below poverty line, to provide necessary technological support for the stabilisation of the activity, to popularise small scale sericulture farming ie, homestead sericulture farming system, to effectively utilise wasteland of Institution/Estates/Government etc.

The project includes five major components viz. Assistance for Establishment of Mulberry Kissan Nurseries, Assistance to Individual Sericulturists, Establishment of Chawki Rearing-cum- Input Service Centres, Setting up of Disinfection Squad and Establishment of Silk Reeling Infrastructure. These five components are expected to complement each other and serve as a strong back bone to the scheme implementation by way of creating a congenial circumstance for wholesome development of the sericulture industry in the rural sector involving BPL families. The total project cost is 14.32 crores. The basic unit is designated as a Field Operating Unit (FOU), consisting of four BPL beneficiaries together managing one acre mulberry cultivation and silkworm rearing. Since the BPL beneficiaries can’t be expected to possess own land, the cultivation will be taken up in leased land. A financial assistance to the tune of nearly Rs. 1lakh is proposed under various heads for each FOU. Later Self Help Groups are constituted by such five FOUS, forming a group of 20 members. It is expected that the SERISREE project will be a turning point in Kerala’s sericulture.

Under the project it is expected to bring in 1000 acres of fresh mulberry plantations and a matching cocoon production. The entire activity is proposed to create employment opportunities to the tune of 59 lakhs man days in three years.

Kerala State Sericulture Co-operative Federation (Serifed) was established as a nodal agency for implementing sericulture activities in the State. Sericulture in Kerala is based on small-holdings, low investment and family labor oriented; moderate income generating and a subsidy oriented one. The major strategies of Serifed during 2008-09 are promotion of new mulberry varieties suitable for Kerala conditions, cluster approach of sericulture development of potential areas, active involvement of local self Government institutions, development of 100% Bivoltine sericulture, implementation of women empowerment scheme etc.

Research updates 04

gkrajeshrajesh@gmail.com — Wed, 02 Jan 2008 06:38:00 +0000

Nine assorted papers on mulberry, silkworm physiology, molecular biology and seri medicine.

1. Micromorphological Characterization of Ten Mulberry Cultivars (Morus spp)
Magda Biasiolo, Maria Teresa Da Canal, and Noemi Tornadore
Economic Botany Volume 58, Issue 4 (December 2004) pp. 639–646
The micromorphological features of the vegetative and reproductive structures of ten mulberry cultivars grown at the Specialized Sericultural Section of the Agricultural Zoology Experimental Institute of Padua, northeastern Italy, were examined by SEM in order to determine the charactetistics that were the most valuable taxonomically. The observed specimens (leaves, flowers, seeds, and pollen grains) showed micromorphological differences regarding leaf hairiness, quantity of waxes, quality of epidermis cuticle and tepal hairiness. The effects of differing environments in altering the floral sex ratios of this basically monoecious group of plants were also investigated. However, no significant differences between the micromorphology of the seeds and the pollen grains of these selected cultivars were detected. The authors are hopeful that the information gained in this study may prove useful in the future creation of an exhaustive and final catalogue “descriptor” of cultivated varieties belonging to the genus Morus L.
2. Developmental Profile of Annexin IX and its Possible Role in Programmed Cell Death of the Bombyx mori Anterior Silk Gland
Yu Kaneko, Keiko Takaki, Masafumi Iwami, and Sho Sakurai
Zoological Science Volume 23, Issue 6 (June 2006) pp. 533–542
During pupal metamorphosis, the anterior silk gland (ASG) of the silkworm, Bombyx mori, undergoes programmed cell death (PCD), which is triggered by 20-hydroxyecdysone (20E). Annexin IX (ANX IX) has been identified as a 20E-inducible gene in dying ASGs, and we show here that its expression is down-regulated in tissues destined to die but not in tissues that survive pupal metamorphosis. ANX IX expression was high in the ASGs during the feeding period, when the ecdysteroid titer was low, and decreased in response to the rising ecdysteroid titer that triggered pupal metamorphosis. Before gut purge, in vitro exposure of the ASGs to 20E levels corresponding to the ecdysteroid concentration present at the time of gut purge caused a decrease in ANX IX messenger RNA levels. Expression profiles of EcR and USP, and the 20E concentration-responses of these genes, indicate the importance of the relative abundance of EcR-A and EcR-B1 isoforms in ANX IX regulation. These results suggest an involvement of ANX IX in the determination of PCD timing by delaying or suppressing the response to the increase in hemolymph ecdysteroid concentration during the prepupal period.

3. Release of Ecdysteroid-Phosphates from Egg Yolk Granules and Their Dephosphorylation during Early Embryonic Development in Silkworm, Bombyx mori
Ryouichi Yamada, Yumi Yamahama, and Haruyuki Sonobe
Zoological Science Volume 22, Issue 2 (February 2005) pp. 187–198
Newly laid eggs of many insect species store maternal ecdysteroids as physiologically inactive phosphoric esters. In the silkworm Bombyx mori, we previously reported the presence of a specific enzyme, called ecdysteroid-phosphate phosphatase (EPPase), which catalyzes the dephosphorylation of ecdysteroid-phosphates to increase the amount of free ecdysteroids during early embryonic development. In this study, we demonstrated that (1) EPPase is found in the cytosol of yolk cells, (2) ecdysteroid-phosphates are localized in yolk granules, being bound to the yolk protein vitellin (Vn), and (3) Vn-bound ecdysteroid-phosphates are scarcely hydrolyzed by EPPase, although free ecdysteroid-phosphates are completely hydrolyzed by EPPase. Thus, we investigated the mechanism by which ecdysteroid-phosphates dissociate from the Vn-ecdysteroid-phosphate complex, and indicated that the acidification of yolk granules causes the dissociation of ecdysteroid-phosphates from the Vn-ecdysteroid-phosphate complex and thereby ecdysteroid-phosphates are released from yolk granules into the cytosol. Indeed, the presence of vacuolar-type proton-translocating ATPase in the membrane fraction of yolk granules was also verified by Western blot analysis. Our experiments revealed that Vn functions as a reservoir of maternal ovarian ecdysteroid-phosphates as well as a nutritional source during embryonic development. This is the first report showing the biochemical mechanism by which maternal Vn-bound ecdysteroid-phosphates function during early embryonic development.

4. Establishment of a Sandwich ELISA System to Detect Diapause Hormone, and Developmental Profile of Hormone Levels in Egg and Subesophageal Ganglion of the Silkworm, Bombyx mori
Norio Kitagawa, Kunihiro Shiomi, Kunio Imai, Teruyuki Niimi, Toshinobu Yaginuma, and Okitsugu Yamashita
Zoological Science Volume 22, Issue 2 (February 2005) pp. 213–221
In the silkworm Bombyx mori, diapause hormone (DH) is produced in the female subesophageal ganglion (SG) and induces embryonic diapause by targeting developing ovaries. DH is processed from a precursor protein consisting of DH, pheromone biosynthesis activating neuropeptide (PBAN) and three other neuropeptides (SGNPs). Because these five neuropeptides share a common sequence, FXPRLamide, at the C-terminus, a direct and specific assay for DH itself is required in order to understand the profile of concentration changes. In this study, we produced a mouse monoclonal antibody (anti-DH[N] mAb) against the N-terminal region of DH and developed a sandwich enzyme-linked immunosorbent assay using the anti-DH[N] mAb and a rabbit polyclonal antibody against the C-terminus of DH. This procedure enabled us to specifically quantify the DH molecule at femtomolar levels (equivalent to 1/10 of SG). We then plotted DH levels in eggs and SGs during embryonic and post-embryonic development. DH was present in late-stage embryos that had been destined for the production of both diapause and nondiapause eggs. DH levels in SG gradually increased in both types during larval development and peaked at the early pupal stage. At the middle pupal stage, DH levels in SG and SG-brain complex decreased markedly in the diapause-egg producing type, thus indicating active release of DH into the hemolymph. From 5th instar larva to adult, no sexual differences in DH levels were observed in SGs or SG-brain complexes from diapause and nondiapause egg-producing types.

5. Functional analysis of four Gloverin-like genes in the silkworm, Bombyx mori. Kawaoka S, Katsuma S, Daimon T, Isono R, Omuro N, Mita K, Shimada T.
Arch Insect Biochem Physiol. 2007 Dec 12; [Epub ahead of print]
To identify genes involved in the innate immunity of the silkworm Bombyx mori, we constructed a cDNA library from the fat body of Escherichia coli-challenged B. mori larvae. Based on the expressed sequence tag (EST) data and whole genome shotgun sequence analysis, we found four Gloverin-like genes, BmGlov1-4, in the Bombyx genome. Northern blot and RT-PCR analysis showed that BmGlov1-4 were induced in the larval fat body after an immune challenge by the injection of E. coli; however, less induction was observed after the injection of a yeast Candida albicans. In silico sequence analysis revealed the presence of a motif homologous to NF-kappaB binding site in the upstream region of each BmGlov gene. Moreover, we expressed recombinant BmGlov1-4 proteins using the baculovirus expression system, and found that all the recombinant BmGlov1-4 significantly inhibited the growth of E. coli. Arch. Insect Biochem. Physiol. 2007. (c) 2007 Wiley-Liss, Inc.

6. Solubilization of the Ecdysone Binding Protein from Anterior Silk Gland Cell Membranes of the Silkworm, Bombyx mori.
M Elmogy, M Iwami, and S Sakurai
Zoolog Sci. 2007; 24: 971
We previously provided preliminary evidence for the presence of a putative membrane ecdysone receptor (mEcR) anchored in the plasma membranes of anterior silk glands (ASGs) in Bombyx mori. This receptor may act in concert with the conventional EcR in 20E-dependent programmed cell death of these glands. We report here, for the first time, the solubilization of mEcR from ASG membranes using the zwitterionic detergent CHAPS in the presence of NaCl. Our results show by ligand binding assay that mEcR solubilized this way is functionally active and retains 75% of its native binding activity. We also defined experimental conditions that yielded protein/detergent complexes with partial binding activity, which makes it possible to purify the membrane-bound ecdysone binding protein.

7. A silkworm baculovirus model for assessing the therapeutic effects of antiviral compounds: characterization and application to the isolation of antivirals from traditional medicines.
Y Orihara, H Hamamoto, H Kasuga, T Shimada, Y Kawaguchi, and K Sekimizu
J Gen Virol. 2008; 89: 188
Ganciclovir, foscarnet, vidarabine and ribavirin, which are used to treat viral infections in humans, inhibited the proliferation of a baculovirus (Bombyx mori nucleopolyhedrovirus) in BmN4 cells, a cultured silkworm cell line. These antiviral agents inhibited the proliferation of baculovirus in silkworm body fluid and had therapeutic effects. Using the silkworm infection model, the antiviral activity of Kampo medicines was screened and it was found that cinnamon bark, a component of the traditional Japanese medicine Mao-to, had a therapeutic effect. Based on the therapeutic activity, the antiviral substance was purified. Nuclear magnetic resonance analysis of the purified fraction revealed that the antiviral activity was due to cinnzeylanine, which has previously been isolated from Cinnamomum zeylanicum. Cinnzeylanine inhibits the proliferation of herpes simplex virus type 1 in Vero cells. These results suggest that the silkworm-baculovirus infection model is useful for screening antiviral agents that are effective for treating humans infected with DNA viruses.

8. Antidiabetic Properties of 2,5-Dihydroxy-4,3'-Di(beta-D-Glucopyranosyloxy)-trans-Stilbene from Mulberry (Morus bombycis Koidzumi) Root in Streptozotocin-Induced Diabetic Rats.
Heo SI, Jin YS, Jung MJ, Wang MH.
J Med Food. 2007 Dec;10(4):602-7.
We investigated the antidiabetic properties of 2,5-dihydroxy-4,3-di(beta-D-glucopyranosyloxy)-trans-stilbene (DGTS) isolated from Morus bombycis Koidzumi in streptozotocin (STZ)-induced diabetic rats. The DGTS prevented the increase in aspartate aminotransferase, alanine aminotransferase, and blood urea nitrogen levels in serum of diabetic rats. At doses of 200-800 mg/kg, DGTS improved hyperglycemia in the rats, and the hypoglycemic effect of DGTS was comparable to that of tolbutamide. The histological observations showed that DGTS prevented atrophy of pancreatic beta-cells and vascular degenerative changes in the islets. DGTS reversed STZ-induced diabetes and had antioxidant activity in assays of FeCl(2)/ascorbic acid-induced lipid peroxidation in the rats. Levels of cytochrome P450 2E1 mRNA, as measured by reverse transcription-polymerase chain reaction, were lower in the livers of the DGTS-treated rats than those of the control group. These results suggest that DGTS might be beneficial in the treatment of type 1 diabetes

9. Structural Disorder in Silk Proteins Reveals the Emergence of Elastomericity. Dicko C, Porter D, Bond J, Kenney JM, Vollrath F.
Biomacromolecules. 2007 Dec 14; [Epub ahead of print]
Spider silks combine basic amino acids into strong and versatile fibers where the quality of the elastomer is attributed to the interaction of highly adapted protein motifs with a complex spinning process. The evaluation, however, of the interaction has remained elusive. Here, we present a novel analysis to study silk formation by examining the secondary structures of silk proteins in solution. Using the seven different silks of Nephila edulis as a benchmark system, we define a structural disorder parameter (the folding index, gamma). We found that gamma is highly correlated with the ratio of glycine present. Testing the correlation between glycine content and the folding index (gamma) against a selected range of silks, we find quantitatively that, in order to achieve specialization with changes in mechanical performance, the spider's silks require higher structural flexibility at the expense of reduced stability and consequently an increased conversion-energy cost. Taken together, our biophysical and evolutionary findings reveal that silk elastomericity evolved in tandem with specializations in the process of silk spinning.

SILK SOCKS, A NEW BIOTECH PRODUCT

gkrajeshrajesh@gmail.com — Mon, 10 Dec 2007 09:15:00 +0000

It is reported by The “Times on line” that researchers at Shinshu University have succeeded in creating a silk thread that is stronger, softer and more durable than conventional silk by genetically modifying silkworms with spider genes. It is also reported that a Japanese manufacturer is already experimenting with the thread, and spider socks, stockings and even fishing lines are expected to appear on the market within a few years. The news item claims that the fiber is going to have far reaching consequences in the textile industry: “It is a miracle substance - lighter than feathers, stronger than steel, one of the toughest fibres found in nature. Now scientists in Japan have found a way of harnessing the remarkable power of spiders’ webs to make anything from tights and fishing nets to bulletproof vests”. The genetically modified silkworm caterpillars spin cocoons, 10 per cent of which consist of spider proteins. The team is hopeful to increase the proportion of spider thread material to 50 per cent. The spider used as gene donor is Nephila clavata, the golden orb spider.

The research team is led by Masao Nakagaki, a professor of insect genetics (Faculty of Textile Science and Technology "Silkworm Genetics and Pathology, Department pf Applied Biology, Shinshu University "). Efforts to harness spider silk were afoot for a number of years through genetically modified goats by extruding it from the udders of female goats. However silkworm is the most apropriate bio reactor for this purpose, given its five thousand years old relation with man and the unique genetic make up of the organism enabling it to be the supplest creature for genetic manipulations. Though the silkworm was heralded as a most important laboratory tool by such veteran genetists as Prof. Y. Tazima, it was yet to be used as one. The creature was named by Carl Linnaeus himself, studied by stalwarts such as Darwin, Pasteur and Malphigi. In fact the ‘Malphigion tubules’ were first identified in silkworms.
Fritz Vollrath, a German zoologist is an authority on ‘nephilia’ silk. In “Discovery” site you can read about his findings on ‘nephilia’ silk properties. (Link: ttp://discovermagazine.com/2001/sep/featbiology/) Also his detailed account on micro scopic analy sis and surface properties of ‘nephilia’ silk can be read from www.blackwell-synergy.com/doi/pdf/10.1046/j.1365-2818.1998.00285.x
A large collection of ‘Nephila’ photographs is available at the site: Photographers direct http://www.photographersdirect.com/stockimages/n/nephila.asp
Stress–strain curves of washed and degummed single-filament silkworm silk and ‘Nephila ‘spider dragline silk are compared to illustrate the extra stress bearing capacity of ‘nephilia’ silk (See the link: http://images.google.co.in/imgres?imgurl=http://www.nature.com/nature/journal/v418/n6899/images/418741a-f2.2.jpg&imgrefurl=http://www.nature.com/nature/journal/v418/n6899/fig_tab/418741a_F2.html&h=621&w=600&sz=72&hl=en&start=10&um=1&tbnid=6CEyhfaSIg4POM:&tbnh=136&tbnw=131&prev=/images%3Fq%3Dsilkworm%26svnum%3D10%26um%3D1%26hl%3Den%26sa%3DN )

Research updates 03

gkrajeshrajesh@gmail.com — Tue, 04 Dec 2007 08:49:00 +0000

Seven papers on silkworm molecular biology, silk nanotechnology and biomaterial properties.

Inactivation of pyocyanin synthesis genes has no effect on the virulence of Pseudomonas aeruginosa PAO1 toward the silkworm, Bombyx mori.
Chieda Y, Iiyama K, Lee JM, Kusakabe T, Yasunaga-Aoki C, Shimizu . S (Laboratory of Insect Pathology and Microbial Control, Institute of Biological Control, Faculty of Agriculture, Graduate School, Kyushu University, Fukuoka, Japan.)
FEMS Microbiol Lett. 2007 Nov 19; [Epub ahead of print]
The contribution of pyocyanin to the virulence of Pseudomonas aeruginosa against the silkworm Bombyx mori was studied. First, purified pyocyanin was injected into the hemocoel of B. mori. Acute toxicity was observed only when a high dose of pyocyanin was injected. The lethal dose 50% value of pyocyanin was found to be 9.52 mug per larva. Next, mutant strains of phzM and phzS, which encode putative phenazine-specific methytransferase and flavin-containing monooxygenase, respectively, were created, and their virulence was compared with that of the PAO1 parent strain. Although the ability to produce pyocyanin was completely lost in the phz-mutant strains, they maintained the same level of virulence as the PAO1 parent strain. In addition, the complementation of the corresponding gene in trans in the mutant strains did not have any effect on the virulence of those mutant strains. These results indicated that pyocyanin does not act as a virulence factor in B. mori after invasion, which was different from the results obtained in other Lepidopteran host models.
PMID: 18031534 [PubMed - as supplied by publisher]
Related Links
Effect of superoxide dismutase gene inactivation on virulence of Pseudomonas aeruginosa PAO1 toward the silkworm, Bombyx mori. [Appl Environ Microbiol. 2007] PMID: 17220257
Functional analysis of genes for biosynthesis of pyocyanin and phenazine-1-carboxamide from Pseudomonas aeruginosa PAO1. [J Bacteriol. 2001] PMID: 11591691
Pathogenicity of gacA mutant of Pseudomonas aeruginosa PA01 in the silkworm, Bombyx mori. [FEMS Microbiol Lett. 2005] PMID: 15727838
Effect of rpoS mutation on the stress response and expression of virulence factors in Pseudomonas aeruginosa. [J Bacteriol. 1999] PMID: 10383954
Structural and functional analysis of the pyocyanin biosynthetic protein PhzM from Pseudomonas aeruginosa. [Biochemistry. 2007] PMID: 17253782

Silver nanoparticles incorporated electrospun silk fibers.
M Kang, R Jung, HS Kim, JH Youk, and HJ Jin
J Nanosci Nanotechnol. 2007; 7: 3888.
We present a simple and mass-producible method of incorporating silver nanoparticles on the surface of electrospun silk non-woven membranes for the fabrication of antimicrobial wound dressings. Nanofibrous silk membranes with fiber diameters of 460 +/- 40 nm were electrospun from an aqueous Bombyx mori fibroin solution. The electrospun membranes incorporating silver nanoparticles were prepared by dipping the membranes in aqueous silver nitrate (AgNO3) solution (0.5 or 1.0 wt%) followed by photoreduction. Field emission scanning and transmission electron microscopy showed that silver nanoparticles were generated on the electrospun silk fibroin nanofibers as well as inside them. The interaction between the silver nanoparticles and amide groups in the silk fibroin molecules was characterized using X-ray photoelectron spectroscopy.
Link: http://highwire.stanford.edu/cgi/medline/pmid;18047081

Controlled release from multilayer silk biomaterial coatings to modulate vascular cell responses.
Wang X, Zhang X, Castellot J, Herman I, Iafrati M, Kaplan DL. (Department of Biomedical Engineering, Tufts University, Medford, MA 02155, USA.)
Biomaterials. 2007 Nov 27; [Epub ahead of print]
A multilayered silk fibroin protein coating system was employed as a drug carrier and delivery system to evaluate vascular cell responses to heparin, paclitaxel, and clopidogrel. The results demonstrated that the silk coating system was an effective system for drug-eluting coatings, such as for stent applications, based on its useful micromechanical properties and biological outcomes. Cell attachment and viability studies with human aortic endothelial cells (HAECs) and human coronary artery smooth muscle cells (HCASMCs) on the drug-incorporated silk coatings demonstrated that paclitaxel and clopidogrel inhibited smooth muscle cell (SMC) proliferation and retarded endothelial cell proliferation. Heparin-loaded silk multilayers promoted HAEC proliferation while inhibiting HCASMC proliferation, desired outcomes for the prevention of restenosis. The preservation of the phenotype of endothelial cells on silk and heparin-loaded silk coatings was confirmed with the presence of endothelial markers CD-31, CD-146, vWF and VE-Cadherin using immunocytochemistry assays. A preliminary in-vivo study in a porcine aorta showed integrity of the silk coatings after implantation and the reduction of platelet adhesion on the heparin-loaded silk coatings.
Related Links
Silk coatings on PLGA and alginate microspheres for protein delivery. [Biomaterials. 2007] PMID: 17583788
Platelet-derived growth factor receptor antagonist STI571 (imatinib mesylate) inhibits human vascular smooth muscle proliferation and migration in vitro but not in vivo. [J Invasive Cardiol. 2007] PMID: 17541129
Vascular endothelial growth factor and heparin in a biologic glue promotes human aortic endothelial cell proliferation with aortic smooth muscle cell inhibition. [Surgery. 1996] PMID: 8751615
Nanolayer biomaterial coatings of silk fibroin for controlled release. [J Control Release. 2007] PMID: 17628161
Survival of endothelial cells in vitro on Paclitaxel-loaded coronary stents. [J Biomater Appl. 2005] PMID: 15788425
Performance evaluation of a silk protein-based matrix for the enzymatic conversion of tyrosine to L-DOPA.
Acharya C, Kumar V, Sen R, Kundu SC. (Department of Biotechnology, Indian Institute of Technology, Kharagpur, India.)
Biotechnol J. 2007 Nov 22; [Epub ahead of print]
L-DOPA (3,4-dihydroxyphenyl-L-alanine), one of the most important intermediates in the melanin biosynthesis pathway, is used for the treatment of Parkinson's disease. With a view of developing a cheaper and more effective method for the bioconversion of tyrosine to L-DOPA, the potential and performance of a novel fibrous matrix prepared from Bombyx mori silk protein fibroin were evaluated for the immobilization of tyrosinase. Cross-linkage between fibroin and tyrosinase using glutaraldehyde was evident from Fourier transform infra red spectroscopy. Maximum product formation occurred when 1000 U enzyme was immobilized on 20 mg fibroin. The optimum conditions for maximal L-DOPA production using immobilized tyrosinase were 40 degrees C and pH 5.5, conditions that caused a 50% loss of free enzyme activity. Immobilized tyrosinase also showed to have a higher degree of stability during storage and it retained 80% of its original activity after repeated reuses. The efficiency of this immobilized tyrosinase system to produce L-DOPA was high, as evident from a high effectiveness factor, between 0.7 and 0.8, thereby making this method feasible for the large-scale production of L-DOPA.
Related Links
Production of L-DOPA by tyrosinase immobilized on modified polystyrene. [Appl Biochem Biotechnol. 2003] PMID: 14665734
Tyrosinase-catalyzed modification of Bombyx mori silk fibroin: grafting of chitosan under heterogeneous reaction conditions. [J Biotechnol. 2006] PMID: 16621091
L-DOPA production by immobilized tyrosinase. [Appl Biochem Biotechnol. 2000] PMID: 10849837
Innovative effect of illite on improved microbiological conversion of L-tyrosine to 3,4 dihydroxy phenyl L-alanine (L-DOPA) by Aspergillus oryzae ME2 under acidic reaction conditions. [Curr Microbiol. 2006] PMID: 17039388
Fast enzymatic preparation of L-dopa from tyrosine and molecular oxygen: a potential method for preparing [15O]L-dopa. [Int J Rad Appl Instrum [A]. 1990] PMID: 2176194
Silkworm powder containing manganese superoxide dismutase regulated the immunity and inhibited the growth of Hepatoma 22 cell in mice.
Yue WF, Deng W, Li XH, Roy B, Li GL, Liu JM, Wu XF, Sun HX, Yao ML, David WC, Miao YG. (Institute of Sericulture and Apiculture, College of Animal Sciences, Zhejiang University, Hangzhou, 310029, P.R. China, miaoyg@zju.edu.cn.)
Mol Biol Rep. 2007 Nov 22; [Epub ahead of print]
The effects of SOD contained silkworm powder on immune regulation and inhibition against Hepatoma 22 tumor cells in vivo were investigated. The activity of natural killer cell (NK) and the ConA-stimulated spleen proliferation were measured. The results found that the SOD-contained silkworm powder caused an enhancement on NK cell activity, which implied this material modulated the immune system in mice in vivo. The NK cell activities of Hepatoma 22 tumor modeled mice treated with silkworm powder including SOD were increased significantly compared to a modeled control and silkworm powder without SOD, reaching 36.18%. In addition, the ConA-stimulated spleen proliferation of SOD treated mice was higher than that of the controls. The treatment of SOD contained silkworm powder presented 40.3% of average inhibition rate to Hepatoma 22 tumor, showing stronger inhibition against tumor. There were no significant difference in body weight between modeled control and SOD silkworm powder feeding in Hepatoma 22 tumor modeled mice, suggesting the SOD silkworm powder is safety as an inhibitant to tumor. In conclusion, these findings demonstrate that administration of silkworm powder containing SOD results in activation of NK cells and immunity, suggesting the silkworm powder containing SOD plays a positive role in tumor inhibition.
Related Links
Manganese superoxide dismutase expressed in silkworm larvae, Bombyx mori L enhances the NK activity and splenocyte proliferation against Sarcoma 180 tumor cells in vivo. [Mol Biol Rep. 2007] PMID: 17934870
Anti-oxidation and immune responses in mice upon exposure to manganese superoxide dismutase expressed in silkworm larvae, Bombyx mori L. [Cell Biol Int. 2007] PMID: 17452112
Immunity promotion and proteomic identification in mice upon exposure to manganese superoxide dismutase expressed in silkworm larvae. [J Proteome Res. 2007] PMID: 17385907
Effects of silkworm larvae powder containing manganese superoxide dismutase on immune activity of mice. [Mol Biol Rep. 2007] PMID: 17605091
CpG oligodeoxynucleotides inhibit tumor growth and reverse the immunosuppression caused by the therapy with 5-fluorouracil in murine hepatoma. [World J Gastroenterol. 2005] PMID: 15754409
High-level expression of orange fluorescent protein in the silkworm larvae by the Bac-to-Bac system.
Liu JM, David WC, Ip DT, Li XH, Li GL, Wu XF, Yue WF, Zhang X, Miao YG. (Institute of Sericulture and Apiculture, College of Animal Sciences, Zhejiang University, Hangzhou, 310029, P.R. China, miaoyg@zju.edu.cn.)
Mol Biol Rep. 2007 Nov 23; [Epub ahead of print]

This novel orange fluorescent protein (OFP) emits brilliant orange fluorescent light. OFP has high fluorescence quantum yield, fast maturation rate, and stability, which imply this protein should be the most favorable biotechnological tools used to investigate the function of target gene by visualizing, monitoring, and quantifying in living cells. B. mori, silkworm has been used as an important bioreactor for the production of recombinant proteins through baculovirus expression system (BES). In this paper, we used infection technique which introduced the baculovirus DNA into silkworms using a cationic lipofectin reagent instead of directly injecting the virus, and demonstrated a high-level expression of the orange fluorescent protein (OFP) gene in the Bombyx mori, silkworm larvae. When recombinant rBacmid/BmNPV/OFP DNA ranging from 50-100 ng/larval was injected, a sufficient OFP expression in hemolymph was harvested. The recombinant viruses could be obtained from the hemolymph of infected larvae and stored as seed which could be used for the large-scale expression. This procedure omitted the costly and labor-consumed insect cell culture. Further investigation of OFP should provide us with more insight in unlocking the mystery of the mechanisms of autocatalytic bioluminescence and its utilization in biotechnology.
Related Links
Expression of spider flagelliform silk protein in Bombyx mori cell line by a novel Bac-to-Bac/BmNPV baculovirus expression system. [Appl Microbiol Biotechnol. 2006] PMID: 16158284
A highly efficient method for the generation of a recombinant Bombyx mori nuclear-polyhedrosis-virus Bacmid and large-scale expression of foreign proteins in silkworm (B. mori) larvae. [Biotechnol Appl Biochem. 2007] PMID: 17428194
Cloning and expression of manganese superoxide dismutase of the silkworm, Bombyx mori by Bac-to-Bac/BmNPV Baculovirus expression system. [Appl Microbiol Biotechnol. 2006] PMID: 16804693
A new technique for producing recombinant baculovirus directly in silkworm larvae. [Biotechnol Lett. 2007] PMID: 17091380
An innovative technique for inoculating recombinant baculovirus into the silkworm Bombyx mori using lipofectin. [Res Microbiol. 2004] PMID: 15249063
Sonication-induced gelation of silk fibroin for cell encapsulation.
Wang X, Kluge JA, Leisk GG, Kaplan DL. (Department of Biomedical Engineering, Tufts University, 4 Colby Street, Medford, MA 02155, USA).
Biomaterials. 2007 Nov 19; [Epub ahead of print]
Purified native silk fibroin forms beta-sheet-rich, physically cross-linked, hydrogels from aqueous solution, in a process influenced by environmental parameters. Previously we reported gelation times of days to weeks for aqueous native silk protein solutions, with high ionic strength and temperature and low pH responsible for increasing gelation kinetics. Here we report a novel method to accelerate the process and control silk fibroin gelation through ultrasonication. Depending on the sonication parameters, including power output and time, along with silk fibroin concentration, gelation could be controlled from minutes to hours, allowing the post-sonication addition of cells prior to final gel setting. Mechanistically, ultrasonication initiated the formation of beta-sheets by alteration in hydrophobic hydration, thus accelerating the formation of physical cross-links responsible for gel stabilization. K(+) at physiological concentrations and low pH promoted gelation, which was not observed in the presence of Ca(2+). The hydrogels were assessed for mechanical properties and proteolytic degradation; reported values matched or exceeded other cell-encapsulating gel material systems. Human bone marrow derived mesenchymal stem cells (hMSCs) were successfully incorporated into these silk fibroin hydrogels after sonication, followed by rapid gelation and sustained cell function. Sonicated silk fibroin solutions at 4%, 8%, and 12% (w/v), followed by mixing in hMSCs, gelled within 0.5-2h. The cells grew and proliferated in the 4% gels over 21 days, while survival was lower in the gels with higher protein content. Thus, sonication provides a useful new tool with which to initiate rapid sol-gel transitions, such as for cell encapsulation.
PMID: 18031805 [PubMed - as supplied by publisher]
Related Links
Structure and properties of silk hydrogels. [Biomacromolecules. 2004] PMID: 15132662
Mechanisms of silk fibroin sol-gel transitions. [J Phys Chem B. 2006] PMID: 17064118
Structure and properties of regenerated Antheraea pernyi silk fibroin in aqueous solution. [Int J Biol Macromol. 2007] PMID: 17173967
Conformation transition kinetics of Bombyx mori silk protein. [Proteins. 2007] PMID: 17436322 New oral dosage form for elderly patients: preparation and characterization of silk fibroin gel. [Chem Pharm Bull (Tokyo). 1995] PMID: 7728934

RESEARCH UPDATES 02

gkrajeshrajesh@gmail.com — Wed, 03 Oct 2007 13:31:00 +0000

9 papers on silkworm, 4 papers on silk proteins and 5 papers on medicinal applications of sericulture

A. SILKWORM

1. Determination of phosphorylated amino acid residues of Rab8 from Bombyx mori.
Uno T, Nakada T, Okamaoto S, Nakamura M, Matsubara M, Imaishi H, Yamagata H, Kanamaru K, Takagi M.
Arch Insect Biochem Physiol. 2007 Oct;66(2):89-97.
The Rab family of small GTPases are key regulators of membrane trafficking. Partially purified Rab8 from Bombyx mori (BRab8) was phosphorylated by protein kinase C in mammalian cells in vitro. To determine which of the seven serines and four threonines are phosphorylated, we generated deletion and site-directed mutants of BRab8, inserted them in Escherichia coli, partially purified the encoded fusion proteins by affinity chromatography, and examined their phosphorylation by protein kinase C in vitro. We found that Ser-132 of BRab8 was specifically phosphorylated by protein kinase C. In addition, Western blotting using an antiserum against BRab8 and in-gel staining for phosphorylated proteins revealed that BRab8 is phosphorylated in vivo.

http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17879235&ordinalpos=6&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum

2. The nicotinic acetylcholine receptor gene family of the silkworm, Bombyx mori.
Shao YM, Dong K, Zhang CX.
BMC Genomics 2007, 8:324doi:10.1186/1471-216

Nicotinic acetylcholine receptors (nAChRs) mediate fast synaptic cholinergic transmission in the insect central nervous system. The insect nAChR is the molecular target of a class of insecticides, neonicotinoids. Like mammalian nAChRs, insect nAChRs are considered to be made up of five subunits, coded by homologous genes belonging to the same family. The nAChR subunit genes of Drosophila melanogaster, Apis mellifera and Anopheles gambiae have been cloned previously based on their genome sequences. The silkworm Bombyx mori is a model insect of Lepidoptera, among which are many agricultural pests. Identification and characterization of B. mori nAChR genes could provide valuable basic information for this important family of receptor genes and for the study of the molecular mechanisms of neonicotinoid action and resistance. RESULTS: We searched the genome sequence database of B. mori with the fruit fly and honeybee nAChRs by tBlastn and cloned all putative silkworm nAChR cDNAs by reverse transcriptase-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE) methods. B. mori appears to have the largest known insect nAChR gene family to date, including nine alpha-type subunits and three beta-type subunits. The silkworm possesses three genes having low identity with others, including one alpha and two beta subunits, alpha9, beta2 and beta3. Like the fruit fly and honeybee counterparts, silkworm nAChR gene alpha6 has RNA-editing sites, and alpha4, alpha6 and alpha8 undergo alternative splicing. In particular, alternative exon 7 of Bma8 may have arisen from a recent duplication event. Truncated transcripts were found for Bma4 and Bma5. CONCLUSIONS: B. mori possesses a largest known insect nAChR gene family characterized to date, including nine alpha-type subunits and three beta-type subunits. RNA-editing, alternative splicing and truncated transcripts were found in several subunit genes, which might enhance the diversity of the gene family.

FullText:http://www.biomedcentral.com/1471-2164/8/324 and http://www.ncbi.nlm.nih.gov/entrez/utils/fref.fcgi?PrId=3196&itool=AbstractPlus-def&uid=17868469&db=pubmed&url=http://www.biomedcentral.com/1471-2164/8/324

3. A germline transgenic silkworm that secretes recombinant proteins in the sericin layer of cocoon.
Tomita M, Hino R, Ogawa S, Iizuka M, Adachi T, Shimizu K, Sotoshiro H, Yoshizato K. (Yoshizato Project, Cooperative Link of Unique Science and Technology for Economy Revitalization, Hiroshima Prefectural Institute of Industrial Science and Technology, 3-10-32 Kagamiyama, Higashihiroshima, Hiroshima, 739-0046, Japan.)
Transgenic Res. 2007 Aug;16(4):449-65. Epub 2007 Apr 6.
A silk thread of the silkworm, Bombyx mori, is composed of the insoluble inner fibroin and the hydrophilic outer sericin layer, which are synthesized in the posterior and middle silk gland (MSG), respectively. This study aimed to develop a novel sericin 1 gene (ser1) promoter-driven recombinant expression system using transgenic silkworms, in which recombinant proteins are synthesized in MSG and secreted into the sericin layer. To obtain a high level of gene expression, we tested whether a baculovirus-derived enhancer, hr3, and a trans-regulator, IE1, are capable of stimulating the transcriptional activity of the ser1 promoter, using a transient gene expression system. The results showed that hr3 and IE1 cooperatively increased the ser1 promoter activity more than 30-fold. Then, transgenic silkworms were generated which expressed the EGFP with the signal peptide in MSG under the control of the hr3-linked ser1 promoter and IE1 gene. The silkworms exclusively secreted the EGFP into the sericin layer of cocoons as predicted. The expressed EGFP was extractable from cocoons through a simple procedure with neutral pH buffer solution. The expression system developed in this study enables us to produce recombinant proteins in bulk that can be easily extracted and purified.

http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17415674&ordinalpos=20&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum

4. Generation of a transgenic silkworm that secretes recombinant proteins in the sericin layer of cocoon: production of recombinant human serum albumin
Ogawa S, Tomita M, Shimizu K, Yoshizato K.(Yoshizato Project, Cooperative Link of Unique Science and Technology for Economy Revitalization, Hiroshima Prefectural Institute of Industrial Science and Technology, 3-10-32 Kagamiyama, Higashihiroshima, Hiroshima 739-0046, Japan.)
J Biotechnol. 2007 Feb 20;128(3):531-44. Epub 2006 Nov 17.
In this study we produced germline transgenic silkworms that spin cocoons containing recombinant human serum albumin (rHSA) in the sericin layer. A piggyBac-based transformation vector was constructed that carried HSA cDNA driven by sericin-1 gene promoter, viral enhancer hr3, and gene encoding viral trans-activator IE1. Isolated silk glands were bombarded with the vector and transplanted into host larvae. Three days later, the transplants were immunohistochemically analyzed, which showed that middle silk gland (MSG) cells expressed rHSA and secreted it into the MSG lumen. Then, silkworm eggs were injected with the vector and developed to larvae. The obtained transgenic silkworms spun silk threads whose sericin layers contained rHSA at 3.0microg/mg of cocoons. Most (83%) of the rHSA in cocoons was extracted with phosphate buffered saline, which was then subjected to ammonium sulfate precipitation and affinity chromatography. Finally, we obtained 2.8mg of 99%-pure rHSA from 2g of cocoons. Measurements of circular dichroism spectra of rHSA, and equilibrium dissociation constants of rHSA to warfarin and naproxen indicated that rHSA was conformationally and functionally identical to natural plasma HSA. Germline transgenic silkworms will be useful for producing various recombinant proteins in the sericin layer of cocoons.

http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17166611&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVAbstractPlus

5. Silk gland specific secretory expression of egfp gene in silkworm Bombyx mori with rAcMNPV system.
Guo XY, Guo TQ, Wang SP, Wang JY, Lu CD. (Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, PR China.)
Arch Virol. 2005 Jun;150(6):1151-60. Epub 2005 Feb 10.
To evaluate the possibility of establishing an in vivo baculovirus expression system in a silk gland specific secretory way, the recombinant Autographa californica nucleopolyhedrovirus (AcserpegfpDeltaEGT) carrying the reporter gene egfp downstream of silkworm ser1 promoter and signal peptide coding sequence was generated. The purified recombinant baculovirus AcserpegfpDeltaEGT was injected into the haemocoel of newly ecdysed 5thHendekl) instar silkworm larvae at the amount of 10(6) pfu per larva. At 5 days post injection, green fluorescence derived from EGFP could be observed with fluorescent microscope in only the silk gland but not other tissues after dissection of the silkworm. By making an opening on the silk gland wall, green fluorescence could be observed in the outflow of silk gland indicating the secretion of EGFP and the effectiveness of ser1 signal peptide. Western blotting assay confirmed that EGFP exists in the water-soluble part of cocoon silk too. We also established a simple protocol to purify EGFP from the secreted silk proteins.

http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=15703851&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVAbstractPlus

6. Analysis of tissue-specific region in sericin 1 gene promoter of Bombyx mori.
Liu Y, Yu L, Guo X, Guo T, Wang S, Lu C. (College of Biomedical Engineering and Instrument Science, Zhejiang University, Hangzhou 310027, China.)
Biochem Biophys Res Commun. 2006 Mar 31;342(1):273-9. Epub 2006 Feb 6
The gene encoding sericin 1 (Ser1) of silkworm (Bombyx mori) is specifically expressed in the middle silk gland cells. To identify element involved in this transcription-dependent spatial restriction, truncation of the 5' terminal from the sericin 1 (Ser1) promoter is studied in vivo. A 209bp DNA sequence upstream of the transcriptional start site (-586 to -378) is found to be responsible for promoting tissue-specific transcription. Analysis of this 209bp region by overlapping deletion studies showed that a 25bp region (-500 to -476) suppresses
the ectopic expression of the Ser1 promoter. An unknown factor abundant in fat body nuclear extracts is shown to bind to this 25bp fragment. These results suggest that this 25bp region and the unknown factor are necessary for determining the tissue-specificity of the Ser1 promoter.

http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16480950&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVAbstractPlus

7. Expression in Escherichia coli and purification of bioactive antibacterial peptide ABP-CM4 from the Chinese silk worm, Bombyx mori.
Li BC, Zhang SQ, Dan WB, Chen YQ, Cao P. (Jiangsu Province Key Laboratory for Molecular and Medical Biotechnology, Life Sciences College, Nanjing Normal University, Jiangsu, Nanjing, PR China.)
Biotechnol Lett. 2007 Jul;29(7):1031-6. Epub 2007 Mar 21.
The antibacterial peptide CM4 (ABP-CM4), isolated from Chinese Bombys mori, is a 35-residue cationic, amphipathic alpha-helical peptide that exhibits a broad range of antimicrobial activity. To explore a new approach for the expression of ABP-CM4 in E. coli, the gene ABP-CM4, obtained by recursive PCR (rPCR), was cloned into the vector pET32a to construct a fusion expression plasmid. The fusion protein Trx-CM4 was expressed in soluble form, purified by Ni(2+)-chelating chromatography, and cleaved by formic acid to release recombinant CM4. Purification of rCM4 was achieved by affinity chromatography and reverse-phase HPLC. The purified of recombinant peptide showed antimicrobial activities against E. coli K(12)D(31), Penicillium chrysogenum, Aspergillus niger and Gibberella saubinetii. According to the antimicrobial peptide database (http://aps.unmc.edu/AP/main.html), 116 peptides contain a Met residue, but only 5 peptides contain the AspPro site, indicating a broader application of formic acid than CNBr in cleaving fusion protein. The successful application to the expression of the ABP-CM4 indicates that the system is a low-cost, efficient way of producting milligram quantities of ABP-CM4 that is biologically active.

http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17375264&ordinalpos=21&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum

8. Expression of the antibacterial peptide CM4-like gene of Chinese silkworm Bombyx mori in Escherichia coli and its antibacterial activity analysis
Li BC, Chen YQ, Liu P, Zhang SQ. (The Life Science School of Nanjing Normal University, Nanjing 210097. jslbc@126.com)
Fen Zi Xi Bao Sheng Wu Xue Bao. 2007 Apr;40(2):98-102
[Article in Chinese] According to the amino acid sequence of CM4 and the bias for preferred condons of E. coli, the CM4-like gene was obtained by a recursive PCR (rPCR) strategy using two lapping oligonucleotides. The synthesized gene was coloned into the expression vector pET32(a) and transformed into E. coli BL21 (DE3). Recombinant CM4-like gene expression was driven by the T7 promoter on the vector upon addition of IPTG and high level of expression was achieved. The solube protein was purified by Ni-chelating agarose and treated with formic acid. After cleavege, the recombinant peptide was purified by another Ni(2+)-NTA-Agarose affinity chromatography and cation-exchange chromatography. Results of agarose diffuse assay and liquid turbidity analysis indicated that the recombinant peptide exhibited the antibacterial activity.

http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17580662&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVAbstractPlus

9. Structural Basis of Ligand Binding and Release in Insect Pheromone-binding Proteins: NMR Structure of Antheraea polyphemus PBP1 at pH 4.5.
Damberger FF, Ishida Y, Leal WS, Wuthrich K.
J Mol Biol. 2007 Aug 17; [Epub ahead of print]
The NMR structure of the Antheraea polyphemus pheromone-binding protein 1 at pH 4.5, ApolPBP1(A), was determined at 20 degrees C. The structure consists of six alpha-helices, which are arranged in a globular fold that encapsulates a central helix alpha7 formed by the C-terminal polypeptide segment 131-142. The 3D arrangement of these helices is anchored by the three disulfide bonds 19-54, 50-108 and 97-117, which were identified by NMR. Superposition of the ApolPBP1(A) structure with the structure of the homologous pheromone-binding protein of Bombyx mori at pH 4.5, BmorPBP(A), yielded an rmsd of 1.7 A calculated for the backbone heavy-atoms N, C(alpha) and C' of residues 10-142. In contrast, the present ApolPBP1(A) structure is different from a recently proposed molecular model for a low-pH form of ApolPBP1 that does not contain the central helix alpha7. ApolPBP1 exhibits a pH-dependent transition between two different globular conformations in slow exchange on the NMR chemical shift timescale similar to BmorPBP, suggesting that the two proteins use the same mechanism of ligand binding and ejection. The extensive sequence homology observed for pheromone-binding proteins from moth species further implies that the previously proposed mechanism of ligand ejection involving the insertion of a C-terminal helix into the pheromone-binding site is a general feature of pheromone signaling in moths.

http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17884092&ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum

B. SILK PROTEINS

1. Structure and properties of regenerated Antheraea pernyi silk fibroin in aqueous solution
Tao W, Li M, Zhao C.(School of Material Engineering, Stem Cell Research Laboratory of Jiangsu Province, Suzhou University, Campus Box 64, No. 178 East Gan-Jiang Road, Suzhou 215021, China.)
Int J Biol Macromol. 2007 Apr 10;40(5):472-8. Epub 2006 Nov 24.
Antheraea pernyi silk fibroin fibers were dissolved by aqueous lithium thiocyanate to obtain regenerated A. pernyi silk fibroin solution. By means of circular dichroism, (13)C NMR and Raman spectroscopy, the molecular conformation of regenerated A. pernyi silk fibroin in aqueous solution was investigated. The relationship of environmental factors and sol-gel transformation behavior of regenerated A. pernyi silk fibroin was also studied. The molecular conformations of regenerated A. pernyi silk fibroin mainly were alpha-helix and random coil in solution. There also existed a little beta-sheet conformation. It was obviously different with Bombyx mori silk fibroin, whose molecular conformation in solution was only random coil but no alpha-helix existence. With the increase of temperature and solution concentration and with the decrease of solution pH value, the gelation velocity of regenerated A. pernyi silk fibroin solution increased. Especially, it showed that A. pernyi silk fibroin was more sensitive to temperature than B. mori silk fibroin during the sol-gel transformation. The velocity increased obviously when the temperature was above 30 degrees C. During the sol-gel transformation, the molecular conformation of regenerated A. pernyi silk fibroin changed from random coil to beta-sheet structure. The results of these studies provided important insight into the preparation of new biomaterials by silk fibroin protein.

http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17173967&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVAbstractPlus

2. Tyrosinase-catalyzed modification of Bombyx mori silk fibroin: grafting of chitosan under heterogeneous reaction conditions.
Freddi G, Anghileri A, Sampaio S, Buchert J, Monti P, Taddei P. (Stazione Sperimentale per la Seta, via Giuseppe Colombo 83, Milano, Italy. freddi@ssiseta.it)
J Biotechnol. 2006 Sep 1;125(2):281-94. Epub 2006 Apr 18
The capability of mushroom tyrosinase to catalyze the oxidation of tyrosine residues of Bombyx mori silk fibroin was studied under heterogeneous reaction conditions, by using a series of silk substrates differing in surface and bulk morphology and structure, i.e. hydrated and insoluble gels, mechanically generated powder and fibre. Tyrosinase was able to oxidize 10-11% of the tyrosine residues of silk gels. The yield of the reaction was very low for the powder and undetectable for fibres. FT-Raman spectroscopy gave evidence of the oxidation reaction. New bands attributable to vibrations of oxidized tyrosine species (o-quinone) appeared, and the value of the I853/I829 intensity ratio of the tyrosine doublet changed following oxidation of tyrosine. The thermal behaviour of SF substrates was not affected by enzymatic oxidation. o-Quinones formed by tyrosinase onto gels and powder were able to undergo non-enzymatic coupling with chitosan. FT-IR and FT-Raman spectroscopy provided clear evidence of the formation of silk-chitosan bioconjugates under heterogeneous reaction conditions. Chitosan grafting caused a beta-sheet --> random coil conformational transition of silk fibroin and significant changes in the thermal behaviour. Chitosan grafting did not occur, or occurred at an undetectable level on silk fibres. The results reported in this study show the potential of the enzymatically initiated protein-polysaccharide grafting for the production of a new range of bio-based, environmentally friendly polymers.

3. Chemical and physical properties of sulfated silk fabrics.
Taddei P, Arosio C, Monti P, Tsukada M, Arai T, Freddi G.(Centro di Studio sulla Spettroscopia Raman, Dipartimento di Biochimica G. Moruzzi, Università di Bologna, via Belmeloro 8/2, Bologna 40126, Italy.)
Biomacromolecules. 2007 Apr;8(4):1200-8. Epub 2007 Mar 6.
Silk fabrics were treated with chlorosulphonic acid in pyridine for different times. The amount of sulfur bound to silk increased during the first 2 h of reaction and then reached a plateau. The amino acidic pattern of sulfated silk remained essentially unchanged for short reaction times (< or ="2"> or =2 h). Spectroscopic analyses performed by FT-IR and FT-Raman showed the appearance of new bands attributable to various vibrations of sulfated groups. The IR bands at 1049 and 1014 cm-1, due to organic sulfate salts, were particularly intense. Bands assigned to alkyl sulfates and sulfonamides appeared in the 1300-1180 cm-1 range. Organic covalent sulfates displayed a weak but distinct IR band at 1385 cm-1. Both IR and Raman spectra revealed that silk fibroin mainly bound sulfates through the hydroxyl groups of Ser and Tyr, while involvement of amines could not be proved. Changes observed in the amide I and II range indicated an increase of the degree of molecular disorder of sulfated silk. Accordingly, the I850/I830 intensity ratio between the two Tyr bands at 850-830 cm-1 increased from 1.41 to 1.52, indicating a more exposed state of Tyr residues in sulfated silk. TGA, DSC, and TG analyses showed that sulfated silk attained a higher thermal stability. A thermal transition attributable to sulfated silk fibroin fractions appeared at about 260 degrees C in the DSCthermograms.

http://www.ncbi.nlm.nih.gov/sites/entrez?=pubmed&Cmd=ShowDetailView&TermToSearch=17338562&ordinalpos=22&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum

4. A study on the flow stability of regenerated silk fibroin aqueous solution.
Wang H, Zhang Y, Shao H, Hu X.(State Key Laboratory for Modification of Chemical Fibers and Polymer Materials, College of Material Science and Engineering, Donghua University, Shanghai 200051, PR China).
Int J Biol Macromol. 2005 Jul;36(1-2):66-70
The flow stability of silk fibroin (SF) aqueous solutions with different concentrations under different temperatures was investigated. It was found that the flow stability decreased quickly with the increase of solution concentration and temperature. X-ray diffraction, Fourier transform infrared (FTIR) and Raman spectroscopy analysis showed that silk fibroin in aqueous solution was mainly in random coil and alpha-helix conformation. However, it turned into alpha-helix and beta-sheet conformation after gelation, and both silk I and silk II crystalline structures appeared accordingly. The investigation implies that the original dilute regenerated SF aqueous solution should be stored under low temperature and concentrated just before spinning.

http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=15916801&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVAbstractPlus

C. MEDICINAL APPLICATIONS
1. Development and evaluation of silk fibroin-based nerve grafts used for peripheral nerve regeneration.
Yang Y, Ding F, Wu J, Hu W, Liu W, Liu J, Gu X.
Biomaterials. 2007 Sep 18; [Epub ahead of print]
Slk fibroin (SF), derived from natural silk long used as a textile material, has recently become an important biomaterial for tissue engineering applications. We have previously reported on good in vitro biocompatibility of SF fibers with peripheral nerve tissues and cells. In the present study, we developed a novel biomimetic design of the SF-based nerve graft (SF graft) which was composed of a SF-nerve guidance conduit (NGC) inserted with oriented SF filaments. The SF-NGC prepared via well-established procedures exhibits an eggshell-like microstructure that is responsible for its superior mechanical and permeable properties beneficial to nerve regeneration. The SF graft was used for bridge implantation across a 10-mm long sciatic nerve defect in rats, and the outcome of peripheral nerve repair at 6 months post-implantation was evaluated by a combination of electrophysiological assessment, FluoroGold retrograde tracing and histological investigation. The examined functional and morphological parameters show that SF grafts could promote peripheral nerve regeneration with effects approaching those elicited by nerve autografts which are generally considered as the gold standard for treating large peripheral nerve defects, thus raising a potential possibility of using these newly developed nerve grafts as a promising alternative to nerve autografts.

http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6TWB-4PPFTBC-4&_user=989289&_coverDate=09%2F19%2F2007&_rdoc=1&_fmt=&_orig=search&_sort=d&view=c&_acct=C000049901&_version=1&_urlVersion=0&_userid=989289&md5=2be7c99e0c8bc73d65966ad569342c95

2. Silk-fibroin-coated liposomes for long-term and targeted drug delivery.
Gobin AS, Rhea R, Newman RA, Mathur AB. (University of Texas MD Anderson Cancer Center, Laboratory of Reparative Biology and Bioengineering, Plastic Surgery, Houston, TX 72230, USA.)
Int J Nanomedicine. 2006;1(1):81-7
Many barriers to drug delivery into a tumor site require careful consideration when designing a new drug. In this study, the adhesive targeting and drug specificity of modified liposomal vesicles on human-scar-producing cells, keloid fibroblasts, were investigated. Keloids express abundant levels of mucopolysaccharides and receptor tyrosine kinase (RTK). In this report, the structural properties, drug release kinetics, and therapeutic availability of silk-fibroin-coated, emodin-loaded liposomes (SF-ELP), compared with uncoated, emodin-loaded liposomes (ELP), were investigated. SF-ELP had a highly organized lamellae structure, which contributed to 55% of the liposomal diameter. This modified liposomal structure decreased emodin release rates by changing the release kinetics from a swelling and diffusional process to a purely diffusional process, probably due to steric hindrance. SF-ELP also increased adhesion targeting to keloid fibroblasts. Increased retention of SF-ELP is most likely due to the interaction of the fibrous protein coating around the ELP with the pericellular molecules around the cell. SF-ELP also decreased survival rate of keloids that expressed high levels of RTK. These results demonstrated that SF-ELP enhanced emodin delivery by improved diffusion kinetics and specific cell targeting.

http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17722265&ordinalpos=13&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum

3. Nanolayer biomaterial coatings of silk fibroin for controlled release
Wang X, Hu X, Daley A, Rabotyagova O, Cebe P, Kaplan DL. (Department of Biomedical Engineering, Tufts University, Medford, Massachusetts 02155, USA.)
J Control Release. 2007 Aug 28;121(3):190-9. Epub 2007 Jun 14
An all-aqueous, stepwise deposition process with silk fibroin protein for the assembly of nanoscale layered controlled release coatings was exploited. Model compounds, Rhodamine B, Even Blue and Azoalbumin, representing small molecule drugs and therapeutically relevant proteins were incorporated in the nanocoating process and their loading and release behavior was quantified. In addition, the structure and morphology of the coatings were characterized. Release studies in vitro showed that control of beta-sheet crystal content and the multilayer structure of the silk coatings correlated with the release properties of the incorporated compounds. In particular, higher crystallinity and a thicker silk capping layer suppressed the initial burst of release and prolonged the duration of release. These novel coatings and deposition approach provide a unique option to regulate structure and morphology, and thus release kinetics. The results also suggest these systems as a promising framework for surface engineering of biomaterials and medical devices to regulate the release of drugs, when considered with the all-aqueous process involved, the conformal nature of the coatings, the robust material properties of silk fibroin, and the degradability and biocompatibility of this family of protein.

FullText:http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17628161&ordinalpos=14&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum

4. Silk fibroin mediated delivery of liposomal emodin to breast cancer cells.
Cheema SK, Gobin AS, Rhea R, Lopez-Berestein G, Newman RA, Mathur AB. (University of Texas M.D. Anderson Cancer Center, Departments of Biomedical Engineering and Plastic Surgery, Unit 602, P.O. Box 301402, Houston, TX 72230-1402, United States.)
Int J Pharm. 2007 Aug 16;341(1-2):221-9. Epub 2007 Apr 3.
The efficacy of a drug is dependent on its mode of delivery and its potency at the tumor site. In this study, the drug delivery and efficacy of silk fibroin coated liposomes (SF-ELP), encapsulating a receptor tyrosine kinase inhibitor, emodin, on Her2/neu over-expressing breast cancer cells, was investigated. This study demonstrates that SF-ELP was more efficacious in suppressing the growth of Her2/neu over-expressing breast cancer cells MDA-MB-453 and BT-474 as compared to uncoated emodin loaded liposomes (ELP). Reduced levels of phosphorylated Her2/neu correlated with growth inhibition observed in the MDA-MB-453 cells, treated with both ELP and SF-ELP. ELP treatment of MDA-MB-453 breast cancer cells resulted in inhibition of the PI3K pathway whereas SF-ELP treatment inhibited both the PI3K and MAPK pathways, which contributed to the enhanced growth inhibitory effects of Her2/neu over-expressing breast cancer cells. Coating of ELP with silk fibroin did not alter the target specificity of emodin, on the other hand the emodin efficacy was enhanced. Higher uptake of emodin delivered by SF-ELP lead to increased cell death as compared to emodin delivery via ELP. Silk fibroin coating around the liposome imparts an extra layer that emodin has to extravasate in order to release from the encapsulating liposome. This increases retention of the drug in the cell for a longer time and protects emodin from quick release and metabolism. Longer intracellular retention may lead to the longer availability of emodin for down-modulation of various Her2/neu pathways. This study demonstrates that silk fibroin coating enhanced emodin delivery in Her2/neu over-expressing breast cancer cells thereby increasing the overall efficacy of the drug.

http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17499461&ordinalpos=19&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum

5. Food-grade mulberry powder enriched with 1-deoxynojirimycin suppresses the elevation of postprandial blood glucose in humans
Kimura T, Nakagawa K, Kubota H, Kojima Y, Goto Y, Yamagishi K, Oita S, Oikawa S, Miyazawa T. (Food & Biodynamic Chemistry Laboratory, Graduate School of Agricultural Science, Tohoku University, Sendai 981-8555, Japan. kmr@affrc.go.jp)
J. Agric. Food Chem., 55 (14), 5869 -5874, 2007. 10.1021

Mulberry 1-deoxynojirimycin (DNJ), a potent glucosidase inhibitor, has been hypothesized to be beneficial for the suppression of abnormally high blood glucose levels and thereby prevention of diabetes mellitus. However, DNJ contents in commercial mulberry products were as low as about 0.1% (100 mg/100 g of dry product), implying that the bioavailability of DNJ might not be expected. We carried out studies in two directions: (1) production of food-grade mulberry powder containing a maximally high DNJ content; (2) determination of the optimal dose of the DNJ-enriched powder for the suppression of the postprandial blood glucose through clinical trials. The following method was used: (1) DNJ concentrations in mulberry leaves from different cultivars, harvest seasons, and leaf locations were determined using hydrophilic interaction chromatography with evaporative light scattering detection. (2) Healthy volunteers received 0, 0.4, 0.8, and 1.2 g of DNJ-enriched powder (corresponding to 0, 6, 12, and 18 mg of DNJ, respectively), followed by 50 g of sucrose. Before and 30-180 min after the DNJ/sucrose administration, plasma glucose and insulin were determined. The following results were obtained: (1) Young mulberry leaves taken from the top part of the branches in summer contained the highest amount of DNJ. After optimization of the harvesting and drying processes for young mulberry leaves (Morus alba L. var. Shin ichinose), DNJ-enriched powder (1.5%) was produced. (2) A human study indicated that the single oral administration of 0.8 and 1.2 g of DNJ-enriched powder significantly suppressed the elevation of postprandial blood glucose and secretion of insulin, revealing the physiological impact of mulberry DNJ (effective dose and efficacy in humans). This study suggests that the newly developed DNJ-enriched powder can be used as a dietary supplement for preventing diabetes mellitus.

Full text: http://pubs.acs.org/cgi-bin/abstract.cgi/jafcau/2007/55/i14/abs/jf062680g.html

RESEARCH UPDATES-01

gkrajeshrajesh@gmail.com — Sat, 15 Sep 2007 08:56:00 +0000

We start publishing latest research findings in sericulture and related fields for the benefit of researchers and students in the field. Our intention is to help you by reducing your efforts in finding latest research information. We painstakingly hunt for articles and present before you. Make it a habit to check this site once a week, you get it all here. To begin with the information is provided under four categories viz. Mulberry, Silkworm, Non mulberry sericulture and Silk. Title, details of publication and a link to the full article are provided on a weekly basis. Each weekly posting of RESEARCH UPDATES will be linked.
In a few cases you may need subscription to the journal to access the full article. We hope it will be of use to all subscribers of ‘the silkworm’. We are keen to receive your comments.

A. Mulberry
1. Light, Temperature, Seed Burial, and Mulch Effects on Mulberry Weed (Fatoua villosa) Seed Germination
GINA M. PENNY and JOSEPH C. NEAL
Weed Technology Volume 17; Issue 2 (April 2003) pp. 213–218
Abstract
Fatoua villosa (mulberry weed) is a new and invasive weed of container nurseries and landscapes in the southeastern United States. Studies were conducted to determine the effects of light, planting depth, mulch depth, and temperature on mulberry weed seed germination and seedling emergence. Light stimulated mulberry weed seed germination, with less than 5% of seeds germinating in the dark compared with 48 to 60% germinating in the light. In all emergence studies, the highest number of seedlings emerged when seeds were placed on the soil surface, with emergence decreasing as planting or mulch depth increased. Planting depths of 1.8 cm or mulch depths of 3.7 cm reduced mulberry weed emergence by 90%. These data suggest that mulch would control mulberry weed effectively. To study the effects of temperature on germination, two seed batches collected locally in October 1998 and August 1999 were used. Maximum germination of seeds collected in 1998 occurred at 25 C, with germination decreasing at higher temperatures and no germination at lower than 15 C or over 40 C. For seeds collected in 1999, maximum germination occurred from 19 to 29 C, with germination decreasing with temperatures above 29 C or below 19 C. At temperatures of 15 and 42 C germination, percentages were 71 and 11%, respectively. Seedlings germinated at 15 C developed slowly but otherwise appeared normal. For both seed lots, seedlings were stunted and chlorotic at 38 C. That mulberry weed seed germinated over a wide range of temperatures suggests its potential to emerge throughout most of spring, summer, and autumn in the southeastern United States.
Link to full article (you may need subscription): http://www.bioone.org/perlserv/?request=get-pdf&doi=10.1614%2F0890-037X%282003%29017%5B0213%3ALTSBAM%5D2.0.CO%3B2
2. Micromorphological Characterization of Ten Mulberry Cultivars (Morus spp)
Magda Biasiolo, Maria Teresa Da Canal, and Noemi Tornadore(Biology Department, Geobotany Section, Padua University, V. G. Colombo 3, 35121 Padova, Italy; E-mail: tornado@civ.bio.unipd.it)
Economic Botany Volume 58, Issue 4 (December 2004)
Abstract
The micromorphological features of the vegetative and reproductive structures of ten mulberry cultivars grown at the Specialized Sericultural Section of the Agricultural Zoology Experimental Institute of Padua, northeastern Italy, were examined by SEM in order to determine the charactetistics that were the most valuable taxonomically. The observed specimens (leaves, flowers, seeds, and pollen grains) showed micromorphological differences regarding leaf hairiness, quantity of waxes, quality of epidermis cuticle and tepal hairiness. The effects of differing environments in altering the floral sex ratios of this basically monoecious group of plants were also investigated. However, no significant differences between the micromorphology of the seeds and the pollen grains of these selected cultivars were detected. The authors are hopeful that the information gained in this study may prove useful in the future creation of an exhaustive and final catalogue “descriptor” of cultivated varieties belonging to the genus Morus L.
Keywords: White mulberry, cultivars, SEM, leaf, flower, seed, pollen grain micromorphology
Link to full article (you may need subscription): http://www.bioone.org/perlserv/?request=get-pdf&doi=10.1663%2F0013-0001%282004%29058%5B0639%3AMCOTMC%5D2.0.CO%3B2
B. Silkworm
1. Developmental Profile of Annexin IX and its Possible Role in Programmed Cell Death of the Bombyx mori Anterior Silk Gland
Yu Kaneko, Keiko Takaki, Masafumi Iwami, and Sho Sakurai
Zoological Science Volume 23, Issue 6 (June 2006) pp. 533–542
Abstract
During pupal metamorphosis, the anterior silk gland (ASG) of the silkworm, Bombyx mori, undergoes programmed cell death (PCD), which is triggered by 20-hydroxyecdysone (20E). Annexin IX (ANX IX) has been identified as a 20E-inducible gene in dying ASGs, and we show here that its expression is down-regulated in tissues destined to die but not in tissues that survive pupal metamorphosis. ANX IX expression was high in the ASGs during the feeding period, when the ecdysteroid titer was low, and decreased in response to the rising ecdysteroid titer that triggered pupal metamorphosis. Before gut purge, in vitro exposure of the ASGs to 20E levels corresponding to the ecdysteroid concentration present at the time of gut purge caused a decrease in ANX IX messenger RNA levels. Expression profiles of EcR and USP, and the 20E concentration-responses of these genes, indicate the importance of the relative abundance of EcR-A and EcR-B1 isoforms in ANX IX regulation. These results suggest an involvement of ANX IX in the determination of PCD timing by delaying or suppressing the response to the increase in hemolymph ecdysteroid concentration during the prepupal period.
Keywords: annexin, Bombyx, 20-hydroxyecdysone, EcR, programmed cell death
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2. Release of Ecdysteroid-Phosphates from Egg Yolk Granules and Their Dephosphorylation during Early Embryonic Development in Silkworm, Bombyx mori
Ryouichi Yamada, Yumi Yamahama, and Haruyuki Sonobe
Zoological Science Volume 22, Issue 2 (February 2005) pp. 187–198
Abstract
Newly laid eggs of many insect species store maternal ecdysteroids as physiologically inactive phosphoric esters. In the silkworm Bombyx mori, we previously reported the presence of a specific enzyme, called ecdysteroid-phosphate phosphatase (EPPase), which catalyzes the dephosphorylation of ecdysteroid-phosphates to increase the amount of free ecdysteroids during early embryonic development. In this study, we demonstrated that (1) EPPase is found in the cytosol of yolk cells, (2) ecdysteroid-phosphates are localized in yolk granules, being bound to the yolk protein vitellin (Vn), and (3) Vn-bound ecdysteroid-phosphates are scarcely hydrolyzed by EPPase, although free ecdysteroid-phosphates are completely hydrolyzed by EPPase. Thus, we investigated the mechanism by which ecdysteroid-phosphates dissociate from the Vn-ecdysteroid-phosphate complex, and indicated that the acidification of yolk granules causes the dissociation of ecdysteroid-phosphates from the Vn-ecdysteroid-phosphate complex and thereby ecdysteroid-phosphates are released from yolk granules into the cytosol. Indeed, the presence of vacuolar-type proton-translocating ATPase in the membrane fraction of yolk granules was also verified by Western blot analysis. Our experiments revealed that Vn functions as a reservoir of maternal ovarian ecdysteroid-phosphates as well as a nutritional source during embryonic development. This is the first report showing the biochemical mechanism by which maternal Vn-bound ecdysteroid-phosphates function during early embryonic development.
Keywords: ecdysteroids, phosphatase, V-ATPase, vitellin, embryonic development
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3. Establishment of a Sandwich ELISA System to Detect Diapause Hormone, and Developmental Profile of Hormone Levels in Egg and Subesophageal Ganglion of the Silkworm, Bombyx mori
Norio Kitagawa, Kunihiro Shiomi, Kunio Imai, Teruyuki Niimi, Toshinobu Yaginuma, and Okitsugu Yamashita
Zoological Science Volume 22, Issue 2 (February 2005) pp. 213–221
Abstract
In the silkworm Bombyx mori, diapause hormone (DH) is produced in the female subesophageal ganglion (SG) and induces embryonic diapause by targeting developing ovaries. DH is processed from a precursor protein consisting of DH, pheromone biosynthesis activating neuropeptide (PBAN) and three other neuropeptides (SGNPs). Because these five neuropeptides share a common sequence, FXPRLamide, at the C-terminus, a direct and specific assay for DH itself is required in order to understand the profile of concentration changes. In this study, we produced a mouse monoclonal antibody (anti-DH[N] mAb) against the N-terminal region of DH and developed a sandwich enzyme-linked immunosorbent assay using the anti-DH[N] mAb and a rabbit polyclonal antibody against the C-terminus of DH. This procedure enabled us to specifically quantify the DH molecule at femtomolar levels (equivalent to 1/10 of SG). We then plotted DH levels in eggs and SGs during embryonic and post-embryonic development. DH was present in late-stage embryos that had been destined for the production of both diapause and nondiapause eggs. DH levels in SG gradually increased in both types during larval development and peaked at the early pupal stage. At the middle pupal stage, DH levels in SG and SG-brain complex decreased markedly in the diapause-egg producing type, thus indicating active release of DH into the hemolymph. From 5th instar larva to adult, no sexual differences in DH levels were observed in SGs or SG-brain complexes from diapause and nondiapause egg-producing types.
Keywords: Diapause hormone, FXPRLamide neuropeptide family, bivoltinism, Bombyx mori, subesophageal ganglion, sandwich enzyme-linked immunosorbent assay
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4. Effects of Applaud on the Growth of Silkworm (Lepidoptera: Bombycidae)
Maria E. Vassarmidaki, Paschalis C. Harizanis, and Sergios Katsikis
Journal of Economic Entomology Volume 93, Issue 2 (April 2000) pp. 290–292
Abstract
An experiment was carried out to evaluate the effect of the insecticide Applaud (buprofezin 25% WP) on the silkworm Bombyx mori (L.). This insecticide belongs to the class of insect growth regulators (IGR). The larvae were fed on leaves treated with 3 different concentrations (0.5, 1, 2 g/liter) of Applaud on the 1st d of each instar. Analysis of data with the Tukey–Kramer test at 1% significant level revealed that mortality and larval duration did not differ among the treatments. On the contrary, the larval weight, which was estimated just before mounting (procedure during which the mature larva climbing on a branch or other material to spin the cocoon), differed among the treatments. Also, cocoon weight, shell weight, and cocoon sericin and fibroin content were different among the treatments, except the shell cocoon ratio. Maximum weight was observed in the controls and minimum in the last instar treatments. Our data suggest that supplementation of Applaud through food to larvae does not affect their mortality rate. On the contrary, it affects larval growth and cocoon parameters.
Keywords: Bombyx mori, buprofezin, larval duration, larval weight, cocoon parameters
Link to full article (you may need subscription): http://www.bioone.org/perlserv/?request=get-pdf&doi=10.1603%2F0022-0493%282000%29093%5B0290%3AEOAOTG%5D2.0.CO%3B2
5. Stress induction of Bm1 RNA in silkworm larvae: SINEs, an unusual class of stress genes
Richard H. Kimura, Prabhakara V. Choudary, Koni K. Stone, and Carl W. Schmid
Cell Stress & Chaperones Volume 6, Issue 3 (July 2001) pp. 263–272
This study surveys the induction of RNA polymerase III (Pol III)–directed expression of short interspersed element (SINE) transcripts by various stresses in an animal model, silkworm larvae. Sublethal heat shock and exposure to several toxic compounds increase the level of Bm1 RNA, the silkworm SINE transcript, while also transiently increasing expression of a well-characterized stress-induced transcript, Hsp70 messenger RNA (mRNA). In certain cases, the Bm1 RNA response coincides with that of Hsp70 mRNA, but more often Bm1 RNA responds later in recovery. Baculovirus infection and exposure to certain toxic compounds increase Bm1 RNA but not Hsp70 mRNA, showing that SINE induction is not necessarily coupled to transcription of this particular heat shock gene. SINEs behave as an additional class of stress-inducible genes in living animals but are unusual as stress genes because of their high copy number, genomic dispersion, and Pol III–directed transcription.
Link to full article (you may need subscription): http://www.bioone.org/perlserv/?request=get-pdf&doi=10.1379%2F1466-1268%282001%29006%3C0263%3ASIOBRI%3E2.0.CO%3B2
6. The Bmdsx transgene including trimmed introns is sex-specifically spliced in tissues of the silkworm, Bombyx mori
Shunsuke Funaguma, Masataka G. Suzuki, Toshiki Tamura, and Toru Shimada
Journal of Insect Science Volume 5, Issue 17 (May 2005) pp. 1–6
Abstract
Orthologue of the sex-determining gene doublesex (dsx) and known to be sex-specifically expressed in various tissues of the silkworm, Bombyx mori. Its pre-mRNA is sex-specifically spliced and encodes female-specific or male-specific polypeptides. The open reading frame of Bmdsx consists of 5 exons, of which exons 3 and 4 are female-specific and its pre-mRNA was known to undergo default processing to generate the female-type mRNA. Previous reports have shown that the mechanism of splicing of the doublesex gene is different in Drosophila melanogaster and Bombyx mori. However, intron 4 is so long that it is difficult to identify the intronic cis-element(s) required for male-specific splicing of Bmdsx pre-mRNA using Bmdsx minigenes whose introns are shortened in various manners. As a first step toward discovery of the cis-element, the Bmdsx mini gene, which consisted of exon 1 and 5 and internally shortened introns 2 to 4, was constructed, and transgenic silkworms expressing this construct were generated. Bmdsx pre-mRNA transcribed derived from transgene was sex-specifically spliced. This result shows that the mini gene contained the information necessary for the correct regulation of alternative splicing.
Keywords: alternative splicing, Bmdsx, Bombyx mori, piggyback, long intron
Link to full article (you may need subscription): http://www.bioone.org/perlserv/?request=get-document&doi=10.1672%2F1536-2442%282005%29005%5B0001%3ATBTITI%5D2.0.CO%3B2
7. ESTABLISHMENT AND CHARACTERIZATION OF A CONTINUOUS CELL LINE FROM PUPAL OVARIES OF JAPANESE OAK SILKWORM ANTHERAEA YAMAMAI GUERIN-MENEVILLE
SHIGEO IMANISHI, HAJIME INOUE, TAKESHI KAWARABATA, KOYU HARA, MASAKO FUNAKOSHI, CHISA YASUNAGA-AOKI, and KAZUHIKO MITSUDA
In Vitro Cellular & Developmental Biology - Animal Volume 39, Issue 1 (January 2003) pp. 1–3
Abstract
Pupal ovaries of the wild oak silkworm Antheraea yamamai Guerin-Meneville were cultured in MGM-448 (Modified Grace Medium-448) medium containing 10% fetal bovine serum. After the primary culture was set up in 1988, a continuous cell line was obtained in 1991, designated as NISES-Anya-0611 (Anya-0611). The population doubling time was 54 hrs. and 19 min. at 96 passages and 88 hrs. and 29 min. at 387 passages. Spindle-shaped and spherical cells coexisted in the cell group. The cell line karyotype line was typical of lepidopteran cell lines, consisting of numerous small chromosomes. The cell line was distinguished from other lepidopteran cell lines by comparing malic enzyme, phosphoglucose isomerase, phosphoglucose mutase, and isocitric dehydrogenase isozyme patterns. The cell line was highly infected to the Antheraea yamamai nuclear polyhedrosis virus (Anya NPV). The luciferase gene of recombinant Bm NPV (BmNPVP6ETL) was able to express in the cell line, too, so that luciferase recombinant products were able to be detected in the cell body and in supernatant. The Anya NPV clone group was isolated on the cell seat using plaque purification.
Keywords: insect, wild silkworm, primary culture, karyotype, isozyme patterns, nuclear polyhedrosis virus
Link to full article (you may need subscription): http://www.bioone.org/perlserv/?request=get-pdf&doi=10.1290%2F1543-706X%282003%29039%3C0001%3AEACOAC%3E2.0.CO%3B2
8. Effects of Wolbachia in the uzifly, Exorista sorbillans, a parasitoid of the silkworm, Bombyx mori
H.P. Puttaraju and B.M. Prakash
Journal of Insect Science Volume 5, Issue 30 (November 2005) pp. 1–7
Abstract
The uzifly, Exorista sorbillans (Diptera: Tachinidae), a parasitoid of the silkworm, Bombyx mori L. (Lepidoptera: Bombycidae), harbours Wolbachia (Rickettsia) endosymbionts. Administration of 0.05 mg/ml oxytetracycline to the adult uziflies removed Wolbachia endosymbionts and resulted in different reproductive disorders, such as i) reduction in fecundity of uninfected females, ii) cytoplasmic incompatibility in crosses between infected males and uninfected females, iii) sterility in the crosses between both males and females from uninfected populations, and iv) sex-ratio distortion in uninfected females irrespective of the presence of Wolbachia in males. However, tetracycline treatment did not have much effect on longevity of the uzifly. These results suggest that the interaction of Wolbachia with its uzifly host is one of mutual symbiosis as it controls the reproductive physiology of its hosts.
Keywords: fecundity, cytoplasmic incompatibility, sterility, sex ratio, tetracycline
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9. Apoptosis and Adhesion of Hemocytes During Molting Stage of Silkworm, Bombyx mori
Toshio Okazaki, Noriyuki Okudaira, Kikuo Iwabuchi, Hajime Fugo, and Tatsuo Nagai
Zoological Science Volume 23, Issue 3 (March 2006) pp. 299–304
Abstract
To clarify the regulatory mechanism of the rapid changes in the hemocyte density in the silkworm, Bombyx mori, during ecdysis, we evaluated the relationship between the hemocyte density and the incidence of apoptosis during this stage. We also evaluated the role of the sugar chains on the adhesion of hemocytes by analyzing the effects on the hemocyte density of the injection of enzymes that cut sugar chains and monosaccharides into the body cavity.
The hemocyte density was increased in the molting stage and spinning, and then decreased after the ecdysis. During spinning, the diameter of the granulocytes markedly increased, in which fatty granules in the cytoplasm increased, becoming foamy. They were identified to be apoptotic hemocytes using the Hoechst staining and the Comet assay. The decrease in the hemocyte density during spinning was mainly caused by the apoptosis of granulocytes. Next, we focused on the fluctuation of hemocyte density during the molting stage. Examination of the changes in the hemocyte density induced by injecting glycoside hydrolases, neuraminidase, sialic acid, or monosaccharides into the body cavity during the fourth molt stage and the third day in fifth instar larva demonstrated that the alteration of hemocyte density was regulated by the attachment and detachment of hemocytes via a selectin ligand, sugar chains. As with the injection of glycoside hydrolase, neuraminidase, sialic acid and fucose raised the hemocyte detachment, and it was assumed that the selectin ligands include the sialyl Lewis x like sugar chains, the same as mammalian lymphocytes.
Keywords: Bombyx mori, hemocyte, apoptosis, adhesion, sialyl Lewis x
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10. Immunocytochemical Identification of Neuroactive Substances in the Antennal Lobe of the Male Silkworm Moth Bombyx mori
Masaaki Iwano and Ryohei Kanzaki
Zoological Science Volume 22, Issue 2 (February 2005) pp. 199–211
Abstract
As a first step towards understanding the functional role of neuroactive substances in the first olfactory center of the male silkworm moth Bombyx mori, we carried out an immunocytochemical identification of antennal lobe neurons. Antibodies against γ-aminobutyric acid (GABA), FMRFamide, serotonin, tyramine and histamine were applied to detect their existence in the antennal lobe. In the present immunocytochemical study, we clarified four antenno-cerebral tracts from their origin and projection pathways to the protocerebrum, and revealed the following immunoreactive cellular organization in the antennal lobe. 1) Local interneurons with cell bodies in the lateral cell cluster showed GABA, FMRFamide and tyramine immunoreactivity. 2) Projection neurons passing through the middle antenno-cerebral tract with cell bodies in the lateral cell cluster showed GABA and FMRFamide immunoreactivity. Projection neurons passing through the outer antenno-cerebral tract with cell bodies in the lateral cell cluster showed FMRFamide immunoreactivity. 3) Centrifugal neurons passing through the inner antenno-cerebral tract b with cell bodies located outside the antennal lobe showed serotonin and tyramine immunoreactivity. Our results revealed basic distribution patterns of neuroactive substances in the antennal lobe and indicated that each projection pathway from the antennal lobe to the protocerebrum contains specific combination of neuro-active substances.
Keywords: neuroactive substances, immunocytochemical, antennal lobe, projection pathway, silkworm moth
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11. Gene Expression Profiling in the Silkworm, Bombyx mori, During Early Embryonic Development
Sun-Mee Hong, Si-Kab Nho, Nam-Soon Kim, Jin-Sung Lee, and Seok-Woo Kang
Zoological Science Volume 23, Issue 6 (June 2006) pp. 517–528
Abstract
We prepared a cDNA library for a microarray from eggs of the silkworm, Bombyx mori, at the germ-band formation (24 hours after fertilization) stage. Using a microarray constructed with 2,445 ESTs, we screened gene expression profiles during germ-band formation at six specific time points in the early embryonic stages (from the unfertilized egg to the formation of abdominal leg appendages), and determined 241 of these cDNAs to represent genes that were expressed differentially during the germ-band formation stage. These differentially expressed genes grouped into two clusters. In the early and late clusters, 203 and 38 genes were upregulated, respectively. In the upregulated clusters, we isolated several genes that were associated with development and cell communication, including egalitarian, RAD23b, innexin 2, and senescence-associated protein. Northern blot hybridization revealed that the expression patterns of 14 genes had changed in each of the stages. In this study, we assessed changes in the levels of gene expression in relation to the germ-band formation stages in whole Bombyx embryos.
Keywords: silkworm, Bombyx mori, cDNA microarray, embryogenesis, development, cell communication
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12. A Rapid Increase in cAMP in Response to 20-Hydroxyecdysone in the Anterior Silk Glands of the Silkworm, Bombyx mori
Mohamed Elmogy, Jun Terashima, Masatoshi Iga, Masafumi Iwami, and Sho Sakurai
Zoological Science Volume 23, Issue 8 (August 2006) pp. 715–719
Abstract
In the anterior silk glands (ASGs) of the silkworm, Bombyx mori, intracellular cAMP increases transiently to a very high level shortly after the hemolymph ecdysteroid peak in the prepupal period. In cultured ASGs obtained on the day of gut-purge, cAMP levels were increased by 20-hydroxy-ecdysone (20E), and this increase was enhanced by an inhibitor of phosphodiesterase, but was not affected by α-amanitin, indicating the 20E action may not be mediated via gene expression. The increase in cAMP occurred within 30 seconds of exposure to a physiological concentration of 20E (1 μM), and also by ponasterone A. Our findings indicate a nongenomic action of ecdysteroids in insects, which may be an additional mechanism by which this steroid hormone induces acute responses in tissues and cells.
Keywords: ecdysone, cAMP, silk gland, programmed cell death, Bombyx mori
* Corresponding author. Phone: +81-76-264-6250; Fax : +81-76-264-6255; E-mail: ssakurai@kenroku.kanazawa-u.ac.jp
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13. Brain-Derived Neurotrophic Factor Promotes Neurite Growth and Survival of Antennal Lobe Neurons in Brain from the Silk Moth, Bombyx mori in vitro
Jin Hee Kim, Dong Kyung Sung, Chan Woo Park, Hun Hee Park, Cheolin Park, Soung-Hoo Jeon, Pil Don Kang, O-Yu Kwon, and Bong Hee Lee
Zoological Science Volume 22, Issue 3 (March 2005) pp. 333–342
Abstract
This study was conducted to investigate effects of brain-derived neurotrophic factor on the neurite growth and the survival rate of antennal lobe neurons in vitro, and secretion of brain-derived neurotrophic factor-like neuropeptide from brain into hemolymph in the silk moth, Bombyx mori. In primary culture of antennal lobe neurons with brain-derived neurotrophic factor, it promoted both a neurite extension of putative antennal lobe projection neurons and an outgrowth of branches from principal neurites of putative antennal interneurons with significance (p<0.05). name="cor1">* Corresponding author. Phone: +82-2-3290-3156; Fax: +82-2-3290-3623; E-mail: bhlee@korea.ac.kr
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C. Non mulberry sericulture
1. Impact of Pollen Grains from Bt Transgenic Corn on the Growth and Development of Chinese Tussah Silkworm, Antheraea pernyi (Lepidoptera: Saturniidae)
Wendong Li, Kongming Wu, Xiaoqi Wang, Guirong Wang, and Yuyuan Guo
Environmental Entomology Volume 34, Issue 4 (August 2005) pp. 922–928
Abstract
The tussah silkworm, Antheraea pernyi (Lepidoptera: Saturniidae), is an important natural resource for the silk industry and has been cultured using wild host plants for >2,000 yr in China. To clarify whether there is any risk from pollen of Cry1Ab-containing corn varieties on this insect, the frequency of pollen dispersal and deposition of corn pollen near cornfields and impact on the development of tussah silkworm larvae were studied separately in the field and laboratory. The field survey showed that the pollen density was the highest inside the cornfield with a value of 1,000 grains/cm2. The pollen deposition rapidly declined with distance from the edge of the cornfield as expected in most cases. No significant differences were observed in the amounts of pollen deposited on glass slides positioned at different heights from the ground at each distance. In the laboratory bioassays, there were no significant differences in the larval mortality and weight of Chinese tussah silkworm between treatment with pollen grains from a transgenic corn line and a nontransgenic corn control at a density of 1,000 pollen grains/cm2. Also no significant negative impact was found for efficiency of conversion of digested food (ECD), efficiency of conversion of ingested food (ECI), and efficiency of approximate digestion of food (AD) at the level of 1,000 pollen grains/cm2. The results of this study suggest that the impact on the Chinese tussah silkworm of Bt corn pollen from the hybrid to be commercialized in China is negligible in the natural environment.
Keywords: pollen grains, Bt transgenic corn, Chinese tussah silkworm, environmental impact
Link to full article (you may need subscription): http://www.bioone.org/perlserv/?request=get-pdf&doi=10.1603%2F0046-225X%282005%29034%5B0922%3AIOPGFB%5D2.0.CO%3B2

D. Silk
1. Modeling Of The Stress-Strain Behavior Of Egg Sac Silk Of The Spider Araneus Diadematus
Els Van Nimmen, Kris Gellynck, Tom Gheysens, Lieva Van Langenhove, and Johan Mertens
Journal of Arachnology Volume 33, Issue 2 (August 2005) pp. 629–639
Abstract
Spider silk has attracted the attention of many scientists because of its desirable physical properties. Most of this attention has been devoted to dragline silk, a thread that has high tensile strength, high strain and ultra-low weight. To help understand structure-property relationships in spider silks, the tensile behavior of egg sac (cylindrical gland) silk of Araneus diadematus Clerck 1757 was compared with dragline (major ampullate gland) and silkworm silks. In addition, stress-strain curves of egg sac silk were simulated by a spring-dashpot model, specifically a Standard Linear Solid (SLS) model. The SLS model consists of a spring in series with a dashpot and in parallel with another spring, resulting in three unknown parameters. The average stress-strain curve of fibers from five different egg sacs could be accurately described by the model. Closer examination of the individual stress-strain curves revealed that in each egg sac two populations of fibers could be distinguished based on the parameters of the SLS model. The stress-strain curves of the two populations clearly differed in their behavior beyond the yield point and were probably derived from two different layers within the egg sac. This indicates that silks in the two layers of A. diadematus egg sacs probably have different tensile behavior.
Keywords: Spider silk, tensile behavior, cocoon, cylindrical gland, tubuliform gland, Araneidae
Link to full article (you may need subscription): http://www.bioone.org/perlserv/?request=get-pdf&doi=10.1636%2FCS05-5.1
2. Physical properties of Hydropsyche siltalai (Trichoptera) net silk
Sarah A. BrownA, Graeme D. RuxtonA, and Stuart HumphriesB
Journal of the North American Benthological Society ;Volume 23, Issue 4 (December 2004)
Abstract
Suspension-feeding trichopterans spin a fine-silk capture net that is used to remove suspended matter from the water. The efficiency of these nets has previously been studied by considering the geometry of the web structure but the material from which the nets is constructed has received little attention. We report measurements of the tensile strength and extensibility of net silk from Hydropsyche siltalai. These measurements place caddisfly silk as one of the weakest natural silks so far reported, with a mean tensile strength of 221 ± 22 megaNewtons (MN)/m2. We also show that H. siltalai silk can more than double in length before catastrophic breakage, and that the silk is at least 2 orders of magnitude stronger than the maximum force estimated to act upon it in situ. Possible reasons for this disparity include constraints of evolutionary history and safety margins to prevent net failure or performance reduction.
Keywords: Hydropsyche siltalai, tensile strength, extensibility, breaking strain, stress
Link to full article (you may need subscription): http://www.bioone.org/perlserv/?request=get-pdf&doi=10.1899%2F0887-3593%282004%29023%3C0771%3APPOHST%3E2.0.CO%3B2
3. Spider Dragline Silk: Correlated And Mosaic Evolution In High-Performance Biological Materials
Brook O. Swanson, Todd A. Blackledge, Adam P. Summers, and Cheryl Y. Hayashi
Evolution Volume 60, Issue 12 (December 2006) pp. 2539–2551
Abstract
The evolution of biological materials is a critical, yet poorly understood, component in the generation of biodiversity. For example, the diversification of spiders is correlated with evolutionary changes in the way they use silk, and the material properties of these fibers, such as strength, toughness, extensibility, and stiffness, have profound effects on ecological function. Here, we examine the evolution of the material properties of dragline silk across a phylogenetically diverse sample of species in the Araneomorphae (true spiders). The silks we studied are generally stronger than other biological materials and tougher than most biological or man-made fibers, but their material properties are highly variable; for example, strength and toughness vary more than fourfold among the 21 species we investigated. Furthermore, associations between different properties are complex. Some traits, such as strength and extensibility, seem to evolve independently and show no evidence of correlation or trade-off across species, even though trade-offs between these properties are observed within species. Material properties retain different levels of phylogenetic signal, suggesting that traits such as extensibility and toughness may be subject to different types or intensities of selection in several spider lineages. The picture that emerges is complex, with a mosaic pattern of trait evolution producing a diverse set of materials across spider species. These results show that the properties of biological materials are the target of selection, and that these changes can produce evolutionarily and ecologically important diversity.
Keywords: Biomaterials, biomechanics, independent contrasts, major ampullate silk, phylogenetic signal, tensile test, web
Link to full article (you may need subscription): http://www.bioone.org/perlserv/?request=get-pdf&doi=10.1554%2F06-267.1
4. Spider Silk Proteins – Mechanical Property and Gene Sequence
Anna Rising, Helena Nimmervoll, Stefan Grip, Armando Fernandez-Arias, Erica Storckenfeldt, David P Knight, Fritz Vollrath, and Wilhelm Engström
Zoological Science Volume 22, Issue 3 (March 2005) pp. 273–281
Abstract
Spiders spin up to seven different types of silk and each type possesses different mechanical properties. The reports on base sequences of spider silk protein genes have gained importance as the mechanical properties of silk fibers have been revealed. This review aims to link recent molecular data, often translated into amino acid sequences and predicted three dimensional structural motifs, to known mechanical properties.
Keywords: spider silk, MaSp1, MaSp2, structure, function
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5. The Combing of Cribellar Silk by the Prithine Misionella Mendensis, with Notes on Other Filistatid Spiders (Araneae: Filistatidae)
LARA LOPARDO and MARTÍN J. RAMÍREZ
American Museum Novitates Volume 3563, Issue 1 (May 2007) pp. 1–14
Abstract
We present the first observations of the combing and attaching behavior in the subfamily Prithinae (Filistatidae), taken from Misionella mendensis. We compare its web architecture with that of other prithines (Pritha nana and Pikelinia sp. from Chile) and filistatines (Kukulcania hibernalis and Filistata insidiatrix). The combing behavior of M. mendensis corresponds to the stereotyped type I combing behavior, as is known for other filistatids. However, M. mendensis attaches the cribellar segments in a unique way, splitting the cribellar segment longitudinally and pushing each half to the substrate, attaching the silk with the tarsi of both legs IV simultaneously. These stereotyped movements result in web units of a very characteristic structure. We report the same split attachment behavior in the prithine Pikelinia tambilloi. We scored these observations into a previous dataset for filistatid relationships. Because of the missing observations on attachment behavior in the North American basal genus Filistatinella, the sister group of all other prithines, the evolution of split cribellar strands is a potential synapomorphic characteristic for the Prithinae, or at least the subgroup excluding its basal taxon.Link to full article (you may need subscription): http://www.bioone.org/perlserv/?request=get-pdf&doi=10.1206%2F0003-0082%282007%29529%5B1%3ATCOCSB%5D2.0.CO%3B2

Anthocyanins from mulberry fruits - a challenge for mulberry germplasms

gkrajeshrajesh@gmail.com — Fri, 07 Sep 2007 15:25:00 +0000

Anthocyanins are edible pigments, which hold potential use as natural food colourants. As the safety of synthetic pigments are doubted and in the wake of increasing demand for natural food colourants their significance in food industry increase. Anthocyanins are reported to yield attractive colours such as orange, red and blue. Since they are water-soluble their incorporation into aqueous food systems is easy. Apart from the colouring property anthocyanins are also known to possess antioxidant property and improve visual acuity. They also possess antineoplastic, radiation –protective, vasotonic, vasoprotective, anti-inflammatory, chemo and hepato - protective properties.
Xueming Liu and his co workers at the Sericultural Research Institute, Guangdong Academy of Agricultural Sciences, China in 2004 developed a cheap and industrially feasible method for purification of anthocyanins from mulberry fruit which could be used as a red food colourant of high colour value (of above 100). They found that out of 31 Chinese mulberry cultivars tested the total anthocyanin yield varied from 147.68 mg. to 2725.46 mg. per litre of fruit juice. Extraction and purification was done by using acidified ethanol as effluent solvent and cross-linked polystyrene copolymer - macro porous resin as adsorbant. The results indicated that total sugars, total acids and vitamins remained intact in the residual juice after removal of anthocyanins and that the residual juice could be fermented in order to produce products such as juice, wine and sauce.
In many parts of the globe mulberry is grown for its fruit. The fruit is known to have many medicinal properties and used for making jam, wine etc. As the genera Morus has been domesticated over thousands of years and constantly been subjected to heterosis breeding (mainly for improving leaf yield), it would not be impossible for evolving breeds suitable for berry production. The finding offers possible industrial use of mulberry as a source of anthocyanins as natural food colourant, which could enhance the overall profitability of sericulture. Anthocyanin content was found to depend on climate and area of cultivation and it was higher on a sunny day. This finding holds promise for tropical sericulture countries for profiting from industrial anthocyanin production from mulberry through better anthocyanin recovery.
This offers a challenging task to the mulberry germplasms resources across the globe, in exploration and collection of fruit yielding mulberry species; their Characterization, cataloguing and evaluation for anthocyanin content by using traditional as well as modern means and bio technology tools; developing an information system about these cultivars or varieties; training and global coordination of utilization of these genetic stocks and finally in evolving suitable breeding strategies to improve the anthocyanin content in potential breeds by collaboration with various research stations in the field of sericulture, plant genetics & breeding, biotechnology and pharmacology.

Reference: Liu, Xueming et. al. (2004): Quantification and purification of Mulberry anthocyanins with macroporous resins.; Journal of Bio medicine and Biotechnology; 2004:5 326-331, http://www.jbb.hindawi.com/.

Franklin on SILK

gkrajeshrajesh@gmail.com — Mon, 03 Sep 2007 12:54:00 +0000

Benjamin Franklin, one of the most important Founding Fathers of the United States said
“…….It is the happiest of all inventions of clothing…… The wear of silken garments continues so much longer, from the strength of the materials, as to give it greatly the preference …….Mulberry trees may be planted in hedge grows, on walker avenues or for shade near a house, where nothing else is wanted to grow……”

SDS PAGE - What each chemical ingredient does?

gkrajeshrajesh@gmail.com — Sun, 19 Aug 2007 10:49:00 +0000

The importance of Poly Acrylamide Gel electrophoresis in protein research is un questioned. The composition of the gel is complex and the procedure is more an art than science. Precision is the key word in its making. This article provides information regarding the importance of various ingredients of the PAG

Poly acrylamide gel (PAG) had been known as a potential embedding medium for sectioning tissues as early as 1954. Two independent groups Davis and Raymond employed PAG in electrophoresis in 1959. It possesses several electrophoretically desirable features that made it a versatile medium. Poly acrylamide gel separates protein molecules according to both size and charge. It is a synthetic gel, thermo-stable, transparent, strong, relatively chemically inert, can be prepared with a wide range of average pore sizes, can withstand high voltage gradients, feasible to various staining and destaining procedures and can be digested to extract separated fractions or dried for autoradiography and permanent recording. DISC electrophoresis utilizes gels of different pore sizes. The name DISC was derived from the discontinuities in the electrophoretic matrix and coincidentally from the discoid shape of the separated zones of ions (Anbalagan, 1999). There are two layers of gel namely staking gel or spacer gel and resolving gel or separating gel.
Staking gel or spacer gel: It is a large pore poly acrylamide gel (4%). This gel is prepared with Tris buffer pH 6.8 of about 2 pH units lower than that of electrophoresis buffer. These conditions provide an environment for Kohlrausch reactions, as a result, proteins are concentrated to several fold and a thin starting zone of the order of 19 microns is achieved in a few minutes. This gel is cast over the resolving gel. The height of the staking gel region was always maintained more than double the height and the volume of the sample to be applied.
Resolving gel or Separating Gel: This is a small pore polyacryl amide gel (3 - 30%). The Tris buffer used is of pH 8.8. In this gel, macro molecules separate according to their size. In the present experiment, 8%, 10% and 12% Resolving gel were used for separating different range of proteins. 8% gel for 24 – 205 kD proteins, 10% gel for 14-205 kD proteins and 12% gel for 14-66 kD proteins
The chemical ingredients of the gel are the following
Tris (tris (hydroxy methyl) aminomethane) (C4H11NO3; mw: 121.14). It has been used as a buffer because it is an innocuous substance to most proteins. Its pKa is 8.3 at 20°C and reasonably a very satisfactory buffer in the pH range 7.0 – 9.0.
Glycine (Amino Acetic Acid) (C2H5NO2; mw: 75.07). Glycine has been used as the source of trailing ion or slow ion because its pKa is 9.69 and mobility of glycinate are such that the effective mobility can be set at a value below that of the slowest known proteins of net negative charge in the pH range. The minimum pH of this range is somewhere around 8.0.
Acrylamide (C3H5NO; mw: 71.08). It is a white crystalline powder. While dissolving in water, autopolymerisation of acrylamide takes place. It is a slow spontaneous process by which acrylamide molecules join together by head on tail fashion. But in presence of free radicals generating system, acrylamide monomers are activated into a free-radical state. These activated monomers polymerise quickly and form long chain polymers. This kind of reaction is known as Vinyl addition polymerisation. A solution of these polymer chains becomes viscous but does not form a gel, because the chains simply slide over one another. Gel formation requires hooking various chains together. Acrylamide is a neuro toxin. It is also essential to store acrylamide in a cool dark and dry place to reduce autopolymerisation and hydrolysis.
Bisacrylamide (N,N’-Methylenebisacrylamide) (C7H10N2O2; mw: 154.17). Bisacrylamide is the most frequently used cross linking agent for poly acryl- amide gels. Chemically it is thought of having two-acrylamide molecules coupled head to head at their non-reactive ends. Bisacrylamide was preserved at 4°C.
Sodium Dodecyl Sulphate (SDS) (C12H25NaO4S; mw: 288.38). SDS is the most common dissociating agent used to denature native proteins to individual polypeptides. When a protein mixture is heated to 100°C in presence of SDS, the detergent wraps around the polypeptide backbone. It binds to polypeptides in a constant weight ratio of 1.4 g/g of polypeptide. In this process, the intrinsic charges of polypeptides becomes negligible when compared to the negative charges contributed by SDS. Thus polypeptides after treatment becomes a rod like structure possessing a uniform charge density, that is same net negative charge per unit length. Mobilities of these proteins will be a linear function of the logarithms of their molecular weights.
Ammonium per sulphate (APS) (N2H8S2O8; mw: 228.2). APS is an initiator for gel formation. APS was stored at 4°C.
TEMED (N, N, N’, N’-tetramethylethylenediamine) (C6H16N2; mw: 116.21). Chemical polymerisation of acrylamide gel is used for SDS-PAGE. It can be initiated by ammonium per sulphate and the quarternary amine, N, N, N’, N’-tetramethylethylenediamine (TEMED). The rate of polymerisation and the properties of the resulting gel depends on the concentration of APS and TEMED. Increasing the amount of APS and TEMED results in a decrease in the average polymer chain length, an increase in gel turbidity and a decrease in gel elasticity. Decreasing the amount of initiators shows the reverse effect. It is recommended that lowest catalysts concentrations that will allow polymerisation in the optimal period of time should be used. APS and TEMED are used, approximately in equimoloar concentrations in the range of 1 to 10 mM. TEMED was stored at 4°C.
The following chemicals are used for processing of the gel and the protein samples visualized in it.
Bromo Phenol Blue (BPB) (3’, 3’’, 5’, 5’’-Tetrabromophenolsulph- onephthalein) (C19H10Br4O5S; mw: 669.99). BPB is the universal marker dye. Proteins and nucleic acids are mostly colourless. When they are subjected to electrophoresis, it is important to stop the run before they run off the gel. BPB is the most commonly employed tracking dye, because it is viable in alkali and neutral pH, it is a small molecule, it is ionisable and it is negatively charged above pH 4.6 and hence moves towards anode. Being a small molecule it moves ahead of most proteins and nucleic acids. As it reaches the anodic end of the electrophoresis medium electrophoresis is stopped. It can bind with proteins weakly and give blue colour.
Glycerol (C3H8O3; mw: 92.09). It is a preservative and a weighing agent. Additon of glycerol (20-30 or 50%) is often recommended for the storage of enzymes. Glycerol maintains the protein solution at very low temperature, without freezing. It also helps to weigh down the sample into the wells without being spread while loading.
Coomassie Brilliant Blue (CBB) (C45H44N3NaO7S2; mw: 825.97). CBB is the most popular protein stain. It is an anionic dye, which binds with proteins non-specifically. The structure of CBB is predominantly non-polar. So is usually used (0.025%) in methanolic solution (40%) and Acetic Acid (7%). Proteins in the gel are fixed by acetic acid and simultaneously stained. The excess dye incorporated in the gel can be removed by destaining with the same solution containing no dye. The proteins are detected as blue bands on a clear background. As SDS is also anionic in nature, it is reported to interfere with staining process. Therefore, large volume of staining solution is recommended. Approximately 10 times the volume of the gel.
Butanol (C4H10O; mw: 74.12). Water saturated butanol is used as an overlay solution on the resolving gel.
Beta Mercapto Ethanol (HS-CH2CH2OH; mw: 78.13). BME was procured from LKB, Bromma, Sweden and was stored at 4°C.
References

1. Anbalagan K (1999). An Introduction to Electrophoresis. Ed. Anbalagan K. pub. The electrophoresis institute, Biotech-Yercaud, Salem, India.
2. Sambrook J and Russel DW (2001). Molecular Cloning: A Laboratory Manual. Cold Spring Harbour, New York.

SILK dumping- the theory and practice. Is the contingent protection substantiated?

gkrajeshrajesh@gmail.com — Mon, 09 Jul 2007 09:44:00 +0000

The recent imposition of anti dumping duties on Chinese silk imports to India has stirred up the global silk scenario. The issue whether there is dumping of specific grade raw silks by China is debated with in and out side the countries concerned. This article takes a glimpse at the economic theory behind dumping and anti dumping strategies, as the bulk of the current debates is found to be deficient in the theory behind the phenomenon. The discussion is published in two parts.

Part-1

Dumping is usually understood to mean that a product is exported at a price lower than the price at which the identical or a similar product is sold by the same producers on the exporting country’s domestic market. Dumping can be either a result of monopoly or an instrument to create or strengthen monopoly.
The conditions mandatory for dumping to take place are
1. Presence of an imperfect market where price discrimination between markets is possible. (Because in imperfect markets firms are price setters not price takers) 2. Segmented markets where there is no arbitrage easily possible between markets.
Only if these two conditions are satisfied is it possible for the exporting firm to engage in dumping. For any firm, price discrimination in favor of exports is more common because the share of exports is usually lesser than the domestic demand.
The classic view at dumping identifies the following types
· Private long term dumping and price discrimination
· Subsidized and public dumping
· Dumping in the interest of the exporting country
· Short term dumping
Private long term dumping and price discrimination
Private long-term dumping and price discrimination can result from the profit maximizing policies of a discriminating monopolist. The behavior of the monopolist will be based on the elasticity of demand existing in the domestic and foreign markets. Suppose the domestic demand is less elastic compared to the foreign demand he will restrict the supply of goods in the domestic market (to raise the prices) and dump the product in the foreign market to take advantage of the price elasticity of demand existing there. This concept can be graphically represented.

figure:1

In the figure 1; Dh D’h and Df D’f represent the domestic and foreign demand curves respectively. Mh M’h and Mf M’f represent marginal domestic and marginal foreign revenue curves. The horizontal summing of the two marginal revenue curves gives the aggregate marginal revenue curve Ma M’a. The domestic producers marginal cost curve is C C’. The discriminating monopolist will produce at the point where his aggregate marginal revenue is equal to his marginal cost. Total output will be OR, sales at home- O Wh, and sales abroad- O Wf. The producer has equated his marginal revenue in both markets to his marginal cost, hence satisfying the condition for his private optimum. The price at home is O Ph and price of export is O Pf. In the diagram it is assumed that at the relevant points the elasticity of demand at home is lower than that abroad, so that the domestic price is higher, and thus there is dumping. The essence of private dumping is price discrimination.
Subsidized and public dumping
This is a case of competitive market, not monopoly. The case of an export subsidy is represented in figure 2. The domestic supply curve, which is equal to marginal costs is represented by Sh S’h. Domestic demand curve is Dh D’h. Here price is equal to marginal cost of production. The foreign demand is assumed to be perfectly elastic as represented by the foreign demand curve Df D’f. An export subsidy of Df S is installed which raises the domestic price to OS. Consumption falls and production rises. For the higher domestic price to be possible there have to be either transport costs or a domestic tariff. Domestic consumers pay the price OS and foreign consumers pay a lower O Df. Thus there is dumping.

figure:2
The case of public dumping is more or less the same. Here we are concerned with export marketing arrangements in which there are private competitive producers and a marketing board intervenes to protect exports, rather than maximizing the profit of producers. The board will charge a higher price to the domestic consumer and the profit margin will be used to provide export subsidy.
Dumping in the interest of the exporting country
As distinct from the private interest of the monopolist, dumping can also be in the national interest of the exporting country. National policy in the form of tariffs can make private long term dumping possible. If the foreign demand curve is perfectly elastic then a price equal to the marginal cost can be put for both the domestic and foreign markets. If the foreign demand curve is downward sloping the national optimum demands that in the export market marginal cost to be made equal to marginal revenue and in the domestic market marginal cost to be made equal to average revenue. Thus export price exceeds the domestic price (reverse dumping). In this situation by imposing an optimum export tax the country forces the exporters to bring down the export prices which in effect can lead to dumping.
Short term dumping
Short term dumping is of two types namely sporadic and predatory. Sporadic dumping is the fall in foreign supply prices due to variations in production abroad due to various technical reasons etc. Though a fall in prices is always beneficial to the consumers, it increases the risk of domestic producers.
Predatory dumping is not just a manifestation of monopoly but a technique to maintain it. A foreign dominant supplier may temporarily reduce his supply price so as to force out a domestic producer trying to enter or extent his share of the market, the foreign supplier being able to sustain a price war longer than the domestic supplier. Predatory dumping is effective only if the foreign exporter is able to sustain a monopoly. It is not sufficient for him that he is able to force out a domestic supplier. If he has to compete with other foreign suppliers he may not subsequently be able to raise his price.
Anti Dumping - Meaning and concept
Anti dumping is a measure to rectify the situation arising out of the dumping of goods and its trade distortive effect. Thus the purpose of anti dumping duty is to re establish fair trade. Many countries have anti dumping regulation of some kind. One outcome of the Kennedy round of GATT negotiations was an agreement establishing an international anti dumping code which now governs the anti dumping policies of signatory countries. Dumping refers to private dumping and is considered to occur “if the export price of the product exported from one country to other is less than the comparable price for the like product when destined for consumption on the exporting country. Action could be taken if there is material injury to the domestic producers and sufficient evidence of the injury. Countervailing duties can be imposed to offset subsidies.
The important concern is with short term dumping, which creates uncertainty and may be an instrument of monopoly. The concern is often with producer rather than the consumer interests. If the import price falls there will be domestic income re distribution effect, which should be prevented. Another concern is that of fairness. Dumping seems unfair when it is believed that the export price is below the foreign suppliers average cost of production.
Anti dumping duty and customs duty.
Customs duties fall in the realm of trade and fiscal policies of the Government while anti dumping and anti subsidy measures are there as trade remedial measures. The object of anti dumping and allied duties is to offset the injurious effects of international price discrimination while customs duties have implication for the government revenue and for overall development of the economy. Anti dumping duties are not necessarily in the nature of a tax measure in as much as the authority is empowered to suspend these duties in case of an exporter offering a price undertaking. Thus such measures are not always in the form of duties/ tax. Anti dumping and anti subsidy duties are levied against exporter/ country in as much as they are country specific and exporter specific as against the customs duties which are general and universally applicable to all imports irrespective of the country of origin and the exporter.
Anti dumping (AD) and countervailing (CV) procedures- implications for developing countries
Contrary to their design as temporary means to offset unfair competition, these trade defense measures are in practice used as a long term strategy for various economic difficulties. Used (abused) as a substitute for positive adjustment measures ad and CV actions are also utilized to deal with structural problems. Applied as an instrument for tackling the negative consequences of trade liberalization they became a common tool to protect domestic producers from foreign competition. Faced with the need to protect sensitive domestic industries from increased imports or price slumps, countries often decide to use AD/ CV measures instead of (the more costly) safeguard measures provided for in the GATT 1994.
The WTO era saw a notable rise in AD and CV proceedings. Anti dumping investigations more than doubled and countervailing investigations increased six fold. Countries that appreciated their exchange rate regimes also seem to have used anti dumping to limit current account deficits caused by external shocks.
The almost exclusive restriction of AD initiations to the Big four (Australia, Canada, European Union and United States) was replaced by a broadened field of applicants. Argentina, Brazil, India, Mexico and South Africa became active users, responsible for a significant number of new investigations. Together they account for about one quarter of the AD investigations initiated since 1995. The United States and the European Union initiated two thirds of all CV investigations. Altogether developed countries were behind more than 80% of the overall number for CV investigations initiated in the WTO period.
Anti dumping and countervailing actions has a variety of negative implications.
· They can create substantial distortions with damaging effects on trade and competition.
· The imposition (or even the mere threat) of a duty may lead exporting firms to change production and seek alternative sources of supply.
· The exclusive focus on certain domestic producer neglects costs imposed on consumers due to price increases.
· The existence of these trade defense measures encourages rent seeking behavior by import competing firms.

To be continued.........

Exclusive interview with Dr. Panomir Tzenov, Executive Director of Bulgarian National Center of Agricultural Sciences (NCAS)

gkrajeshrajesh@gmail.com — Wed, 04 Jul 2007 04:35:00 +0000

Dr. PANOMIR IVANOV TZENOV was born in Vratza, Bulgaria in 1961. He took his MSc from The University of Zootechnics and Veterinary Medicine – Stara Zagora in Animal breeding engineering – sericulture. He obtained his PhD. in sericulture in 1996.
Dr. Tzenov started his career as a technologist in silkworm egg production at the “Silkworm breeding and egg production enterprise”, Vratza, under the Sericulture Experiment Station (SES) Vratza. He became the head of the station in 1987. From 1994 to 2003 he served as The Director of SES, Vratza and as National Director of the project “Rehabilitation of sericulture”, financed by FAO during 2000 – 2002. Presently he holds the position of Executive director of Bulgarian National Center of Agricultural Sciences (NCAS), Sofia.
He is member of various reputed national and international forums such as International Working Group on Sericulture Germplasm” under FAO, International Working group on the global silk handcraft cottage industries and silk enterprises development” under FAO, The Publishing council at the National Center of Agricultural Sciences, Bulgaria etc. He is the founder president of Black, Caspian Seas and Central Asia Silk Association (BACSA).
He has contributed immensely to general sericulture, silkworm breeding, egg production, rearing technology and cocoon and silk processing. He has 164 published papers to his credit and holds author’s certificates for two commercial silkworm hybrids.
Dr. Tzenov is widely traveled and has participated in more than 30 scientific conferences across Asia and Europe.

Inspite of his busy schedule he has found time to give an exclusive interview to 'The Silkworm' for which The Silkworm is thankful.

Tell me about BACSA, its objectives, membership, mode of operation etc.
The Black, Caspian Seas and Central Asia Silk Association (BACSA) was established after the “International Workshop on Revival and Promotion of Sericultural Industries and Small Enterprise Development in the Black, Caspian Seas and Central Asia Region”, organized by FAO in cooperation with the Government of Uzbekistan and held at Tashkent, Uzbekistan from 11 to 15 April 2005 in order to promote sericulture production in the region countries. The association’s main tasks are to: Generate sericulture projects from external resources, including bilateral and multilateral cooperation; Sensitize respective governments and prospective donors; Promote local and regional joint efforts which allow the cooperation between the countries of the Black, Caspian seas region and Central Asia to develop concrete actions that fortify the sustainable development of the sericulture in the region; Promote making agreements for international scientific-technical cooperation and business relations between the countries involved and Promote market studies, training, and dispersion of sericultural germplasm, and silkworm eggs.

For the operation of the association there are, chosen democratically by the members a President, two Vice-presidents, national coordinators for each member country, members of an Executive Committee. The members of the Executive Committee are directly the people in charge of coordination of all the raised activities for their country, within the regional context. The Executive Committee it is the bridge between the country, the national coordinator and the other countries of the association, to execute the actions defined in the region.

The Committee gathers at least once a year and has the following functions: to evaluate the work made by each national coordinator in activities of coordination in her/his country with respect to the BACASA, to recommend the names of the people in the association to receive training abroad. to evaluate and to watch the handling of the "Rotary Funds" and "Research Funds" that will be probably created and to give the recommendations on orientation and better use of these resources, to present/display the research proposals that require financing on the part of the "Research Fund", to approve the necessary resources for this aim and to give recommendations and suggestions on all publications and written material that takes place within the frame of the BACASA and to advise to the association’s President on the advances and progresses that must take place in the development of the activities and give recommendations her/him on the modifications and corrections that are due to make for the final succes of the projects.
Is sericulture a dying enterprise? Does it have a future?
The status of sericulture depends on many factors. In some countries it is really a dying enterprise, due mainly to their high economical development, combined with lack of any governmental support. On the other hand the selling of raw silk/silk allied products by the Chinese at too low prices during the period 1995 – 2005 led to a collapse of the sericulture industry in many countries. The Chinese sold 1 kg of raw silk for US$ 16-18/kg, 3 A quality. That means the fresh cocoon purchasing price should be about US$ 0. 50 – 0.80. Very few countries have so poor farmers who are ready to produce cocoons at this price. On the other hand in many countries the sericulture farmers are supported by the government. In the last year the raw silk price in China jumped to about US$ 43/kg. Therefore the increasing price of the raw silk at the international market, combined with stable state policy to support the sericulture development are the two key factors for the bright future of sericulture. The availability of local silk market is also very important.
Silkworm rearing within the European union countries is considered as one of the protected and promoted activities, being subsidized by Euro 133 per box of 20.000 eggs. This subsidy creates a considerably high income to the farmer, since it approximately duplicates his total income, added to the cocoon value. The replacement of traditional crops by perennial mulberry cultivation is additionally subsidized by the EU by means of the initial installation cost and the income loss for twenty years.
Considering the above I can make the final conclusion that the sericultural industry has a good future.
What is the relevance of sericulture as an agro industry in the developing and underdeveloped countries?
Apart from giving a high-value product, sericulture is a highly women/old people- focused, labour intensive, rural based, income leveler. From one hectare of irrigated mulberry, from the stage of mulberry cultivation to the stage of weaving and marketing, about twelve persons get employment throughout the year. Since the producers of silk cocoons are mostly small farmers while the costly silk final products can be purchased only by the affluent, it transfers income from the rich to the poor, from the urban to rural families. Internationally also, the silk producing countries are mainly the developing countries while silk products have a high demand in the EU countries, Japan and the USA.
Sericulture is also a drought-proffer because, the mulberry plant whose roots go deep in the soil, does not die during prolonged drought and its leaves will sprout whenever there is rain. In the tropical countries, even with 40% rainfall, there will be two crops of mulberry instead of the normal four or five which means, even in the worst drought year, there will be 40% of normal income for the sericulture families. In some temperate /subtropical countries where the summer is very dry and autumn rearing is impossible without irrigation, is possible to get at least one spring cocoon crop without any irrigation of mulberry.
Sericulture thus scores on every point: One of the highest income from one unit of land; Use of less water and drought resistance; Income leveler - transfer of income from rich to the poor nationally and internationally; Generating high rural employment; Gender benign in favor of women-employment; Silkworm rearing is indoors and has no arduous work-drudgery; A natural fibre and ecologically harmless; Hygroscopic, absorbs body moisture and therefore comfortable to wear; No synthetic substitute and highly demanded in EU countries and the USA; No competitor to food crops.
However, in new areas initial promotion and investment is needed in the establishment stage. This is because, while mulberry can be grown without much difficulty, the art and science of silkworm rearing, marketing arrangements for cocoons, reeling and weaving have to be organized in the initial stages till the industry takes root.
The reeled silk yarn is not perishable and being a low-volume, high-value product it has a ready market and can be transported easily. In fact across countries it is air-freighted and not sent by surface transport as transport cost is a small fraction of the total cost. The more difficult task is the teaching of rearing and organizing of reeling to create a market for cocoons produced by the farmer. Once these two aspects are taken care of, sericulture will entrench itself and will develop. Silk being a natural fiber and hygroscopic, it is comfortable to wear in all seasons as it absorbs body moisture. Sericulture thus fits neatly into the economy of any developing country with a significant rural sector, for quite a few decades to come.
Silkworm has long been identified as a laboratory tool (courtesy Dr. Tazima) but yet to be exploited as one. Why is it so?
Recently the silkworm was used as a laboratory tool for the medicine purposes.
The silkworm genome project is underway. What are its implications to the scientific field and agricultural (sericulture) field?
Scientific field: getting new scientific information about the animal genome.
Agricultural field: creation of new silkworm breeds and hybrids combining precious characters, for example sturdiness with high productivity.
While considering the long history of domestication, the bulk of breeding experiments done, viability to genetic manipulation, genomic information, versatility of sericigenous fauna etc., do you think paucity of competent researchers has been the worst handicap for the sector? What are your suggestions for a remedy?
I do not think that paucity of competent researchers has been the worst handicap for the sector. There are many other reasons, the main ones – insufficient financing, considering the sericulture as a minor sector etc.

Dr. Panomir Tzenov can be contacted at panomir@yahoo.com. For more information on BACSA visit http://www.bacsa-silk.org/index.php?lang=bu

Good news for silk

gkrajeshrajesh@gmail.com — Wed, 27 Jun 2007 15:33:00 +0000

The provisional production figures released by the Central Silk Board (CSB) revealed that domestic raw silk production is showing an upswing with a record of 18760 tons of raw silk during fiscal 2007. The upward move represents 8.41 percent surge over the previous year figures of 17305 tons; however, the record production was much more than the peak production of 17351 tons achieved in 2001-02. During 2002-03 and 2003-04, the drought conditions in the southern region had deterring effect in silk production as farmers went to the extent of even uprooting the mulberry plantations; whereas the ill effects of the severe drought appear to thin out as mulberry silk production had gathered momentum for the fourth successive year.
Last year, the mulberry silk production, including that of bi-voltine silk was 16805 tons up from 15445 tons from the previous year. The redeeming factor of last year's production however was the signs of superior bi-voltine silk galloping in growth by 12.77 percent to cross the 1000 ton mark.
However, the increase in production only proves that bi-voltine silk had come to stay and was finding increased acceptance at farmer's level every year. For more related news visit http://www.bharattextile.com/newsitems

Uzi egg on larval body!

gkrajeshrajesh@gmail.com — Sun, 17 Jun 2007 16:54:00 +0000

Heat shock proteins – a forgotten link in Silkworm breeding for robustness

gkrajeshrajesh@gmail.com — Fri, 15 Jun 2007 12:30:00 +0000

Silkworm is one of the most thermal-sensitive organisms. Intensive and careful domestication over centuries has apparently deprived the insect of opportunities to acquire thermo tolerance. Among many factors attributed to poor performance of the bivoltine strains under tropical conditions the major aspect is that many quantitative characters decline sharply when temperature is higher than 28°C. The risk of hybridization of polyvoltine to bivoltine could not be taken due to the delay in fixation of economic characters. The long and hard struggle to evolve robust-productive silkworm hybrids has not so far met with satisfactory results.

The front ranking breeders in the field agrees to the fact that it is a difficult task to breed such bivoltine breeds, which are suitable to high temperature environment and yet productive. Therefore means other than the conventional breeding methods are to be adopted to attain the goal. With the aid of modern biotechnological tools it may be possible to quantify the factors responsible for the expression of temperature tolerance. Resistance to high temperature has been recognized as a heritable character in silkworm and the possibility for temperature tolerant silkworm races were suggested by Kato as early as 1989. Thorough understanding of the phenomenon of temperature tolerance in silkworm is an essential pre requisite for attaining any results in this direction.

Heat Shock Proteins

It is known that rapid heat hardening can be elicited by a brief exposure of cells to sub-lethal high temperature, which in turn provides protection from subsequent and more severe temperature. In 1962, Ritossa reported that heat and the metabolic inhibitor dinitrophenol induced a characteristic pattern of puffing in the chromosomes of Drosophila. This discovery eventually led to the identification of the heat-shock proteins (Hsp) or stress proteins whose expression these puffs represented. Beginning in the mid-1980's, investigators recognized that many Hsps function as molecular chaperones and thus play a critical role in protein folding, intracellular trafficking of proteins, and coping with proteins denatured by heat and other stresses. Accordingly, the study of stress proteins has undergone explosive growth.

Heat-shock proteins are classified into families on the basis of sequence homology and typical molecular weight as Hsp 110, Hsp 100, Hsp 90, Hsp 70, Hsp 40, Hsp 10 and small heat- shock protein families. In eukaryotes many families comprise multiple members that differ in inducibility, intra cellular localisation and function.

Extensive studies have been conducted on the heat- shock response in insects such as Drosophila, Chironomous, Lymantria dispar, the tobacco hornworm-Manduca sexta, the desert ant-Cataglyphis, the fleshfly-Sarcophaga crassipalpis, the locust Locusta migratoria etc. There are reports on the activity of hest shock proteins in silkworm. Evegnev et. al. (1987) studied heat shock response in Bombyx mori cells. Temperature elevation induced active transcription of heat shock mRNAs in infected cells. But at the level of translation headstock treatment failed to induce hsp synthesis and was not able to inhibit production of polyhedrin in such cells.

Joy and Gopinathan in 1995 reported the appearance of 93, 70, 46 and 28 kDa protein bands consequent to high temperature exposure in Bombyx mori. in both bivoltine and multivoltine strains, but with variying kinetics. Lee et.al., in 2003 cloned a genomic DNA fragment containing a promoter region for the gene encoding an HSC70-4 homologue, the structure of which was deduced from the partial cDNA sequences that were registered in a Bombyx mori EST date base. The deduced amino acid sequence with 649 residues was 89% and 96% identical to those from Drosphilla melanogaster hsc-4 and Manduca sexta HSC-70-4 respectively. The expression analysis by reverse transcription PCR demonstrated that mRNA transcription occurred in all tissues examined and was not stimulated by heat shock. Thus HSC70-4, the molecular chaperon is ubiquitously expressed in every tissue of Bombyx mori.

Considering the enormous investigations conducted on HSPs in a plethora of organisms ranging from bacteria to man, it is felt that there is an acute shortage of literature on the heat shock response of the silkworm Bombyx mori. There is dire necessity for 1. Understanding the molecular mechanism of temperature tolerance in silkworm. 2. Identification of the various families of HSPs synthesized and the threshold temperature, which induce their expression. 3. Understanding the differential expression pattern of various HSPs in bivoltine and polyvoltine races and 4. To locate the genes responsible for the heat inducible HSPs and subsequent steps to introgress the same into the bivoltine genome either by conventional breeding or by use of molecular techniques.

The silk factories.......

gkrajeshrajesh@gmail.com — Wed, 13 Jun 2007 11:44:00 +0000