Kenya Forensics Online Resource

Forensic Biology

2012-05-31T00:23:00.000-07:00

COLLECTION OF BIOLOGICAL EVIDENCE.

The strength of any forensic evidence presented in a court of law highly depends on how effective its collection was and the sensitivity with which the evidence was handled after that. This previously ignored part of court proceedings seems to be catching up as evidenced with the professionalism witnessed in the police department lately.

Care is being taken during collection of this evidence just as required. Just to remind ourselves, biological evidence tends to deteriorate quite fast and get contaminated hence measures should be taken to prevent that and ensure its integrity.

The success in finding biological material depends upon the search method employed and also the integrity and state of the scene. It is important to adhere to the following guidelines while collecting the evidence:

Wear the appropriate gear. These include full overalls, shoe covers, gloves and face masks.
Seclude the area by marking it with tape to minimize human traffic.
Start collecting evidence from outwards going inwards to capture as much evidence as possible.
A range of light sources can be operated at fixed wavelengths and be used to detect stains.
· Take as many photographs as possible at different angles( Thats a whole topic all together)

Methods used for collection will vary depending on the type of sample. For example,

Searching a crime scene or items recovered from a crime scene for blood can be aided by use of luminol (3-aminophthalhydrazide)The chemical can be sprayed onto a wide area and will become oxidized and luminescent in the presence of haemoglobin, which is found in red blood cells. It is necessary to darken the area being searched in order that the luminescent are detected.
Dry stains and contact marks on large immovable items are normally collected using a sterile swab that has been moistened with distilled water, in other cases scraping or cutting the material may be appropriate.
Lifting from the surface using high quality adhesive tape is an alternative method for collecting epithelial cells.
Liquid blood can be collected using a syringe. It can also be applied to FTA paper that is impregnated with chemicals to prevent action of microbial agents and stabilize the DNA.
Smaller movable objects such as weapons, which might contain biological material should be packaged at the scene and examined in there controlled environment of a forensic laboratory.

TO BE CONTINUED…

Techniques in Making Shoe Impressions

2012-05-30T09:20:00.000-07:00

Topic

Crime scene investigators make shoe impressions with plaster of paris and dental stone.

Introduction

The most common method of entering and leaving an area where a crime is committed is by walking. Criminalists often gain information by examining the surfaces on which a perpetrator walked. Footwear or bare feet can leave superficial prints on firm surfaces such as flooring.

However, walking across a soft surface like damp soil leaves impressions or indentations.

Footwear impressions can provide crucial clues in an investigation. Prints or impressions can show the points of entry and exit at the crime scene.

In addition, shoes carry materials from one environment to another. This material can provide information about where this person has been.

An impression can tell a lot about the shoe that made it. Every shoe is unique, with its own pattern of wear and tear. Therefore an impression of footwear can be compared to a particular pair of shoes to help connect those shoes to the crime scene. Impressions can also tell something about the height and weight of the wearer. Heavy individuals make deeper impressions than light ones. In addition, a tall person has a longer stride than a short person.

There are several techniques for making casts of shoe impressions. In this experiment, you will compare the effectiveness of two materials in making casts: plaster of Paris and dental stone.

Time Required

45 minutes on day 1

45 minutes on day 2

Materials

a) 2 pounds (lbs)(0.91 kilograms [kg]) of dental stone in a large

b) Ziploc™ bag

c) 3 lbs (1.36 kg) of plaster of paris in a large ziplock bag

d) 3 to 5 cups of water in a bottle

e) 5 to 10 twigs or craft (Popsicle™) sticks soaked in water

f) 3 or 4 long strips of cardboard (about 4 inches [in.] by 24 in.

g) [10.2 centimeters (cm) by 61 cm])

h) large spoon

i) talcum powder

j) magnifying glass

k) scrub brush

l) paper towels

m) access to water

n) science notebook

Procedure, Day 1

1. Follow your teacher to an outdoor area where there are some shoe impressions.

2. Closely examine two footprints. Carefully remove any rocks, twigs, or other debris from the footprints.

3. Sprinkle a little talcum powder over these two footprints to help hold the soil in place and secure the impressions.

4. Build a retaining wall around each impression using strips of cardboard (see Figure 1).

(This wall will prevent the plaster of paris or dental stone from escaping when it is poured into the impression.)

5. Make a cast of one footprint using dental stone. To do so:

a. Pour about 1 cup of water into the Ziploc™ bag of dental stone.

b. Knead the bag of dental stone for 2 to 3 minutes (min) to mix thoroughly.

c. Examine the consistency of the dental stone. Ideally, it should be similar to pancake batter. If it is too thick, add a little more water. (Be careful; if you add too much water, your mixture will be too thin.)

d. The dental stone becomes firm very quickly, so work rapidly to pour the mixture. Avoid pouring the material directly into the impression, which can distort it. Instead, hold the spoon just above the impression and slowly pour the mixture into the spoon so that it falls into the impression. When the impression is about half full, remove the spoon and pour the remaining mixture directly. The poured cast should be 2 to 3 in. (about 5 to 7.5 cm) thick.

e. Use a stick or pencil to etch your name and the date in the upper surface of dental stone.

f. Let the cast sit for 30 min to harden. During this time, carry out step 6 below.

g. After 30 min or more, insert the handle of the spoon into the ground about 1/2 in. (1.2 cm) from the impression and gently pry up the cast.

h. The cast may have soil or grass on it; do not remove it at this time.

i. Take the cast indoors and allow it to harden for 24 hours.

6. Make a cast of the other shoe impression using plaster of paris. To do so:

a. Pour about 1 cup of water into the Ziploc™ bag of plaster of paris.

b. Knead the bag of plaster of paris and water for 2 to 3 min to mix thoroughly.

c. Examine the consistency of the mixture. Ideally, it should be similar to pancake batter. If it is too thick, add a little more water. (Be careful; if you add too much water, your mixture will be too thin.)

d. Pour about 1/2 in. (1.2 cm) of plaster of paris into the impression, using the spoon as you did earlier to avoid damaging the impression. Add some reinforcing materials such as twigs or craft (Popsicle™) sticks that have been soaked in water (see Figure 2). (Dry sticks will absorb water from the plaster of paris and make it brittle.) Quickly finish pouring the plaster of paris into the impression to a thickness of at least 3 in.

e. Follow directions e through i in step 5.

Procedure, Day 2

1. Examine each shoe cast. In your science notebook, note any significant differences in the casts.

2. Using a scrub brush, gently clean each cast with water. Pat dry each cast with paper towels.

3. Examine each shoe cast again. In your notebook, note whether or not washing damaged either cast.

4. Closely examine each shoe cast with a magnifying glass. Search for nicks, cuts, or scrapes in the casts that indicate identifying markings on the shoes. Record these in your science notebook.

Analysis

1. Why might an investigator make casts of shoe impressions?

2. in your opinion, which cast was easier to pour, the dental stone or plaster of paris? Explain your answer.

3. Which material held up best to washing? Explain your answer.

4. Which material provided the most detail when viewed under the magnifying glass? Explain your answer.

5. Describe any nicks, scratches, wear patterns, or other unique marks found in either cast.

6. What information might a forensic investigator acquire from examining a shoe cast?

7. Complete a Venn diagram (see Figure 3) comparing the two casting materials.

What is special about DNA?

2012-05-30T09:16:00.006-07:00

DNA is a chemical that occurs inside every cell of a person’s body. The DNA is contained in 22 pairs of structures known as chromosomes, shaped like an X, plus an extra pair – the sex chromosomes – which determine whether someone is male or female. In this final pair, women have two X chromosomes, but men have one X and one Y chromosome. Each chromosome consists of two long strings of chemical letters, twisted together in the famous shape of the double-helix. The chemical letters occur in pairs as rungs on this twisted chemical ladder. The four chemical letters of the genetic code spell out instructions to the cell about how to make the proteins that allow the human body to grow and function normally. The parts of the DNA sequence that contain the instructions for making proteins are known as genes.

DNA is useful to identify an individual because everyone’s genetic code is thought to be unique, unless they have an identical twin. The string of chemical letters in a person’s DNA can therefore act like a unique bar code to identify them. Because a person inherits half their DNA from their mother and half from their father, it can also be used to identify their relatives. Close relatives have a DNA sequence that is more alike than distant relatives or than someone who is unrelated.

Biological identifiers such as DNA, fingerprints, iris scans and digital photographs are known as ‘biometrics’. In recent years there has been a lot of interest in developing biometrics to track and identify individuals as they enter or leave different countries or as they use public or private services, such as banks, computers, workplaces or hospitals.

Unlike iris scans and photographs, DNA and fingerprints can be left wherever a person goes: for example, on a glass or cup that they have been drinking from. This means that they can be used to track individuals – i.e. to find out whether they have been at a particular place, such as a crime scene or meeting place – where there might not be a scanner or a camera.

DNA differs from fingerprints in two main ways:

2012-05-30T09:16:00.003-07:00

• Because DNA has a biological function, some of the information in a person’s DNA may be relevant to their health or other physical characteristics, such as their eye colour.

• Because DNA is shared with relatives, a person’s DNA can be used to help identify their parents or children and perhaps more distant relatives.

However, DNA profiles used by the police are not based on the whole sequence of someone’s DNA, but only on parts of it. This means that the information contained in them is more limited than that contained in a person’s whole genetic make-up.

Resting Membrane Potential of Nerves

2012-05-30T09:15:00.001-07:00

The resting membrane potential of large nerve ﬁbers when not transmitting nerve signals is about –90 millivolts. That is, the potential inside the ﬁber is 90 millivolts more negative than the potential in the extracellular ﬂuid on the outside of the ﬁber. In the next few paragraphs, we explain all the factors that determine the level of this resting potential, but before doing so, we must describe the transport properties of the resting nerve membrane for sodium and potassium.

Active Transport of Sodium and Potassium Ions through the Membrane—the Sodium-Potassium (Na+ -K+) Pump. First, let us recall that all cell membranes of the body have a powerful Na+ -K+ that continually pumps sodium ions to the outside of the cell and potassium ions to the inside, as illustrated on the left-hand side in. Further, note that this is an electrogenic pump because more positive charges are pumped to the outside than to the inside (three Na+ ions to the outside for each two K+ ions to the inside), leaving a net deﬁcit of positive ions on the inside; this causes a negative potential inside the cell membrane The Na+ -K+ also causes large concentration gradients for sodium and potassium across the resting nerve membrane Leakage of Potassium and Sodium Through the Nerve Membrane. The right side of Figure below shows a channel protein in the nerve membrane through which potassium and sodium ions can leak, called a potassium-sodium (K+ -Na+) “leak” channel. The emphasis is on potassium leakage because, on average, the channels are far more permeable to potassium than to sodium, normally about 100 times as permeable. As discussed later, this differential in permeability is exceedingly important in determining the level of the normal resting membrane potential.

Origin of the Normal Resting Membrane Potential

The figure shows the important factors in the establishment of the normal resting membrane potential of–90 millivolts. They are as follows.

1. Contribution of the Potassium Diffusion Potential.

In the figure below1A, we make the assumption that the only movement of ions through the membrane is diffusion of potassium ions, as demonstrated by the open channels between the potassium symbols (K+) inside and outside the membrane. Because of the high ratio of potassium ions inside to outside, 35:1, the Nernst potential corresponding to this ratio is –94 millivolts because the logarithm of 35 is 1.54, and these times –61millivolts is –94 millivolts. Therefore, if potassium ions were the only factor causing the resting potential, the resting potential inside the ﬁber would be equal to –94 millivolts, as shown in the ﬁgure.

2. Contribution of Sodium Diffusion through the Nerve Membrane.

The figure shows the addition of slight permeability of the nerve membrane to sodium ions, caused by the minute diffusion of sodium ions through the K+ -Na leak channels. The ratio of sodium ions from inside to outside the membrane is 0.1, and this gives a calculated Nernst potential for the inside of the membrane of +61 millivolts. But also shown in Figure 1B is the Nernst potential for potassium diffusion of –94 millivolts. How do these interact with each other, and what will be the summated potential? This can be answered by using the Goldman equation described previously. Intuitively, one can see that if the membrane is highly permeable to potassium but only slightly permeable to sodium, it is logical that the diffusion of potassium con tributes far more to the membrane potential than does the diffusion of sodium. In the normal nerve ﬁber, the permeability of the membrane to potassium is about 100 times as great as its permeability to sodium. Using this value in the Goldman equation gives a potential inside the membrane of –86 millivolts, which is near the potassium potential shown in the figure.

3. Contribution of the Na -K Pump.

In Figure 1C, the Na+ -K+ pump is shown to provide an additional contribution to the resting potential. In this ﬁgure, there is continuous pumping of three sodium ions to the outside for each two potassium ions pumped to the inside of the membrane. The fact that more sodium ions are being pumped to the outside than potassium to the inside causes continual loss of positive charges from inside the membrane; this creates an additional degree of negativity (about –4 millivolts additional) on the inside beyond that which can be accounted for by diffusion alone. Therefore, as shown in Figure 1C, the net membrane potential with all these factors operative at the same time is about –90 millivolts.

In summary, the diffusion potentials alone caused by potassium and sodium diffusion would give a membrane potential of about –86 millivolts, almost all of this being determined by potassium diffusion. Then, an additional –4 millivolts is contributed to the membrane potential by the continuously acting electrogenic Na+ -K+ pump, giving a net membrane potential of –90 millivolts.

CELL DIFFERENTIATION

2012-05-30T09:12:00.001-07:00

A special characteristic of cell growth and cell division is cell differentiation, which refers to changes in physical and functional properties of cells as they proliferate in the embryo to form the different bodily structures and organs. The description of an especially interesting experiment that helps explain these processes follows.

When the nucleus from an intestinal mucosal cell of a frog is surgically implanted into a frog ovum from which the original ovum nucleus was removed, the result is often the formation of a normal frog. This demonstrates that even the intestinal mucosal cell, which is a well-differentiated cell, carries all the necessary genetic information for development of all structures required in the frog’s body.

Therefore, it has become clear that differentiation results not from loss of genes but from selective repression of different genetic operons. In fact, electron micrographs suggest that some segments of DNA helixes wound around histone cores become so condensed that they no longer uncoil to form RNA molecules. One explanation for this is as follows: It has been supposed that the cellular genome begins at a certain stage of cell differentiation to produce a regulatory protein that forever after represses a select group of genes. Therefore, the repressed genes never function again. Regardless of the mechanism, mature human cells produce a maximum of about 8000 to 10,000 proteins rather than the potential 30,000 or more if all genes were active.

Embryological experiments show that certain cells in an embryo control differentiation of adjacent cells. For instance, the primordial chorda-mesoderm is called the primary organizer of the embryo because it forms a focus around which the rest of the embryo develops.

It differentiates into a mesodermal axis that contains segmentally arranged somite’s and, as a result of inductions in the surrounding tissues, causes formation of essentially all the organs of the body.

Another instance of induction occurs when the developing eye vesicles come in contact with the ectoderm of the head and cause the ectoderm to thicken into a lens plate those folds inward to form the lens of the eye. Therefore, a large share of the embryo develops as a result of such inductions, one part of the body affecting another part, and this part affecting still other parts.

Thus, although our understanding of cell differentiation is still hazy, we know many control mechanisms by which differentiation could occur.

APOPTOSIS—PROGRAMMED CELL DEATH

2012-05-30T09:07:00.001-07:00

The 100 trillion cells of the body are members of a highly organized community in which the total number of cells is regulated not only by controlling the rate of cell division but also by controlling the rate of cell death. When cells are no longer needed or become a threat to the organism, they undergo a suicidal programmed cell death, or apoptosis. This process involves a speciﬁc proteolytic cascade that causes the cell to shrink and condense, to disassemble its cytoskeleton, and to alter its cell surface so that a neighboring phagocytic cell, such as a macrophage, can attach to the cell membrane and digest the cell.

In contrast to programmed death, cells that die as a result of an acute injury usually swell and burst due to loss of cell membrane integrity, a process called cell necrosis. Necrotic cells may spill their contents, causing inﬂammation and injury to neighboring cells. Apoptosis, however, is an orderly cell death that results in disassembly and phagocytosis of the cell before any leakage of its contents occurs, and neighboring cells usually remain healthy.

Apoptosis is initiated by activation of a family of proteases called caspases. These are enzymes that are synthesized and stored in the cell as inactive procaspases. The mechanisms of activation of caspases are complex, but once activated; the enzymes cleave and activate other procaspases, triggering a cascade that rapidly breaks down proteins within the cell. The cell thus dismantles itself, and its remains are rapidly digested by neighboring phagocytic cells.

A tremendous amount of apoptosis occurs in tissues that are being remodeled during development. Even in adult humans, billions of cells die each hour in tissues such as the intestine and bone marrow and are replaced by new cells. Programmed cell death, however, is precisely balanced with the formation of new cells in healthy adults. Otherwise, the body’s tissues would shrink or grow excessively. Recent studies suggest that abnormalities of apoptosis may play a key role in neurodegenerative diseases such as Alzheimer’s disease, as well as in cancer and auto-immune disorders. Some drugs that have been used successfully for chemotherapy appear to induce apoptosis in cancer cells.

ROLE OF DNA IN SOLVING CRIMES (An Excerpt from Gene Watch UK Briefing January 2011)

2012-05-30T09:01:00.002-07:00

People can leave traces of their DNA at a crime scene because it is inside every cell of their body. DNA can be extracted from blood, semen, saliva or hair roots left at a crime scene using a chemical process. Tiny amounts of DNA can sometimes be extracted from a single cell – such as cells shed from someone’s skin when they touch an object using new sensitive techniques (known as ‘low copy number’ DNA).

Police can also collect biological samples from suspects, usually by scraping some cells from inside their cheek.

When biological samples are collected by the police from a crime scene or an individual, they are sent to a laboratory for analysis. The laboratory extracts the DNA, amplifies it using a chemical reaction, and creates a string of numbers based on part of the sequence of chemical letters: this is known as a DNA profile. The DNA profile is not based on the whole sequence of the DNA (which would currently be very expensive) but on parts of it known as ‘short tandem repeats’ (STRs), where the chemical letters of the

DNA are known to be repeated a different number of times in different people. The final DNA profile consists of a string of numbers based on the number of repeats at each of the STRs, plus the results of a test of the sex of the person from whom the sample came.

DNA profiles are not unique but the probability that two people’s DNA profiles match by chance is low. If the DNA profile from an individual matches the DNA profile from a crime scene it is therefore highly likely (but not certain) that the blood, semen or saliva left at the crime scene came from them.

If the police have a number of suspects for a crime a DNA match can help them to identify who was at the crime scene and who wasn’t. The value of this evidence in solving the crime will vary: DNA on a cigarette butt could have been dropped earlier in the day or have been planted by someone who wanted to implicate an innocent person in the crime; in contrast, DNA in semen from a woman who has been raped can show that a particular man was or was not likely to have been involved. However, even a rape case may not be straightforward: for example, if the man argues that the woman agreed to have sex.

When DNA samples are collected at a murder scene, many DNA matches will occur with DNA from the victim or with others who may have been there earlier in the day, not with the perpetrator of the crime. However, these matches can still help to provide important clues that will help to solve the crime. For example, a DNA match between the victim’s blood and a blood stain on someone’s shoes or clothes might be part of the evidence that leads to a criminal’s conviction. In this example, a DNA database is not required because the victim’s DNA can be obtained easily from him or her.

IMAGES of ION TRANSPORT

2012-05-29T22:48:00.000-07:00

ISOLATION AND PURIFICATION OF PROTEINS

2012-05-29T22:43:00.002-07:00

Protein purification is a series of processes intended to isolate a single type of a protein from a complex mixture. Protein purification is vital for the characterization of the function, structure and interactions of the protein of interest. The starting material can be a biological tissue or a microbial culture.

Purification process involves;

· freeing the protein from a matrix that confines it

· separating the protein and non-protein parts of the mixture

· separating the desired protein from all other proteins.

Separation of one protein from all others may exploit differences in protein size, physico-chemical properties, binding affinity and biological activity.

Purification may be preparative or analytical.

Preparative purifications aim to produce a relatively large quantity of purified proteins for subsequent use. Examples include the preparation of commercial products e.g enzymes (e.g. lactase), nutritional proteins (e.g. soy protein isolate), and certain biopharmaceuticals (e.g. insulin).

Analytical purification produces a relatively small amount of a protein for research or analytical purposes, including identification, quantification, and studies of the protein's structure, post-translational modifications and function. Among the first purified proteins were urease and Concanavalin A.

Extraction

Depending on the source, the protein has to be brought into solution by breaking the tissue or cells containing it.

This can be done through cell disruption to release cellular contents by:

· Repeated freezing and thawing

· sonication - applying sound (ultrasound) energy to agitate particles in a sample

· homogenization by high pressure - intensive blending of mutually related substances or groups of mutually related substances to form a constant of different insoluble phases

· filtration - the mechanical or physical operation used to separate solids from fluids

· permeabilization by organic solvents e.g SDS (sodium dodecyl sulfate), Triton X-100 or CHAPS(3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate)

The method of choice depends on how fragile the protein is and how sturdy the cells are.

After extraction process soluble proteins will be in the solvent, and can be separated from cell membranes, DNA etc. by centrifugation.

The extraction process also extracts proteases, which will start digesting the proteins in the solution. If the protein is sensitive to proteolysis, it is usually desirable to proceed quickly, and keep the extract cooled, to slow down proteolysis.

Precipitation and differential solubilization

This is performed by adding increasing amounts of ammonium sulfate - (NH₄)₂SO₄ and collecting the different fractions of precipitate protein.

The hydrophobic groups on the proteins get exposed to the atmosphere and attract other protein hydrophobic groups and get aggregated.

Protein precipitated will be large enough to be visible.

Ammonium sulphate can be removed by dialysis.

Ultracentrifugation

Centrifugation is a process that uses centrifugal force to separate mixtures of particles of varying masses or densities suspended in a liquid, resulting in the pellet and supernatant.

Sucrose gradient centrifugation uses a linear concentration gradient of sugar (sucrose, glycerol, or a silica based density gradient media, like Percoll). A protein sample is layered on top of the gradient and spun at high speeds in an ultracentrifuge. This causes heavy macromolecules to migrate towards the bottom of the tube faster than lighter material. Samples separated by these gradients are referred to as "rate zonal" centrifugations.

After separating the protein/particles, the gradient is then fractionated and collected.

Chromatographic methods

Different proteins interact differently with the column material, and can thus be separated by the time required to pass the column, or the conditions required to elute the protein from the column.

The eluant is usually pooled in different test tubes. Test tubes containing solution with the protein of interest and any other similar proteins is retained.

Usually proteins are detected as they are coming off the column by their absorbance at 280 nm. Many different chromatographic methods exist:

Size exclusion chromatography

Uses porous gels. The principle is that smaller molecules have to traverse a larger volume in a porous matrix. Consequentially, proteins of a certain range in size will require a variable volume of eluent (solvent) before being collected at the other end of the column of gel.

The eluant is usually pooled in different test tubes. Test tubes containing solution with the protein to purify and any other similarly-sized proteins are retained.

Hydrophobic Interaction Chromatography

The Resins used in the column are amphiphiles with both hydrophobic and hydrophilic regions. The hydrophobic part of the resin attracts hydrophobic region on the proteins. The greater the hydrophobic region on the protein the stronger the attraction between the gel and that particular protein

Ion exchange chromatography

Separates compounds according to the nature and degree of their ionic charge. Anion exchange resins have a positive charge and are used to retain and separate negatively charged compounds, while cation exchange resins have a negative charge and are used to separate positively charged molecules.

Before the separation begins a buffer is pumped through the column to equilibrate the opposing charged ions. Upon injection of the sample, solute molecules will exchange with the buffer ions as each competes for the binding sites on the resin. The length of retention for each solute depends upon the strength of its charge. The most weakly charged compounds will elute first, followed by those with successively stronger charges. Because of the nature of the separating mechanism, pH, buffer type, buffer concentration, and temperature all play important roles in controlling the separation e.g. metal binding (nickel, cobalt)

Ion exchange chromatography is frequently used in both analytical and preparative separations.

Affinity chromatography

It’s based upon molecular conformation, which frequently utilizes application specific resins. These resins have ligands attached to their surfaces which are specific for the compounds to be separated. Most frequently, these ligands function in a fashion similar to that of antibody-antigen interactions. This "lock and key" fit between the ligand and its target compound makes it highly specific, frequently generating a single peak, while all else in the sample is unretained.

E.g. Immuno-affinity chromatography (Ab & Ag)

The protein can be eluted by changing the pH or the salinity

High performance liquid chromatography

Also high pressure liquid chromatography - applies high pressure to drive the solutes through the column faster. This means that the diffusion is limited and the resolution is improved. The most common form is "reversed phase" hplc, where the column material is hydrophobic.

The proteins are eluted by a gradient of increasing amounts of an organic solvent, such as acetonitrile. The proteins elute according to their hydrophobicity.

HPLC purification frequently results in denaturation of the purified proteins, thus not applicable to proteins that do not spontaneously refold.

Concentration of the purified protein

Lyophilization

If the solution doesn't contain any other soluble component than the protein in question the protein can be lyophilized (dried). This is commonly done after an HPLC run. This simply removes all volatile components leaving the proteins behind.

Ultrafiltration

Ultrafiltration concentrates a protein solution using selective permeable membranes. The function of the membrane is to let the water and small molecules pass through while retaining the protein.

Analytical

Denaturing-Condition Electrophoresis

A common laboratory technique that can be used both as preparative and analytical method.

The principle of electrophoresis relies on the movement of a charged ion in an electric field.

In practice, the proteins are denatured in a solution containing a detergent (SDS). In these conditions, the proteins are unfolded and coated with negatively charged detergent molecules. The proteins in SDS-PAGE are separated on the sole basis of their size.

In analytical methods, the proteins migrate as bands based on size. Each band can be detected using stains such as Coomassie blue dye or silver stain.

Preparative methods to purify large amounts of protein require the extraction of the protein from the electrophoretic gel. This extraction may involve excision of the gel containing a band, or eluting the band directly off the gel as it runs off the end of the gel.

It provides an improved resolution over size exclusion chromatography, but does not scale to large quantity of proteins.

Non-Denaturing-Condition Electrophoresis

An important non-denaturing electrophoretic procedure for isolating bioactive metalloproteins in complex protein mixtures is termed 'quantitative native continuous polyacrylamide gel electrophoresis (QPNC-PAGE).

QPNC-PAGE is a high-resolution technique applied in biochemistry and bioinorganic chemistry to separate proteins by isoelectric point (the isoelectric point (pI), is the pH at which a particular molecule or surface carries no net electrical charge).

This variant of gel electrophoresis is used to isolate active or native metalloproteins in biological samples.

The QPNC-PAGE procedure is accomplished in a special electrophoresis chamber for separating charged biomolecules. Due to the specific properties of the prepared gel and electrophoresis buffer solution (which is basic and contains Tris-HCl and NaN₃), most proteins of a biological system are charged negatively in the solution, and will migrate from the cathode to the anode due to the electric field.

MODIFICATION OF PROTEINS

Modification of proteins is done to allow manipulation and study of protein function and interactions in any environment.

Includes crosslinking, fragmenting, denaturing, reducing disulfides, or attaching various prosthetic groups (e.g. PEGylation)

Crosslinking Reagents

Use of chemical crosslinkers and bioconjugation reagents for covalent protein crosslinking techniques to conjugate antibodies, immobilize ligands, attach haptens to carrier proteins, and stabilize folded protein structures and protein interaction complexes.

Amine-to-Amine Crosslinkers

Homobifunctional amine-specific protein crosslinking reagents based on NHS-ester and imidoester reactive groups for selective conjugation of primary amines; available in short, long, cleavable, irreversible, membrane permeable and cell surface varieties.

Amine-to-Sulfhydryl Crosslinkers

Heterobifunctional protein crosslinking reagents for conjugation between primary amine (lysine) and sulfhydryl (cysteine) groups of proteins and other molecules; available with different lengths and types of spacer arms.

Carboxyl-to-Amine Crosslinkers

Carbodiimide crosslinking reagents for conjugation of carboxyl groups (glutamate, aspartate, C-termini) to primary amines (lysine, N-termini); also N-hydroxysuccinimide (NHS) for stable activation of carboxylates for amine-conjugation.

Photoreactive Crosslinkers

Aryl azide, diazirine and other photo-reactive (light-activated) chemical crosslinking reagents to conjugate proteins, nucleic acids and other molecular structures involved in receptor-ligand interaction complexes via two-step activation.

Sulfhydryl-to-Carbohydrate Crosslinkers

Crosslinking reagents based on maleimide and hydrazide reactive groups for conjugation and formation of covalent crosslinks between sulfhydryl (cysteine) and aldehyde (oxidized glycoprotein carbohydrate) groups.

Sulfhydryl-to-Hydroxyl Crosslinkers

Crosslinking reagents based on maleimide and isocyanate reactive groups for conjugation and formation of covalent crosslinks between sulfhydryl and hydroxyl groups.

Sulfhydryl-to-Sulfhydryl Crosslinkers

Sulfhydryl-specific crosslinking reagents based on maleimide or pyridyldithiol reactive groups for selective covalent conjugation of protein and peptide thiols (reduced cysteines) to form stable thioether bonds

Chemical modification Reagents

Use chemical agents to modify amino acid side chains on proteins and peptides in order to alter native charges, block or expose reactive binding sites, inactivate functions, or change functional groups to create targets for crosslinking and labeling.

Aminoethylate Reagent

Converts free sulfhydryls into primary amine groups.

Citraconic Anhydride

Citraconic Anhydride (2-methylmaleic anhydride) reversibly blocks primary amines at pH 8. Amines can be unblocked and returned to their native form.

Iodoacetic Acid

For carboxymethylation.

PEGylation Reagents

Use of activated linear and branched derivatives of polyethylene glycol (PEG) for pegylation and PEG-modification of peptides and proteins via primary amines and sulfhydryl groups to increase solubility, prolong stability and reduce immunogenicity.

Amine-Reactive PEG (Linear)

Methyl-PEG-NHS-ester reagents for PEGylation of proteins and molecules having primary amines. Also called MS(PEG)n, where n = 4, 8, 12 or 24 polyethylene glycol units.

Sulfhydryl-Reactive PEG (Branched)

A branched Methyl-PEG-Maleimide reagent for PEGylation of proteins and molecules having free sulfhydryl groups. Also called TMM(PEG)12 or (Methyl-PEG12)3-PEG4-Maleimide.

Carboxy-PEG-Amine Compounds

PEG amino acids, also called CA(PEG)n, where n = 4, 8, 12 or 24 polyethylene glycol units. The PEGylation compounds can be conjugated to molecules or surfaces using the crosslinker EDC or incorporated into AA sequences during peptide synthesis.

Proteases and Protein-Cleaving Reagents

Use of purified and agarose-immobilized proteases for enzymatic proteolysis (cleavage or digestion) of proteins to facilitate amino acid sequencing, peptide analysis and polypeptide structural characterization.

Immobilized Ficin

Immobilized ficin is used primarily to generate Fab and F(ab’)2 fragments from mouse IgG1 antibodies, and immobilization virtually eliminates autolysis of the enzyme and allows tight control of the digestion by removing the ficin at any time.

Immobilized Papain

Immobilized papain is used primarily to generate Fab and Fc fragments from antibodies, and immobilization virtually eliminates autolysis and protease contamination of the sample and allows tight control of the digestion by removing the papain at any time.

Immobilized Pepsin

Immobilized pepsin is used primarily to generate F(ab’)2 fragments from antibodies, and immobilization virtually eliminates autolysis and protease contamination of the sample and allows tight control of the digestion by removing the pepsin at any time.

Protein Denaturants and Chaotropes

Chaotropic and denaturing chemical agents, including urea and guanidine hydrochloride, to disrupt water interactions and promote hydrophobic protein and peptide solubilization, elution, refolding and structural analysis.

Guanidine-HCl and Solution

Particulate free, crystal clear, colorless solution of guanidine hydrochloride with excellent stability. Can be used for washing affinity ligand columns (non-protein ligands), solubilizing inclusion bodies, and other peptide/protein analysis methods.

Urea

A low UV absorbing protein denaturant.

Reducing Agents for Protein Disulfides

Purified powders, convenient solutions and solid-phase resins of disulfide reducing agents, including DTT, BME and TCEP, for stabilization of free sulfhydryls (cysteines) and reduction of disulfide bonds in peptides and proteins.

2-Mercaptoethanol (BME)

2-Mercaptoethanol (B-mercaptoethanol) is a mild reducing agent that is ideal for cleaving disulfide bonds and generating thiols.

2-Mercaptoethylamine-HCl

2-MEA, an reducing agent to dissociate divalent IgG to monovalent IgG by reducing disulfide bonds in the hinge region without separating heavy and light chains.

METHODS OF DETERMINING PROTEIN CONFORMATION

X-ray crystallography

This is the X-ray diffraction pattern. X rays are allowed to strike the protein crystal; the X rays are diffracted by the crystal and impinge on a photographic plate, forming a pattern of spots.

The measured intensity of the diffraction pattern, as recorded on a photographic film, depends particularly on the electron density of the atoms in the protein crystal.

This density is lowest in H atoms, and they do not give a visible diffraction pattern.

C, O and N atoms yield visible diffraction patterns but are present in great number - about 700 or 800 per 100 amino acids. This makes resolution of the structure of a protein containing more than 100 amino acids difficult.

X-ray crystallography is best for structures of rigid proteins that form nice, ordered crystals. Flexible proteins are difficult to study by this method because flexible portions of protein will often be invisible in crystallographic electron density maps, since their electron density will be smeared over a large space.

Resolution is improved by substituting into the side chains of certain amino acids very heavy atoms, (heavy metals) e.g Mercury ions, Platinum chloride.

Measures of the accuracy of a crystallographic structure include resolution, which measures the amount of detail that may be seen in the experimental data, and the R-value, which measures how well the atomic model is supported by the experimental data found in the structure factor file.

The X-ray diffraction technique cannot resolve the complete three-dimensional conformation. Complete resolution is obtained by combination of the X-ray diffraction results with the amino acid sequence analysis. In example proteins; myoglobin, chymotrypsinogen, lysozyme, and ribonuclease.

NMR Spectroscopy

Also Spectrophotometry. Involves the measurement of the degree of absorbance of light by a protein in solution within a specified wavelength.

NMR spectroscopy provides information on proteins in solution, as opposed to those locked in a crystal or bound to a microscope grid, and thus, NMR spectroscopy is the premier method for studying the atomic structures of flexible proteins

It’s useful within the range of visible light only with proteins that contain coloured prosthetic groups (the nonprotein components). Examples of such proteins include the red heme proteins of the blood, the purple pigments of the retina of the eye, green and yellow proteins that contain bile pigments, blue copper-containing proteins, and dark brown proteins called melanins.

Computational methods are used for determining protein structures from NMR data.

Electron Microscopy

A beam of electrons is used to image the molecule directly. Several tricks are used to obtain 3D images. If the proteins can be coaxed into forming small crystals or if they pack symmetrically in a membrane, electron diffraction can be used to generate a 3D density map, using methods similar to X-ray diffraction.

If the molecule is very symmetrical, such as in virus capsids, many separate images may be taken, providing a number of different views. These views are then aligned and averaged to extract 3D information.

Electron tomography, on the other hand, obtains many views by rotating a single specimen and taking several electron micrographs. These views are then processed to give the 3D information.

Electron micrographic experiments may not allow the researcher to see each atom.

Electron micrographic studies often combine information from X-ray crystallography or NMR spectroscopy to sort out the atomic details.

Optical activity

Amino acids, except glycine, exhibit optical activity (rotation of the plane of polarized light). Proteins also are optically active. They are usually levorotatory (i.e., they rotate the plane of polarization to the left) when polarized light of wavelengths in the visible range is used.

Although the optical rotation of a protein depends on all of the amino acids of which it is composed, the most important ones are C and the aromatic amino acids F, Y, and W. The contribution of the other amino acids to the optical activity of a protein is negligible.

STRUCTURE, COMPOSITION, AND FUNCTION OF CONNECTIVE TISSUE

2012-05-29T22:41:00.002-07:00

LECTURE III

Learning Objective: To identify the components of connective tissue and describe their structure, composition & function.

– Connective tissue is made up of specialized cells and extracellular matrix composed of extracellular protein fibers and a ground substance

– Underlies epithelium, supports capillaries, small nerves

Functions of connective tissue

1. Establishing a structural framework

2. Transporting fluids and dissolved materials

3. Protecting delicate organs

4. Supporting, surrounding and interconnecting tissues

5. Storing energy reserves e.g. fat

6. Defending the body from microorganisms

7. Cell growth and differentiation

Structural Elements of Connective Tissue

- Extracellular matrix (ECM) is composed of three structural elements:

A. Protein fibers

B. Ground substance

C. Cells/Fluid

A. Ground Substance

– Is an interstitial (tissue) fluid within which are one or more of the molecules listed below:

a) Hyaluronic acid: a very slippery polysaccharide which serves as a good lubricant for joints.

– Gelatinous, separates cells, traps extracellular fluid; lubricates joints; gives shape to eyeballs; fills body spaces

b) Proteoglycans: Large polysaccharide molecules bound to a protein core.Polysaccharides called glyocosaminoglycans (chondroitin sulfate,keratin sulfate).Protein part attaches to hyaluronic acid. Able to trap large amounts of water.

§ Glycosaminoglycans (GAGs) are attached to proteoglycans

ü They trap water. As GAGs increase, so does viscosity

•chondroitin sulfate – capable of being mineralized; found in cartilage, bones, skin, blood vessels

•dermatin sulfate – harder; found in skin, tendons, blood vessels, heart valves

• keratin sulfate - still harder; bone, cartilage, cornea of the eyes

c. Adhesive molecules: hold proteoglycan aggregates together.

i. Chondronectin in cartilage,

ii. osteonectin in bone,

iii. fibronectin in fibrous connective tissue (Binds cells, collagen and GAG)

iv. Laminin mediates attachment of epithelial cells to basal laminae

– Cells have membrane receptors, integrins, that bind collagen, fibrinectin, laminin and other extracellular structural components

– Functions as a molecular sieve through which nutrients diffuse between blood capillaries and cells

B. Fiber

- Long, rope-like protein extracellular polymers embedded in the ground substance

- Present in variable proportions in the different types of connective tissues

- Fiber provide structural support, adhesion, connect cells

- Three types: collagen, reticular and elastic fibers.

- Collagen and reticular fibers are composed of various types of collagen, elastic fibers are composed mainly of elastin

1. Collagen fibers (white fibers)

- Large fibers made of the protein collagen

- The most abundant fibers (collagen type I)

- Un-branched

- Promote tissue flexibility.

- Localized in tendons, ligaments, cartilages

- Identified in histological sections by staining with various acidic dyes.

2. Elastic fibers (yellow fibres)

- Intermediate fibers made of the protein elastin.

- Branching fibers that allow for stretch and recoil

- can be stretched to 150% of its original length

- Localized in lung, urinary bladder, skin, large blood vessels (aorta)

- Stained using elastin-specific dyes, such as resorcin-fuchsin, aldehyde-fuchsin, or orcein.

3. Reticular fibers

- Small delicate, branched fibers that have same chemical composition of collagen (predominantly collagen type III)

- Branched

- Fill spaces between tissues and organs.

- Localized in liver, spleen, lymph nodes, haematopoietic organs

- Stained using silver salt impregnation

C. Cells

- Fewer, rarely touching, surrounded by a matrix

- Immature forms (-blasts) secrete the matrix and can still divide

- Once the matrix is secreted, the cells mature into -cytes which have decreased cell divisions and secrete less matrix material- chondro- cartilage, osteo- bone, fibro – connective, etc. Fibroblasts – connective tissue proper

Fibroblasts - secrete the proteins needed for fiber synthesis and components of the extracellular matrix
Adipose (adipocytes) - Common in some tissues (dermis of skin); rare in some (cartilage)
Mast cells - Common beneath membranes; along small blood vessels. Can release heparin, histamine, and proteolytic enzymes in response to injury.
Leukocytes (WBC’s) - Respond to injury or infection
Macrophages - Derived from monocytes (a WBC). Phagocytic; provide protection
Chondroblasts - form cartilage
Osteoblasts - form bone
Hematopoietic stem cells - form blood cells
Undifferentiated mesenchyme (stem cells) - Have potential to differentiate into adult cell types

– Have a common mesenchymic origin

Types of Connective Tissues

True (Proper) CT

a) Loose CT

i. Areolar (loose fibrous) connective tissue

ii. Adipose tissue

iii. Reticular connective tissue

a) Dense CT

i. Dense (fibrous) regular connective tissue

ii. Dense (fibrous) irregular connective tissue

Support CT

a) Cartilage

i. Hyaline cartilage

ii. Elastic cartilage

iii. Fibrocartilage

Liquid CT

i. Blood

ii. Lymph

1. True or Proper Connective Tissue

a) Loose Connective Tissue:

i. Areolar tissue-Widely distributed under epithelia

ii. Adipose tissue-Hypodermis, within abdomen, breasts

iii. Reticular connective tissue-Lymphoid organs such as lymph nodes

Areolar (loose fibrous) connective tissue

Description: Gel-like matrix with all three fiber types: Cells: fibroblast, macrophages, mast cells, and some white blood cells.

Function: Wraps and cushions organs: its macrophages phagocytize bacteria: plays important role in inflammation: holds and conveys tissue fluid.

Location: Widely distributed under epithelia of body, e.g forms lamina propria of mucous membrane; packages organs: surrounds capillaries, nerves

ii. Adipose loose connective tissue

Description: Consists of adipocytes; "signet ring" appearing fat cells. They store energy in the form of triglycerides (lipids). have nucleus pushed to the side by large fat droplet

Function: Provides reserve fuel; insulates against heat loss; supports and protects organs

Location: Found in subcutaneous layer, around organs and in the yellow marrow of long bones

iii. Reticular CT

Description: Consists of fine interlacing reticular fibers and reticular cells

Function: Forms the framework (stroma) of organs and binds together smooth muscle tissue cells

Location: Liver, spleen and lymph nodes

b. Dense Connective Tissue:

i. Dense regular connective tissue:

ii. Dense irregular connective tissue:

i. Dense regular Connective Tissue

- Consists of bundles of collagen fibers and fibroblasts

- Forms tendons, ligaments and aponeuroses

- Function = provide strong attachment between various structures

ii. Dense Irregular CT

- Consists of randomly-arranged collagen fibers and a few fibroblasts

- Found in fasciae, dermis of skin, joint capsules, and heart valves

- Function = provide strength

2. Supportive Connective Tissue:

• Cartilage and bone support the rest of the body

• Cartilage

– Grows via interstitial and appositional growth

– Matrix is a firm gel containing chondroitin sulfate

– Cells called chondrocytes

– Cells found in lacunae

– Perichondrium separates cartilage from surrounding tissues

– Three types: hyaline, elastic and fibrocartilage

a) Cartilage

i. Hyaline

Description: Amorphous but firm matrix; collagen fibers form an imperceptible network; chondroblasts produce the matrix and then mature (chondrocytes) lie in lacunae

Function: Supports and reinforces: has resilient cushioning properties; resists compressive stress

Location: Forms most of the embryonic skeleton; covers the ends of long bones in joints cavities; forms costal cartilages of the ribs; cartilages of the nose, trachea, and larynx.

ii. Fibrocartilage

- Matrix similar to but less firm that that in hyaline cartilage

- Thick collagen fibre oredominate

- Found in the pubic symphysis, intervertebral discs, discs in synovial joints (menisci of the knee) & Bone-tendon junction

- Function = support and fusion, and absorbs shocks.

- Deceptively similar to dense CT- Lacunae

iii. Elastic Cartilage

- Threadlike network of elastic fibers within the matrix.

- Similar to hyaline cartilage

- Found in external ear, auditory tubes, and epiglottis.

- Function = gives support, maintains shape, allows flexibility

Fluid connective tissues

• Distinctive collections of cells in a fluid matrix

• Blood

– Formed elements and plasma

• Red blood cells, white blood cells and platelets

– Arteries carry blood away, veins carry to the heart

– Capillaries allow diffusion into the interstitial fluid

• Lymph

– Interstitial fluid entering the lymphatic vessels

Membranes

Membranes are simple organs

- Form a barrier

- Composed of epithelium and connective tissue

- Four types

1. Cutaneous - Covers the body surface

2. Synovial- Incomplete lining within joint cavities

3. Serous-Line sealed internal cavities

-Form transudate

4. Mucous - Line cavities that communicate with the exterior

-Contain lamina propria

THE COMPARISON OF EVIDENCE

2012-05-29T22:30:00.003-07:00

Definition of terms:

Class characteristics

Characteristics that enable an object to be placed into a particular category, for example identifying a trainer as belonging to a certain brand

Test impression

An impression deliberately made using a suspect item in order to compare it with a scene impression.

Scene impression

An impression detected at the scene of a crime, which may be of potential forensic importance.

Individual characteristics

Characteristics that are unique to a particular object (e.g. a tool, tyre or shoe) and, as such, are potentially useful in the identification of scene impressions

COMPARISON BETWEEN AN EVIDENTIAL OBJECT AND A RELEVANT DATABASE

In some instances, the purpose of this type of comparison is to identify a category to which an item of evidence belongs. To achieve this, the class characteristics of the evidential item concerned are established. For example, if footwear impressions or prints are recovered from a crime scene, their sole patterns may be established and then these may be usefully compared with sole patterns held on a footwear database

Through this exercise, it may be possible to identify the manufacturer and, conceivably, the style of the shoe concerned. This type of footwear comparison is particularly relevant to trainers. Similarly, tyre marks left at an incident scene may be compared with an appropriate database of tread pattern designs.

With some specific types of forensic evidence, namely fingerprints and samples of body fluids or tissues used for DNA profiling, the object of comparison with a database is the identification of the individual concerned.

COMPARISON BETWEEN TWO PIECES OF EVIDENCE OBTAINED FROM DIFFERENT PLACES

This type of comparison seeks to determine whether two pieces of apparently similar forensic evidence, for example hairs, textile fibres, paint chips or glass fragments, may share a common origin. Its purpose, therefore, is to determine whether any possible link exists between the two separate locations from which the evidence has been retrieved. This may be between two individuals (as in the case of the victim of an attack and his or her assailant), between an individual and a crime scene, or even between two different crime scenes. This type of comparison may be usefully illustrated by the following hypothetical scenario.

Consider a case in which a car window is broken and the CD player stolen from the vehicle. A suspect is apprehended by the police and, although the CD player is not in the suspect’s possession, there are splinters of glass adhered to the right-hand cuff of his jacket. A comparison is made between shards of glass taken from the car window and those recovered from the suspect. If these samples are found to be indistinguishable, this provides evidence that is consistent with the suspect being at the crime scene.

COMPARISON BETWEEN QUESTIONED SAMPLES, BOTH POSITIVE AND NEGATIVE CONTROLS, AND REFERENCE COLLECTIONS

A crime scene sample that is to be tested to find its evidential value is usually referred to as a questioned sample (or sometimes a disputed sample). Such tests are designed to evaluate a hypothesis. A hypothesis is a supposition that is either true or false and that can be tested by experimentation. For example, if a suspect is detained and found to possess a packet containing a pale brown powder, then the hypothesis may be that the powder is heroin. In order to test this hypothesis, experiments may be carried out that compare the chemical characteristics of this questioned sample with those of a known sample of heroin. Known samples such as this are referred to as positive controls, known or standards. If the questioned sample and the positive control are shown to have characteristics in common, it might be concluded that the questioned sample is indeed heroin. However, this may not be the case. It is possible that the chemicals and/or equipment used in the test were contaminated with heroin. In order to eliminate this possibility, it is necessary to carry out the test in a way that is identical in all respects to the tests to be carried out on the questioned sample and the positive control sample, except that it contains neither of these materials. Such a test is known as a negative control or a blank. In some instances, it is necessary to go to considerable lengths when carrying out the negative control test. For example, when testing for trace levels of explosives, swabs from all surfaces that will come into contact with the sample will be obtained. These will then be tested to show that the equipment was free from explosives. Note that in many applications, the term ‘control’ is used to denote either positive or negative controls; the context makes it clear which type of control is being referred to. There are circumstances in which it is valuable to compare a questioned sample with a number of positive controls. For example, the properties of a liquid retrieved from a scene of suspected arson may be compared with those of a range of flammable liquids, such as different types of petrol, paraffin and diesel fuel. Through comparison, it may be possible to identify the questioned sample via elimination and positive matching. A collection of positive controls used for such a purpose is known as a reference collection.

COMPARISON BETWEEN A SCENE IMPRESSION AND A TEST IMPRESSION

Impressions made by recognizable objects, such as footwear, tyres and tools, are often detected during the examination of crime scenes. If an object suspected of creating the impression(s) in question is subsequently discovered, then that object may be used to create a series of test impressions. A comparison of these test impressions with the scene impression(s) may reveal that both types were created by objects with the same class characteristics. However, in some cases, it may be possible to proceed beyond this stage and identify the suspect item as being the actual one used in the commission of a crime. This can occur when individual characteristics, namely those that are peculiar to a particular individual object, are shown to be visible on the scene impression(s), as well as on the test impressions. Such individual characteristics may be created by some aberration during the manufacturing process but are more likely to be acquired as a result of general wear and tear. Characteristics that are exhibited in evidence and that are capable of identifying a specific item are said to be individualizing.

DID YOU KNOW?

2012-05-29T22:29:00.003-07:00

1. The polymerase chain reaction (PCR) was featured in the motion picture Jurassic Park.

In this story, scientists find fossilized blood-sucking insects. These insects, which had bitten dinosaurs, were caught in tree sap that fossilized into amber.

The scientists in the film extracted dinosaur blood from the fossilized insects and used the DNA in the blood to recreate dinosaurs through cloning.

2. When a human egg and sperm combine, nearly a billion different gene combinations are possible.

3. New Zealand is the world leader in forensic palynology and in the acceptance of pollen and spore evidence in court cases. Palynological evidence is underutilized in many parts of the world, including the United States.

4. Pollen grains can be extracted safely from rocks that are millions of years old. This is a valuable characteristic not only for palynologists, but also for oil companies and archaeologists.

How do you reduce the contamination of evidence?

2012-05-29T22:28:00.003-07:00

1. How do you reduce the contamination of evidence?

The risk of contamination of evidence is minimized by applying the following precautionary steps:

Using chain of custody labels

Opening each package in an area other than where it was originally sealed

Repackaging each item of evidence as soon as it has been analyzed

Assiduously using logging systems

Minimizing the number of people handling the evidence

Storing packaged evidence in a dedicated secure area.

Muscles

2012-05-29T22:27:00.003-07:00

1. Briefly highlight the contraction of the smooth muscles

Calcium ions come from outside the cell. They bind to calmodulin in the cytosol and then bind to an enzyme on myosin called Myosin Light Chain Kinase which uses ATP to transfer a phosphate to myosin. The phosphate activates the myosin which then forms cross bridges with actin. When ca++ is pumped out of the cell, the phosphate gets removed from myosin by another enzyme. Myosin becomes inactive and the muscle relaxes.

2. State the characteristics of smooth muscle

-more actin than myosin

-no striations

-spindle shaped

-no sarcomeres

-fatigue resistant

-activation is involuntary

-innervation is by ANS

-located in blood vessels, gastro-interstinal tract, urinary bladder, respiratory tract, iris of the eye and reproductive organs

-External longitudinal layer-runs parallel to the organs long axis and shorten and widen the lumen when it contracts

-Internal circular layer-runs around the circumference of the organs axis lengthens the tube but narrows the lumen when contracted

3. State the characteristics of skeletal muscles

-Excitability- capacity of the muscle to respond to stimuli

-Extencibility-abilty of the muscle to stretch back to its original length

-Contractility- ability of the muscle to shorten and generate a pulling force

-Elasticity- ability of the muscle to recoil to its original resting length after stretch.

FORENSIC VERSUS HOSPITAL AUTOPSIES

2012-05-29T22:24:00.000-07:00

An autopsy represents a postmortem surgical examination of a human body, but not all autopsies are equivalent. There are two basic types of autopsies: forensic or medico legal autopsies and hospital or medical autopsies. There are several very important differences between the two. The ﬁrst is that forensic autopsies fall under the jurisdiction of the local governmental death investigation ofﬁce (coroner or medical examiner), whereas hospital autopsies do not. As such, forensic autopsies do not require consent from the legal next-of-kin and the coroner or medical examiner may, by law, order an autopsy. Hospital autopsies require a properly executed and witnessed consent signed by the decedent’s (dead person’s) legal next-of-kin before the autopsy can be performed.

Making certain that the person being autopsied is, in fact, the person he/she is supposed to be is of utmost importance, no matter what type of autopsy is being performed. In virtually every hospital autopsy performed, the decedent’s identity is already known and well-documented via identiﬁcation bracelets and within the medical records that accompany the body. In contrast, a very important aspect of every forensic autopsy involves the positive identiﬁcation of each decedent.

Depending on the structure of the death investigation system, this very important task may be the ofﬁcial responsibility of different entities. In certain jurisdictions, the legal responsibility rests with non-pathologist death investigators (coroners or non-pathologist medical examiners), while in other ofﬁces, the responsibility rests with those performing the autopsies. In either case, all persons and ofﬁces involved should work together to ensure positive identity in every case.

Frequently in hospital autopsies, the cause of death is already known, and the autopsy is performed to attempt to answer speciﬁc questions that family members or physicians have regarding the case, such as disease extent, effectiveness of therapy, etc. For these reasons, hospital autopsies are frequently limited to the examination of certain body parts. In addition, the complete medical record is usually available to the pathologist prior to hospital autopsy performance. As a result of this the “background information” being readily available prior to autopsy, a pathologist can perform the autopsy with speciﬁc focus. In contrast, while the cause of death may be obvious in certain forensic cases and a certain amount of background information may be available prior to examination, forensic autopsies are usually performed with the intent of documenting all ﬁndings, rather than being focused on a single issue or two. Frequently in forensic work, all of the important background information, as well as all of the questions that will eventually be asked regarding a speciﬁc case, are not known when the autopsy is being performed.

In general, if resources allow, the most thorough postmortem forensic examination involves a complete autopsy (head, neck, and trunk), although different ofﬁce and practitioner policies and protocols may allow for limited dissections in speciﬁc case types.

Another major difference that exists between hospital and forensic autopsies is the fact that, in many hospital autopsies, the external examination is relatively unimportant, whereas in forensic cases, the external examination can frequently be one of the most important aspects of the case. As such, photography is a key component of the forensic autopsy, whereas it is usually considered optional in the hospital autopsy.

Certain other procedures and tests are common in forensic cases, but quite rare in hospital cases. The two most common are toxicology testing and radiography (X-rays). A signiﬁcant percentage of forensic cases will have positive toxicology tests (drugs are detected), which will, in many cases, be an important factor in determining the cause of death. Likewise, a signiﬁcant percentage of forensic cases also require radiography. Various specialized dissections can occur in either type of autopsy, but are probably more common in forensic cases. These include anterior (front) neck dissections, posterior (back) neck dissections, spinal cord examination, eye removal and examination, dissection of the deep veins of the legs, etc.

“Fixing” organs or tissues in formalin (a preservative solution containing formaldehyde) prior to dissecting them is a procedure that can occur with either type of autopsy, although it is probably more common in hospital cases, where many pathologists were trained to “ﬁx” the organs prior to detailed dissection. In forensic cases, however, it is not unusual in certain cases to choose to ﬁx a particular organ prior to sectioning, with the brain and heart being the most common organs in which this is done. It is important to note here that formalin ﬁxation will change the appearance of tissues, usually causing a darker, less shiny appearance (this is obvious when comparing photographs of “fresh” organs to those of “ﬁxed” organs), and will also cause the tissues to become more ﬁrm, thus making dissection a bit easier.

A ﬁnal difference between hospital and forensic autopsies involves those who actually perform, or assist with, the autopsy. In many hospital-based pathology practices, pathology assistants (PAs) are employed to assist pathologists with many functions. In some practices, they are responsible for performing the “gross” part of the autopsy, with pathologists only performing histologic (microscopic) examinations. However, this is not acceptable practice in forensic autopsies.

BLAST ALONG MOI AVENUE

2012-05-29T07:22:00.001-07:00

Breaking News…

At around 1.15pm on 28^th of May 2012, there was a loud blast along Moi Avenue on a building adjacent to MKU towers. The building houses various stores and customers were busy doing their shopping when the incident occurred.

The cause of the blast apparently has not yet been confirmed but the speculations about a possible electricity failure has been ruled out by the KPLC officials at the scene stating that the nature of the blast amounts to something more than an electrical fault.

The Kenya Prime Minister Hon. Raila Odinga who arrived at the scene spoke about acts of terrorism and bravely stated that the nation will not be intimidated by the terrorist’s effort to destroy the nation. Prime ministers words seemed to bear a lot of weight as he stated that the blast has all the characteristics of acts of terrorism as seen from all over the world.

On the other hand, police commissioner who was also at the scene was quick to state that we should not be quick to state the cause and said he doesn’t see a possibility of an act of terrorism. He however added that that the experts were on their way and investigations are to be started to establish the real cause of the blast.

Another word from the minister of internal security prof George saitoti, said he had no doubt that this and other grenade attacks are part of tactics that the al shaabab are using to intimidate rather hit back at the nation.

Victims from the blast have been rushed to Kenyatta National Hospital where they are receiving treatment and it is said that 35 people have been injured.

And my question is to the forensic experts especially the students in the university. Can you write a detailed report on crime scene investigation procedures stating all the necessary equipment’s and tools to be used?

AN INSIGHT TO FORENSIC SCIENCE DISCIPLINES

2012-05-29T07:21:00.001-07:00

Forensic science is used to help solve crimes and there are many types of forensic methods/ disciplines.

1. Forensic toxicology- refers to the use of toxicology to determine what chemicals are present in the body after earth.

2. Forensic psychology- deals with the study of various human behaviors.

3. Criminalistics- is the application of sciences to situations in an attempt to achieve answers. This is commonly used in the area of fingerprints, tire tracks, or foot impressions.

4. Forensic DNA analysis- is used to determine the DNA sequence from biological samples.

5. Forensic anthropology- the application of physical anthropology in a legal setting

6. Cyber forensics- the gathering of digital data that is used in the court of law.

7. Forensic engineering- the investigation of materials, products, structures or components that fail to operate or do not function as intended.

8. Forensic entomology- the use of insects, and their arthropod relatives that inhabit decomposing remains to aid in legal investigations.

9. Forensic odontology- the study of all dentition of all human beings

This is just but the tip of the iceberg in forensics, there are a whole lot of topics ranging from bugs to DNA and all branches are used in actual criminal investigation.

A comprehensive list on other forensic related disciplines is discussed on Fields of Forensic Science.

Crime scene investigators have special equipment that they use to investigate crime scenes. This equipment is packed into crime scene kits. They include

Fingerprint powders designed to find fingerprint on surfaces, brushes, magnetic powder wands, and placards for making important forensic evidence, tape, scissors, tweezers, tape, pencils and pens.

Chemicals for detecting bodily fluids like luminal and black lights.

Bodily fluids can be the most valuable piece of evidence because it can yield DNA, the holy grail of proof.

FORENSIC SCIENCE DISCIPLINES

2012-05-29T07:20:00.003-07:00

Forensic Pathology

Forensic pathology represents a subspecialty within the medical specialty of pathology dealing speciﬁcally with the investigation of sudden, unexpected, and/or violent deaths. The autopsy is central to the practice of forensic pathology.

Forensic Anthropology

Forensic anthropology is a subspecialty within the scientiﬁc ﬁeld of physical anthropology (the study of human beings in relation to their physical character); in which forensic anthropologists examine skeletal remains (bones). Forensic anthropologists attempt to answer questions about bones, including questions regarding species of origin (human versus nonhuman), gender, age, race, stature, and nutritional status, existence of disease processes, and the presence and character of skeletal trauma. A forensic pathologist may consult with a forensic anthropologist when attempting to address any of the questions above. A frequent instance of consultation occurs when the forensic pathologist is presented with a badly decomposed or skeletonized corpse that is unidentiﬁed

Forensic Entomology

Forensic entomology is a subspecialty within the biological science discipline of entomology (the study of insects) that primarily deals with insect succession patterns in decomposing human bodies. Evaluation of insects (including larval stages, or maggots) found on decomposing bodies can permit scientiﬁc estimation of the time of death .In certain circumstances; information regarding the location of death may also be ascertained. Forensic pathologists do not consult with forensic entomologists in all decomposed cases, but will consult with them on select cases where estimating the time of death may be very important (for example, in homicides with decomposition and insect activity).

Forensic Toxicology

Forensic toxicology is a discipline that involves the identiﬁcation and quantiﬁcation of drugs and other poisons or toxins in body tissues, including blood. “Screening tests” are said to be “qualitative,” where a test is either positive (indicating that the drug/toxin is present) or negative (indicating that the drug/toxin is not present).

When speciﬁc levels of drugs or toxins are determined, the tests are said to be “quantitative.” For a result to have forensic signiﬁcance, two separate methodologies are required, an initial (screening) test, and a conﬁrmatory (quantitative) test. Another function of some toxicology laboratories is drug identiﬁcation. For example, if a bag of white powdery substance is found in the pocket of a dead person, the substance can be submitted to the laboratory for identiﬁcation. Forensic pathologists rely a great deal on the forensic toxicology laboratory. In many jurisdictions, toxicology testing is performed on a majority of the autopsy cases. In a signiﬁcant percentage of forensic autopsy cases, the cause of death is related to the toxicology results.

Forensic Psychiatry

Forensic psychiatry represents a discipline dealing with the evaluation of the mental state of criminals. Occasionally, forensic pathologists will interact with forensic psychiatrists and police investigators to form a “psychiatric proﬁle” of a suspect in a particular murder or series of murders. A “psychological autopsy” is sometimes necessary when attempting to determine the state of mind of a suicide victim.

FORENSIC SCIENCE SALARIES

2012-05-29T07:10:00.001-07:00

Income varies greatly depending on the degree, your actual job, where you work, and how many hours you work.

It’s said; you may never ‘get rich’ but will have a good income, satisfied with your job, knowing you are contributing to justice.

Forensic scientists work different hours depending on job description.

I. Working in the laboratory for almost 40 hours a week.

II. In the fields or digs

III. Others wait for calls and work after their regular shifts

In the USA, the salaries vary as shown below:

Department	Income in $
Crime lab analyst	16.75-21.44/hour
Crime scene analyst	31.87-36.49/hour
criminalists	5000-7000/month
Crime lab director	58,000-76,000/yr
DNA analyst	62,000-75,000/yr
Evidence custodian	86,000-144,000/yr
Firearms examiner	2,000-5,000/month
Fingerprint technician	30,000-46,000/yr
Forensic autopsy Assistant	24.04-29.08/hour
Forensic technician	4,000-5,000/month
Forensic biologist	46,000-64,000/yr
Forensic evidence technician	4,000-5,000/month
Forensic scientist	35,000-52,0000/yr
Law enforcement training officer	22.0-30.00/hour
Latent print examiner	108,000-158,000/yr
Latent print technician	33,000- 50,000/yr
Toxicologist	6000/month
Trace analyst	2,000-3,000/month

In Kenya, that is subject to a lot of influences and the best to say is to urge us to be patient and wait for that day. However, we could compare the salaries to what the present Crime scene

investigators and police earn just to have a rough figure.

FORENSIC EVIDENCE COLLECTION

2012-05-29T07:07:00.001-07:00

Investigators should perform the evidence collection process in a systematic and careful manner.

The process begins with the preliminary crime scene survey/walk-through, followed by a determination of the evidence collection sequence to be used.

The evidence collection sequence may be based on the following information:

• The scene location: interior, exterior, within a vehicle.

• The condition of the evidence: either fragile or stable.

• Weather conditions which might affect the scene or evidence within.

• Scene management considerations which may alter or contaminate the evidence.

• Additional processing techniques that may need to be conducted at the scene with specialized personnel.

Investigators should use the appropriate equipment when collecting evidence. Collection equipment that may come into contact with evidence should be sterile.

The following equipment may be used in the evidence collection process:

• Latex gloves/nitrile gloves (N-DEX, non-latex).

• Forceps.

• Tweezers.

• Scalpels.

• Swabs.

• Paper bags.

• Plastic bags.

• Cardboard boxes.

• Wrapping paper.

• Hand tools.

• Thermometer.

• Plastic 5 gallon bucket with lid.

COLLECTION METHODS

The swabbing collection technique should be used for the recovery of biological evidence in a dried or liquid state. Best practice techniques include the following:

DRIED MATERIAL COLLECTION TECHNIQUE

• With gloved hands, slightly moisten the swab with distilled water. (The swab should be damp but not overly wet.)

• Thoroughly rub the stained area using a single moistened swab for a small stain and multiple swabs for a large stain. When only a small amount of the stain is available, concentrate as much of the stain as possible on the tip of the swab.

• Air-dry the swabs.

• Place each swab into separate package.

• This package may be placed inside a paper envelope.

• Collect a substrate/control sample from an unstained area using the same techniques.

LIQUID MATERIAL COLLECTION TECHNIQUE

A. When suspected biological evidence is found on clothing or other absorbent surfaces transport it to the laboratory in an appropriate container. Wet evidence should not be folded over on itself. Use paper wrapping to prevent contamination during the transfer. This will protect bloodstain patterns and prevent cross-contamination between stains on one item. The item should be air-dried thoroughly in a drying locker and packaged in a container suitable for dried evidence.

B. If the suspected biological evidence is in a liquid form on a fixed surface that cannot be transported (i.e., concrete floor), the substance should be recovered using the following swab technique:

1. With gloved hands, swab the liquid material allowing the swab to absorb as much of the substance as possible. Multiple swabs should be obtained when a large quantity is available.

2. Thoroughly air-dry each swab.

Package the swab inside an appropriate container.

3. Collect a substrate/control sample from an unstained area using the same techniques.

ISOLATION AND PURIFICATION OF PROTEINS

2012-05-27T08:43:00.003-07:00

Purification process involves;

· freeing the protein from a matrix that confines it

· separating the protein and non-protein parts of the mixture

· separating the desired protein from all other proteins.

Separation of one protein from all others may exploit differences in protein size, physico-chemical properties, binding affinity and biological activity.

Purification may be preparative or analytical.

Extraction

Depending on the source, the protein has to be brought into solution by breaking the tissue or cells containing it.

This can be done through cell disruption to release cellular contents by:

· Repeated freezing and thawing

· sonication - applying sound (ultrasound) energy to agitate particles in a sample

· homogenization by high pressure - intensive blending of mutually related substances or groups of mutually related substances to form a constant of different insoluble phases

· filtration - the mechanical or physical operation used to separate solids from fluids

· permeabilization by organic solvents e.g SDS (sodium dodecyl sulfate), Triton X-100 or CHAPS(3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate)

The method of choice depends on how fragile the protein is and how sturdy the cells are.

After extraction process soluble proteins will be in the solvent, and can be separated from cell membranes, DNA etc. by centrifugation.

Precipitation and differential solubilization

This is performed by adding increasing amounts of ammonium sulfate - (NH₄)₂SO₄ and collecting the different fractions of precipitate protein.

The hydrophobic groups on the proteins get exposed to the atmosphere and attract other protein hydrophobic groups and get aggregated.

Protein precipitated will be large enough to be visible.

Ammonium sulphate can be removed by dialysis.

Ultracentrifugation

Centrifugation is a process that uses centrifugal force to separate mixtures of particles of varying masses or densities suspended in a liquid, resulting in the pellet and supernatant.

After separating the protein/particles, the gradient is then fractionated and collected.

Chromatographic methods

Different proteins interact differently with the column material, and can thus be separated by the time required to pass the column, or the conditions required to elute the protein from the column.

The eluant is usually pooled in different test tubes. Test tubes containing solution with the protein of interest and any other similar proteins is retained.

Usually proteins are detected as they are coming off the column by their absorbance at 280 nm. Many different chromatographic methods exist:

Size exclusion chromatography

The eluant is usually pooled in different test tubes. Test tubes containing solution with the protein to purify and any other similarly-sized proteins are retained.

Hydrophobic Interaction Chromatography

Ion exchange chromatography

Ion exchange chromatography is frequently used in both analytical and preparative separations.

Affinity chromatography

E.g. Immuno-affinity chromatography (Ab & Ag)

The protein can be eluted by changing the pH or the salinity

High performance liquid chromatography

The proteins are eluted by a gradient of increasing amounts of an organic solvent, such as acetonitrile. The proteins elute according to their hydrophobicity.

HPLC purification frequently results in denaturation of the purified proteins, thus not applicable to proteins that do not spontaneously refold.

Concentration of the purified protein

Lyophilization

Ultrafiltration

Ultrafiltration concentrates a protein solution using selective permeable membranes. The function of the membrane is to let the water and small molecules pass through while retaining the protein.

Analytical

Denaturing-Condition Electrophoresis

A common laboratory technique that can be used both as preparative and analytical method.

The principle of electrophoresis relies on the movement of a charged ion in an electric field.

In analytical methods, the proteins migrate as bands based on size. Each band can be detected using stains such as Coomassie blue dye or silver stain.

It provides an improved resolution over size exclusion chromatography, but does not scale to large quantity of proteins.

Non-Denaturing-Condition Electrophoresis

This variant of gel electrophoresis is used to isolate active or native metalloproteins in biological samples.

MODIFICATION OF PROTEINS

Modification of proteins is done to allow manipulation and study of protein function and interactions in any environment.

Includes crosslinking, fragmenting, denaturing, reducing disulfides, or attaching various prosthetic groups (e.g. PEGylation)

Crosslinking Reagents

Amine-to-Amine Crosslinkers

Amine-to-Sulfhydryl Crosslinkers

Carboxyl-to-Amine Crosslinkers

Photoreactive Crosslinkers

Sulfhydryl-to-Carbohydrate Crosslinkers

Sulfhydryl-to-Hydroxyl Crosslinkers

Crosslinking reagents based on maleimide and isocyanate reactive groups for conjugation and formation of covalent crosslinks between sulfhydryl and hydroxyl groups.

Sulfhydryl-to-Sulfhydryl Crosslinkers

Chemical modification Reagents

Aminoethylate Reagent

Converts free sulfhydryls into primary amine groups.

Citraconic Anhydride

Citraconic Anhydride (2-methylmaleic anhydride) reversibly blocks primary amines at pH 8. Amines can be unblocked and returned to their native form.

Iodoacetic Acid

For carboxymethylation.

PEGylation Reagents

Amine-Reactive PEG (Linear)

Methyl-PEG-NHS-ester reagents for PEGylation of proteins and molecules having primary amines. Also called MS(PEG)n, where n = 4, 8, 12 or 24 polyethylene glycol units.

Sulfhydryl-Reactive PEG (Branched)

A branched Methyl-PEG-Maleimide reagent for PEGylation of proteins and molecules having free sulfhydryl groups. Also called TMM(PEG)12 or (Methyl-PEG12)3-PEG4-Maleimide.

Carboxy-PEG-Amine Compounds

Proteases and Protein-Cleaving Reagents

Immobilized Ficin

Immobilized Papain

Immobilized Pepsin

Protein Denaturants and Chaotropes

Guanidine-HCl and Solution

Urea

A low UV absorbing protein denaturant.

Reducing Agents for Protein Disulfides

2-Mercaptoethanol (BME)

2-Mercaptoethanol (B-mercaptoethanol) is a mild reducing agent that is ideal for cleaving disulfide bonds and generating thiols.

2-Mercaptoethylamine-HCl

2-MEA, an reducing agent to dissociate divalent IgG to monovalent IgG by reducing disulfide bonds in the hinge region without separating heavy and light chains.

METHODS OF DETERMINING PROTEIN CONFORMATION

X-ray crystallography

This is the X-ray diffraction pattern. X rays are allowed to strike the protein crystal; the X rays are diffracted by the crystal and impinge on a photographic plate, forming a pattern of spots.

The measured intensity of the diffraction pattern, as recorded on a photographic film, depends particularly on the electron density of the atoms in the protein crystal.

This density is lowest in H atoms, and they do not give a visible diffraction pattern.

Resolution is improved by substituting into the side chains of certain amino acids very heavy atoms, (heavy metals) e.g Mercury ions, Platinum chloride.

NMR Spectroscopy

Also Spectrophotometry. Involves the measurement of the degree of absorbance of light by a protein in solution within a specified wavelength.

Computational methods are used for determining protein structures from NMR data.

Electron Microscopy

Electron tomography, on the other hand, obtains many views by rotating a single specimen and taking several electron micrographs. These views are then processed to give the 3D information.

Electron micrographic experiments may not allow the researcher to see each atom.

Electron micrographic studies often combine information from X-ray crystallography or NMR spectroscopy to sort out the atomic details.

Optical activity

STRUCTURE OF SKELETAL MUSCLE:IAHMZ

2012-05-27T02:59:00.001-07:00

Skeletal muscle consists of parallel bundles of musclefibers, each fiber a single, very large, multinucleatedcell, 20 to 100 µm in diameter, formed from many cellsfused together and often spanning the length of the muscle.Each fiber, in turn, contains about 1,000 myofibrils,2 µm in diameter, each consisting of a vast numberof regularly arrayed thick and thin filamentscomplexed to other proteins. A system offlat membranous vesicles called the sarcoplasmicreticulum surrounds each myofibril. Examined underthe electron microscope, muscle fibers reveal alternatingregions of high and low electron density, called theA bands and I bands. The A and I bandsarise from the arrangement of thick and thin filaments, which are aligned and partially overlapping. The I bandis the region of the bundle that in cross section wouldcontain only thin filaments. The darker A band stretchesthe length of the thick filament and includes the regionwhere parallel thick and thin filaments overlap. Bisectingthe I band is a thin structure called the Z disk, perpendicularto the thin filaments and serving as an anchorto which the thin filaments are attached. The Aband too is bisected by a thin line, the M line or M disk,a region of high electron density in the middle of thethick filaments. The entire contractile unit, consistingof bundles of thick filaments interleaved at either endwith bundles of thin filaments, is called the sarcomere.The arrangement of interleaved bundles allows the thickand thin filaments to slide past each other (by a mechanismdiscussed below), causing a progressive shorteningof each sarcomere.The thin actin filaments are attached at one end tothe Z disk in a regular pattern. The assembly includesthe minor muscle proteins α-actinin, desmin, and vimentin.Thin filaments also contain a large proteincalled nebulin(~7,000 amino acid residues), thought tobe structured as an αhelix that is long enough to spanthe length of the filament. The M line similarly organizesthe thick filaments. It contains the proteins paramyosin,C-protein, and M-protein. Another class of proteinscalled titins, the largest single polypeptide chains discoveredthus far (the titin of human cardiac muscle has26,926 amino acid residues), link the thick filaments tothe Z disk, providing additional organization to the overallstructure. Among their structural functions, the proteinsnebulin and titin are believed to act as “molecular rulers,” regulating the length of the thin and thick filaments,respectively. Titin extends from the Z disk to theM line, regulating the length of the sarcomere itself andpreventing overextension of the muscle. The characteristicsarcomere length varies from one muscle tissueto the next in a vertebrate organism, a finding attributedin large part to the different titin variants in thetissues.

The (blue-stained) endomysiumin Figure 1 below, which contains capillariesand nerve fibers, should be noted.

Composition of myofibrils

Myofibrils-composed of:

Contractile proteins
Regulatory proteins
Structural proteins

Contractile proteins: myosin and actin.

• Thick filaments- Myosin (motor protein converts ATP into movement). 300 molecules of -myosin=1 thick filament.

• Thin filaments- are anchored to z-discs and are composed of F-actin and regulatory proteins (tropomyosin and troponin).

- Actin contains a myosin binding site.

Regulatory proteins:

– Tropomyosin and troponin

– Tropomyosin blocks myosin binding to actin by blocking the myosin site on actin in relaxed muscle.

– The tropomyosin is held in place by troponin.

Structural proteins:

– Titin-anchors thick filament to a z-disc and M line

– Other proteins contribute to alignment, stability, and elasticity.

MOLECULAR MECHANISMOF MUSCLE CONTRACTION

2012-05-27T02:56:00.003-07:00

Myosin Thick Filaments Slide along Actin Thin Filaments

The interaction between actin and myosin, like that betweenall proteins and ligands, involves weak bonds.When ATP is not bound to myosin, a face on the myosinhead group binds tightly to actin. When ATPbinds to myosin and is hydrolyzed to ADP and phosphate, a coordinated and cyclic series of conformational changes occurs in which myosin releases the F-actin subunit and binds another subunit farther along the thin filament.

The cycle has four major steps. In step 1, ATP binds to myosin and a cleft in the myosin molecule opens, disrupting the actin-myosin interaction so that the bound actin is released. ATP is then hydrolyzed in step 2, causing a conformational change in the protein to a “high-energy” state that moves the myosin head and changes its orientation in relation to the actin thin filament. Myosin then binds weakly to an F-actin subunit closer to the Z disk than the one just released. As the phosphate product of ATP hydrolysis is released from myosin in step 3, another conformational change occurs in which the myosin cleft closes, strengthening the myosin-actin binding. This is followed quickly by step 4, a “power stroke” during which the conformation of the myosin head returns to the original resting state, its orientation relative to the bound actin changing so as to pull the tail of the myosin toward the Z disk. ADP is then released to complete the cycle. Each cycle generates about 3 to 4 pN (piconewtons) of force and moves the thick filament 5 to 10 nm relative to the thin filament. Because there are many myosin heads in a thick filament,at any given moment some (probably 1% to 3%)are bound to the thin filaments. This prevents the thickfilaments from slipping backward when an individualmyosin head releases the actin subunit to which it wasbound. The thick filament thus actively slides forwardpast the adjacent thin filaments. This process, coordinatedamong the many sarcomeres in a muscle fiber,

brings about muscle contraction.

Types of muscle contraction

Isotonic contraction

- tension produced exceeds the resistance (load), and the muscle fibers shorten, resulting in movement

- Two types of isotonic contractions:

a) Concentric- the muscle tension exceeds the resistance and the muscle shortens

b) Eccentric- the peak tension developed is less than the resistance, and the muscle elongates owing to the contraction of another muscle or the pull of gravity.

2. Isometric contraction

- length of the muscle does not change because the tension produced never exceeds the resistance (load) tension is generated, but not enough to move the load

Terminologies

1. Muscle Tone

- In any skeletal muscle, some motor units are always active, even when the entire muscle is not contracting. Their contractions do not produce enough tension to cause movement, but they do tense and firm the muscle. This resting tension in a skeletal muscle is called muscle tone. A muscle with little muscle tone appears limp and flaccid, whereas one with moderate muscle tone is firm and solid.

2. Hypotonia- decreased muscle tone, flaccid

3. Flaccid paralysis- loss of muscle tone, loss of tendon reflexes, atrophy

4. Hypertonia- increased muscle tone, spasticity or rigidity.

5. Spasticity- increased muscle tone, increased tendon reflexes (e.g. Babinski).

6. Rigidity- increased muscle tone where reflexes are not affected (e.g. tetanus)

SMOOTH MUSCLE TISSUE

2012-05-27T02:54:00.004-07:00

- Cells are not striated

- Fibers smaller than those in skeletal muscle

- Spindle-shaped (about 50 to 200 microns long and only 2 to 10 microns in diameter); single, central nucleus

- More actin than myosin

- No sarcomeres

· Not arranged as symmetrically as in skeletal muscle, thus NO striations.

- Caveolae: indentations in sarcolemma;

· May act like T tubules

- Dense bodies instead of Z disks

· Have noncontractile intermediate filaments

- Is fatigue resistant

- Activation is involuntary

• Locations: Located in blood vessels, respiratory tract, iris of the eye, gastro-intestinal tract, urinary, & reproductive organs

- Grouped into sheets in walls of hollow organs

· An external longitudinal layer - muscle fibers that runs parallel to organ’s long axis. It shortens the tube and widens the lumen when contracted.

· An internal circular layer - muscle fibers that runs around circumference of the organ. It lengthens the tube but narrows the lumen when contracted.

§ Both layers participate in peristalsis. Peristalsis is the directional motion achieved from both of these sheets working together to alternate contraction

- Is innervated by autonomic nervous system (ANS)

- Visceral or unitary smooth muscle

· Only a few muscle fibers innervated in each group

· Impulse spreads through gap junctions

· Whose sheet contracts as a unit

· Often auto-rhythmic

- Multiunit:

o Cells or groups of cells act as independent units

o Arrectorpili of skin and iris of eye

Smooth Muscle Contraction

As in skeletal-muscle cells, contraction in a smooth-muscle cell involves the forming of cross-bridges and thin filaments sliding past thick filaments. However, because smooth muscle is not as organized as skeletal muscle, shortening occurs in all directions. During contraction, the smooth-muscle cell's intermediate filaments help to draw the cell up, like closing a drawstring purse.

Calcium ions regulate contraction in smooth muscle, but they do it in a slightly different way than in skeletal muscle:

1. Calcium ions come from outside of the cell.

2. Calcium ions bind to calmodulin in the cytosol and then to an enzyme on myosin, called myosin light chain kinase.

3. The enzyme uses ATP to transfer a phosphate group to myosin.

4. This phosphate transfer activates myosin.

5. Myosin forms cross-bridges with actin (as occurs in skeletal muscle).

6. When calcium is pumped out of the cell, the phosphate gets removed from myosin by another enzyme.

7. The myosin becomes inactive, and the muscle relaxes.