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Chapter 11 - Protein Characterization Outline: Polyclonal Antibody Monoclonal Antibody Antibody based technology Immunofluorescent Staining NMR Spectroscopy X-ray Crystallography Overview: FIGURE 1: ANTIGEN- ANTIBODY INTERACTIONS The above figure shows the structure of Immunoglobulin, which are found in blood and bodily fluids and used by immune system to identify foreign objects. Immunoglobulin structure has two heavy and two light chains. Fab domains are H-S-S-L, and Fc domains are H-S-S-H bonded. Fab domains show the antigen-antibody interactions. Fab represents fragment antigen binding region and Fc represents fragment crystallizable region. The overall mechanism acts as a lock-key interaction. FIGURE 2: POLYCLONAL VS . MONOCLONAL ANTIBODY Polyclonal antibody is a heterogeneous mixture of antibodies, termed polyclonal, that recognizes different epitope (antigenic determinants) in the antigen. Monoclonal antibody is a homogeneous antibody of nearly any desired specificity against a single epitope. Large amounts of homogeneous antibody of nearly any desired specificity can be obtained by fusing a short lived antibody producing cell with an immortal myeloma cell (hybridoma). ELISA is an enzyme linked immunosorbent assay used to detect the presence of antigen or an antibody. Indirect ELISA is used for detection and quantization of antibody. Sandwich ELISA is used for detection and quantization of antigen. FIGURE 3: INDIRECT VS . SANDWICH ELISA Indirect ELISA Antigen to be tested is added to the cell and washed; serum is added which contains antibodies for antigen added originally which again binds together and washed; a substrate for the enzyme is added and washed; and finally the substrate changes color when reacting with enzyme detecting the amount of antibody. Sandwich ELISA Antibody to be tested is added to the cell and washed; antigen in the well binds to antibody and washed again; a second monoclonal antibody linked to the enzyme binds to the immobilized antigen, washed; and finally a substrate is added and converted by enzyme into colored product. The rate of color formation is proportional to the amount of antigen. FIGURE 4: WESTERN BLOT TECHNIQUE (1) The size (molecular weight) of the protein can be determined via western blot analysis. SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrolysis is a technique widely used to separate proteins according to their electrophoresis mobility (a function of length of polypeptide chain or molecular weight). For this procedure, an electric current is applied to the gel so that the separated proteins transfer through the gel and onto the membrane in the same pattern as they separate on the SDS-PAGE. All sites on the membrane, which do not contain blotted protein from the gel can then be non-specifically "blocked" so that antibody (serum) will not nonspecifically bind to them, causing a false positive result. Often the membrane is cut into strips to facilitate testing of a large number of samples for antibodies directed against the blotted protein (antigen). To detect the antigen blotted on the membrane, a primary antibody (serum) is added at an appropriate dilution and incubated with the membrane. If there are any antibodies present which are directed against one or more of the blotted antigens, those antibodies will bind to the protein(s) while other antibodies will be washed away at the end of the incubation. In order to detect the antibodies which have bound, anti-immunoglobulin antibodies coupled to a reporter group such as the enzyme alkaline phosphatase are added (e.g. Goat anti-human IgG- alkaline phosphatase). This anti-Ig-enzyme is commonly called a "second antibody" or "conjugate". Finally after excess second antibody is washed free of the blot, a substrate is added which will precipitate upon reaction with the conjugate resulting in a visible band where the primary antibody is bound to the protein. FIGURE 5: IMMUNOFLUORESCENT STAINING The above picture shows Actin cytoskeletons of human endothelial cells which glow green in this immunofluorescent micrograph. DNA stained with DAPI is blue in color. DAPI is a fluorescent stain widely used to visualize DNA.The actin filaments has linked them into a highly triangular structure resembling a geodesic dome in these human endothelial cells. Actin takes this form as cells, human endothelial in this case, grip a substrate and spread, forming discrete attachment points on the surface. The geodesic framework functions as a tensegrity structure, giving the cell its architectural integrity. A modification of the basic immunofluorescent staining and flow cytometric analysis protocol can be used for the simultaneous analysis of surface molecules and intracellular antigens at the single-cell level. In this protocol, cells are first activated in vitro, stained for surface antigens as in the surface antigen protocol, then fixed with paraformaldehyde to stabilize the cell membrane and permeabilized with the detergent saponin to allow anti-cytokine antibodies to stain intracellularly. In vitro stimulation of cells is usually required for detection of cytokines by flow cytometry since cytokine levels are typically too low in resting cells. Stimulation of cells with the appropriate reagent will depend on the cell type and the experimental conditions (2). FIGURE 6: QUANTUM DOT RAINBOW BEADS Quantum dots are used as organic dyes replacing traditional dyes in modern biological analysis. The usage of quantum dots for highly sensitive cellular imaging has seen major advances over the past decade. The improved Photostability of quantum dots allows the acquisition of many consecutive focal-plane images that can be reconstructed into highresolution images. QD are used for the localization of the protein in cells or tissues. RAINBOW BEADS are the Polymer beads embedded with quantum dots which fluorescence in five different colors as shown above. FIGURE 7: NMR SPECTROSCOPY (3) Magnetic Resonance Spectroscopy The electromagnetic radiation in this type of spectroscopy is in the region of microwaves (~0.01m) and radio waves (~1-10 m). Due to the relatively low frequency, thus low energy, these radiations will not excite electrons, but they are sufficient to change either electron spins (from -1/2 to +1/2) or nuclear spins. The spins are degenerate in the absence of magnetic field. Hence the absorption of radiation (i.e. resonance) can only be observed when samples are located in the magnetic field. FIGURE 8: NMR MACHINE FROM 60 TO 900 MHZ In a constant magnetic field of 11.74 Tesla all protons absorb near 500 MHz, but the precise position of each resonance peak of the particular proton in the molecule depends on the structure and environment. A more efficient NMR method uses Fourier transform spectroscopy. A sample is placed in a magnetic field and exposed to precisely timed and phased radiofrequency pulses (i.e. radio waves). The resulting timedependent changes in the sample's magnetization (i.e. the distribution of nuclear spins) is digitized and then analyzed mathematically by a Fourier transformation, resulting in a frequency distribution of peaks. NOE NOESY FIGURE 9: NUCLEAR OVERHAUSER EFFECT & NOESY (4) Nuclear Overhauser effect: The change of intensity of one NMR peak when another one is irradiated is called nuclear Overhauser effect (NOE). NOE is appreciable only for very close neighbors. Thus it is useful in determining which nuclei are near each other. NOE identifies pairs of protons that are in close proximity. (A) Protons 2 and 5 are only 4Å apart. (B) NOESY (nuclear Overhauser enhancement spectroscopy) shows that protons 2 and 5 are close. NOESY is the 2D equivalent of transient NOE experiment. The NOESY experiment is crucial for the determination of protein structure. It uses the dipolar interaction of spins (the nuclear Overhauser effect, NOE) for correlation of protons. The intensity of the NOE is in first approximation proportional to 1/r6, with r being the distance between the protons: The correlation between two protons depends on the distance between them, but normally a signal is only observed if their distance is smaller than 5 Å. The NOESY experiment correlates all protons which are close enough. It also correlates protons which are distant in the amino acid sequence but close in space due to tertiary structure. This is the most important information for the determination of protein structures (5). FIGURE 9: X-RAY CRYSTALLOGRAPHY (6) X-ray has a wavelength about 0.1 nm, about 4,000 to 8,000-fold shorter than the visible light. Thus it has much greater resolving power. The key equation for X-ray diffraction is Bragg condition. Bragg's law states that when X-rays hit an atom, they make the electric cloud move as does any electromagnetic wave. Diffraction occurs only when the wavelength is similar to the periodicity of the crystal. Protein structure at 0.1-0.2 nm resolution (atomic scale) can be obtained by analyzing the diffraction patterns. FIGURE 10: SCHEMATIC DIAGRAM ILLUSTRATING THE PRINCIPLE OF DIFFRACTION The oldest and most precise method of X-ray crystallography is single-crystal X-ray diffraction, in which a beam of X-rays strikes a single crystal, producing scattered beams. When they land on a piece of film or detector, these beams make a diffraction pattern of spots; the strengths and angles of these beams are recorded as the crystal is gradually rotated. Each spot is called a reflection, since it corresponds to the reflection of the X-rays from one set of evenly spaced planes within the crystal. FIGURE 11: X-RAY DIFFRACTION PATTERN OF DNA FIBER (7) The X-ray patterns given by DNA fibres show a pair of strong arcs along their vertical axis; Astbury realized that their position indicated a very regular periodicity of 3.4 along the axis of the fibre and that this figure was similar to the thickness of the DNA bases; he therefore suggested that the bases were stacked on top of each other "like a pile of pennies". He was quite right, but the well-known double helix structure had to await much better X-ray pictures (obtained by Wilkins, Franklin and colleagues at King's College, London) and the realization by Crick and Watson (in Cambridge) that the bases were in pairs, joining two backbones running in opposite directions. References: 1. http://www.viswagenbiotech.com/images/sds_western_blot_proprep.jpg 2. http://www.ebioscience.com/ebioscience/appls/FCI.htm 3. http://site.motifolio.com/images/The-basis-of-NMR-spectroscopy-6111167.png 4. http://chem.ch.huji.ac.il/nmr/hebrew/techniques/2d/assigndtbbgc_files/image010.gif 5. http://www.cryst.bbk.ac.uk/PPS2/projects/schirra/html/2dnmr.htm 6. http://www.proteopedia.org/wiki/index.php/X-ray_crystallography 7. http://www.xray.cz/xray/csca/kol2009/abst/klang_files/image008.jpg Chapter 19 – Gene Manipulation Outline: Mutagenesis In-vitro mutagenesis Cassette mutagenesis PCR site-directed mutagenesis Gene Knockout Gene Silencing Overview: FIGURE 1: MUTAGENESIS Mutagenesis is a process by which genetic information of an organism is changed in a stable manner, either naturally or experimentally by the use of chemicals or radiation. Mutagenesis works on three principles 1. Deletions 2. Substitutions 3. Insertions The plasmid with the original gene is cleaved (deletion) at the cleavage site by endonucleases followed by the purification of the original fragment. The new cassette ligate will then by inserted followed by purification of circular DNA, which leads to the production of plasmid with the new gene. FIGURE 2: GENERAL STRATEGY OF IN-VITRO MUTAGENESIS In-vitro mutagenesis is the production of either random or specific mutations in a piece of cloned DNA. Then the DNA is introduced in a cell to assess the results. (i) Plasmid DNA is mutagenized in vitro, then introduced into E. coli by transformation. Mutant clones can be isolated and tested individually or a library of mutant plasmids can be tested using genetic screening. (ii) Creating a mutation by manipulation of a restriction site: Plasmid DNA cut with EcoRI. Treatment with S1 and ligase produces a 4bp deletion mutant. Treatment with DNA polymerase and ligase produces a 4bp insertion mutant. EcoR1 recognizes GAATTC FIGURE 3: CASSETTE MUTAGENESIS Cassette mutagenesis is a process in which a wild type DNA fragment is being replaced by a DNA fragment of desired mutation. (i) Plasmid DNA (a wild type sequence) is cut by two restriction enzymes HindIII and EcoR1. A DNA fragment (cassette) containing the desired mutation is introduced through DNA ligase. The mutant DNA formed consists of the wild type DNA and the new mutated fragment, which is then transformed into E.Coli for a specific mutation. (ii) Cassette mutagenesis using doped oligonucleotides (each bottle is contaminated with small amount of other three precursors i.e. AGCT) to generate numerous mutants in a single experiment. A 30 base pair sequence with mutation at each position can be obtained. It is a very powerful technique which produces thousands of mutants (one or multiple) sequencing each one of them. FIGURE 4: PCR BASED SITE -DIRECTED MUTAGENESIS . TAC (FOR TYROSINE ) WILL PRODUCE TTC ( FOR PHENYLALANINE ) PCR (Polymerase Chain Reaction) is a site directed mutagenesis using double stranded DNA. As shown in the figure above, the parental plasmid consists of double stranded E.Coli plasmid. The two DNA strands are separated by denaturing the parental plasmid and mutagenic primers are added to produce new mutants. Polymerase chain reaction will then produce numerous daughter plasmids without methyl groups and a numerous parental plasmid (wild type) without mutant. Treatment with restriction enzyme Dpnl will cut the parental plasmid at the methylated position which will not transform into any mutation. The daughter plasmid will be the mutated plasmid which will be used to transform into E.Coli to produce desired mutation. FIGURE 5: GENE REPLACEMENT IN YEAST 1) A deletion or mutant gene his3 is inserted into a yeast vector HIS3. 2) Diploid yeast is transformed with exogenous DNA. Recombinant plasmid integrates with XV yeast chromosome and is fairly an automated process. 3) Homologous recombination occurs and a heterozygous yeast cell is generated 4) Intrachromosomal recombination and excision of URA3 will form original gene or produce a new mutant gene. The wild type gene can be replaced or remains the same. FIGURE 6: GENE KNOCKOUT IN YEAST Gene knockout is a genetic technique in which an organism is engineered to carry genes that are made inoperative. The method works in the following ways: 1) A deletion or mutant gene is inserted into a yeast vector (wild type). 2) Diploid yeast is transformed with exogenous DNA. 3) Homologous recombination occurs and a heterozygous yeast cell is generated. 4) The heterozygous yeast undergoes meiosis (Sporulation is the change from diploid to haploid). 5) The 4 haploid spores are tested for viability and other phenotypes. Out of the four haploid spores, two are viable and the other two are inviable. The viable spores are the wild type ones which will be able to survive and the inviable ones will be the knock out ones which will not be able to survive after disruption. Gene knockout in mice ES (embryonic stem cells) heterozygous for X, homozygous for marker (black coat) transplant into blastocoel cavity of 4.5 days embryos (homozygous for white coat). Early embryo implant into a pseudopregnant female. Transgenic mice Cell-type-specific gene knockouts in mice To determine the function of these genes, it is possible to replace an organism’s wild type gene with an inactive gene to create a “gene knockout” It is also possible to introduce additional genes (transgenes) to create a transgenic organism FIGURE 7: PROCEDURE FOR PRODUCING GENE-TARGETED KNOCKOUT MOUSE FIGURE 8: TRANSGENIC PLANTS (1) Transgenic plants are plants possessing a single or multiple genes transferred from a different species. It refers to plants created in lab using recombinant DNA technology. Tumor inducing plasmids can be used to introduce new genes into plant cells. The TDNA segment of Ti plasmids can be integrated into plant genome. FIGURE 9: FORMATION OF A CROWN GALL Crown gall tumors: Formation of a crown gall tumor in plant. Agro bacterium cells enter a wound in the plant, usually at the crown, or the junction of the root and stem. Bacteria have Ti plasmid containing a segment (red) that promotes tumor formation in infected plants, the Ti gene direct the formation of a crown gall, which nourishes the invading bacteria. FIGURE 10: RNAI (RNA INTERFERENCE) MODEL In this model, the proteins Dicer is shown "dicing" a long double-stranded (double stranded RNA) into small pieces called short-interfering RNAs (siRNAs). One strand of these siRNAs is used by the RNAi Silencing Complex to target and destroy a specific gene (mRNA). The efforts here are focused on studying how RNAi works in mammals such as mice and humans. Exactly how mammalian RNAi works is still a mystery. Many proteins are probably involved, and knowing their identities will help unravel the mystery of RNAi. FIGURE 11: RNAI SUPPRESSES GENES The short siRNA pieces unwind into single strand RNAs, which then combine with proteins to form a complex called RISC (RNA-Induced Silencing Complex). The RISC then captures a native mRNA molecule that complements the short siRNA sequence. If the pairing (native mRNA and siRNA piece) is essentially perfect, the native mRNA is cut into useless RNA fragments that aren't translated. If however, the pairing is less than perfect then the RISC complex binds to the mRNA and blocks ribosome movement along the native mRNA also halting translation. The net effect is NO PROTEIN IS MADE. FIGURE 12: EXAMPLE OF RNAI (NATURE 391: 806-11) The above figure shows the effects of mex-3 RNA interference on levels of the endogenous mRNA. Nomarski DIC micrographs show in situ hybridization of 4-cell stage embryos. (A) Negative control showing lack of staining in the absence of the hybridization probe. (B) Embryo from uninjected parent showing normal pattern of endogenous mex-3 RNA (purple staining). (C) Embryo from parent injected with purified mex-3 antisense RNA. These embryos (and the parent animals) retain mex-3 mRNA, although levels may be somewhat less than wild type. (D) Late 4-cell stage embryo from a parent injected with dsRNA corresponding to mex-3; no mex-3 RNA is detected. Gene Silencing • Gene silencing is a general term describing epigenetic processes of gene regulation • The term gene silencing is generally used to describe the "switching off" of a gene by a mechanism other than genetic modification. That is, a gene which would be expressed (turned on) under normal circumstances is switched off by machinery in the cell In two papers published in the online edition of the journal Nature Chemical Biology, researchers at UT Southwestern Medical Center say they have developed a technique that can control gene expression by turning them on or off at the DNA level. In doing so, the research team may have paved the way for the development of new drugs designed to treat many serious diseases. As diagnoses and gene research improves, scientists are consistently finding that nearly all diseases start at the genetic level. "Virtually every disease starts at the level of malfunctioning gene expression, or viral or bacterial gene expression," said Dr. David Corey, professor of pharmacology and biochemistry (2). References: 1. http://www.anselm.edu/homepage/jpitocch/genbio/plantplasmidtechn.JPG 2. http://www.scienceagogo.com/news/20050702030521data_trunc_sys.shtml Chapter 27 – Aging and Associated Diseases Outline: Theories behind aging and longevity Experimental models Diseases associated with aging Alzheimer’s disease Werner Syndrome Huntington’s disease Introduction: The study of aging is not what it used to be. Gerontology was a young science when Congress created the National Institute on Aging (NIA) in 1974 as part of the National Institutes of Health (NIH). At that time, theories of aging abounded, but data was scant. Gerontology lacked, or was just in the early stages of developing, ways to explore the fundamentals of the aging process. Now, nearly 30 years later, the science base has grown in depth, breadth, and detail. And, with this growth have come new insights into the processes and experience of aging. Where gerontologists once looked for a single, all-encompassing theory to explain aging - a single gene, for instance, or the decline of the immune system - they are now finding multiple processes, combining and interacting on many levels. Cells, proteins, tissues, and organ systems are all involved, and gerontologists are now able to discern many more of the mechanisms by which these components cause or react to aging (1). FIGURE 1: US POPULATION OVER THE PERIOD OF TIME FROM THE NATIONAL INSTITUTE ON AGING (2) Improved understanding of the mechanisms of longevity can be used to fight age-related diseases and disabilities and to ensure a healthy, active, and independent life well into a very old age. The dream of extending life span has shifted from legend to laboratory. As gerontologists explore the genes, cells, and organs involved in aging, they are uncovering more and more of the secrets of longevity. As a result, life extension may now be more than the stuff of myth and the retardation of disease and disability, realistic goals. Gerontologists talk about two kinds of life span: 1. Maximum life span (MLS), the greatest age reached by any member of a species. In humans this is 120 years. 2. Average life span (ALS), the average age reached by members of a population. Life expectancy, the number of years an individual can expect to live, is based on average life spans. Average life span and life expectancy in the United States have grown dramatically in this century, from about 47 years in 1900 to about 75 years in 1990. This advance is mostly due to improvements in sanitation, the discovery of antibiotics, and medical care. Now, as scientists make headway against chronic diseases like cancer and heart disease, some think it can be extended even further. Maximum human life span seems to be another matter. There is no evidence that it has changed for thousands of years despite fabled fountains of youth and biblical tales of long-lived patriarchs. However, very recently, the dream of extending life span has shifted from legend to laboratory. As gerontologists explore the genes, cells, and organs involved in aging, they are uncovering more and more of the secrets of longevity. As a result, life extension may now be more than the stuff of myth and the retardation of disease and disability, realistic goals. Theories on Aging: 1. Molecular Gene Theories Codon restriction: translation fidelity impaired due to inability to decode mRNA. Error catastrophe: fidelity declines with increase in abnormal protein. Somatic mutation: accumulation of DNA damage. Gene regulation: changes in gene expression regulating both aging and development. 2. Cellular Theories Free radical: mitochondria ROS, protein DNA damage. Wear and tear: 1. Glycoxidation theory (AGE, advanced glycation end-products) 2. Inflammation theory Senescence: accumulation of senescent cells due to replicative senescence (e.g. telomere shortening) or cellular senescence (e.g. stress) 3. System Theories Rate-of-living: assuming a fixed amount of metabolic potential for every living organism Neuroendocrine: alterations in neuroendocrine control of homeostasis Results in age related physiological changes Immunologic: decline in immune function with age results in increased disease 4. Evolutionary Theories Disposable soma: (Life span theory) soma is disposable following reproduction Antagonistic pleiotropy: genes beneficial at young age are deleterious at old age Mutation accumulation: mutations that affect health at old age Genetic connection and longevity genes: Drosophila melanogaster or fruit flies that routinely live for 70 or 80 days, nearly twice the average of Drosophila life span. Evolutionary biologist Michael Rose has bred the long-lived stocks by selecting and mating flies late in life. FIGURE 2: CALORIE RESTRICTION MODEL OF YEAST To begin the process of genetic selection, Rose first collected eggs lay by middle-aged fruit flies and let them hatch in isolation. The progeny were then transferred to a communal Plexiglas cage to eat, grow, and breed under conditions ideal for mating. Once they had reached advanced ages, the eggs laid by older females (and fertilized by older males) were again collected and removed to individual hatching vials. The cycle was repeated, but with succeeding generations, the day on which the eggs were collected was progressively postponed. After two years and 15 generations, the laboratory had stocks of Drosophila with longer life spans. Rose has bred longer life spans into fruit flies by selecting for other characteristics, such as ability to resist starvation, so the flies' long life spans are not necessarily tied to their fertility late in life. One possibility is that the anti-oxidant enzyme, superoxide dismutase (SOD), is involved. In another laboratory at Irvine, the late Robert Tyler discovered that the longer-lived flies had a somewhat different form of the SOD gene, which was more active than its counterpart in the flies with average life spans. This finding has given a boost to the hypothesis that anti-oxidant enzymes like SOD are linked to aging or longevity. LAG-1 is short for longevity assurance gene. FIGURE 3: XENOHORMESIS HYPOTHESIS In the Xenohormesis hypothesis, Sirtuins enzyme is evolved early in life's history to increase somatic maintenance and survival during times of adversity. The Xenohormesis hypothesis of Howitz and Sinclair proposes that primordial species synthesized polyphenolic molecules to stimulate sirtuins during times of stress. Plants have retained this ability. Survival pathways in fungi and animals have retained the ability to respond to plant stress signaling molecules because they provide useful prediction about the state of the environment and/or food supply. This ability would allow organisms to prepare for and survive adversity when they might otherwise perish (Howitz et al., 2003). FIGURE 4: THE ACTIVITY OF TELOMERE Telomeres cap the ends of chromosomes to protect them from inappropriate “repair” mechanism. Each time a cell divides, the telomeres shed a number of bases, so telomere length gives some indication of how many divisions the cell has already undergone and how many remain before it becomes senescent. Biochemistry and Aging: 1. ROS: Demolishing proteins and damaging nucleic acids, ROS are thought to be the villains in cells. The free radical theory of aging, proposed by Denham Harman at the University of Nebraska, holds that damage caused by ROS is responsible for many of the bodily changes that come with aging. Free radicals have been implicated not only in aging but also in degenerative disorders, including cancer, atherosclerosis, cataracts, and neurodegeneration. 2. AGEs: Glucose attaches to proteins, setting in motion a chain of chemical reactions that ends in the proteins binding together or crosslinking, a process called nonenzymatic glycosylation or glycation, thus altering their biological and structural roles. The process is slow but increases with time. Crosslinks, which have been termed advanced glycosylation end products (AGEs), seem to toughen tissues and may cause some of the deterioration associated with aging. AGEs have been linked to stiffening connective tissue (collagen), hardened arteries, clouded eyes, loss of nerve function, and less efficient kidneys. Diseases associated with Aging: 1. Alzheimer’s Disease Dr. Alois Alzheimer observed and autopsied brain of patient with dementia. Dementia=means out of one’s mind. Clinical diagnosis usually made if three symptoms are present: impairments in short-term memory, in other areas of cognition (e.g., language), and in social or daily functioning. It is the most common form of dementia, a general term for memory loss and other intellectual abilities serious enough to interfere with daily life. Alzheimer’s disease accounts for 50 to 80 percent of dementia cases. Alzheimer autopsied brain of AD patient has shrunken cerebral cortex, with damaged and dead cells. A normal, healthy female brain usually weighs between 1100 and 1400 grams. AD brains shrink below 1000, as the disease destroys brain tissue. Tightly packed ruts & grooves of cortex also change. FIGURE 5: ALZHEIMER’S BRAIN 2. Werner’s Syndrome Thirty years later, Oppenheimer and Kugel (USA) described a similar case of what they termed "Werner's syndrome", an inherited disease in which patients develop symptoms post-puberty that resemble rapid aging. Clinical features of Werner's syndome are now known to include: short stature, thin extremities, graying and loss of hair in their teens, cataracts in their 20's , a change of voice osteoporosis, bone deformities, wrinked, dry skin, diabetes, atherosclerosis, ankle ulcers, malignancies Werner syndrome is a premature aging disease that begins in adolescence or early adulthood and results in the appearance of old age by 30-40 years of age. Its physical characteristics may include short stature (common from childhood on) and other features usually developing during adulthood: wrinkled skin, baldness, cataracts, muscular atrophy and a tendency to diabetes mellitus, among others. The disorder is inherited and transmitted as an autosomal recessive trait. Cells from WS patients have a shorter lifespan in culture than do normal cells. The gene for Werner disease (WRN) was mapped to chromosome 8 and cloned by comparing its sequence to existing sequences in GenBank. However, it has yet to be shown to have real helicase activity (as a DNA unwinds important for DNA replication). The molecular role of WRN in Werner syndrome therefore remains to be proven; as does any role it might have in the aging process in general (3). FIGURE 6: WERNER’S SYNDROME (4) The picture shows Japanese-American Werner patient as a teenager, and at age 48 (Case #1 Epstein et al, 1966, Medicine 45:177). She had eight children, two of whom were also affected. At 48, she had hair loss and graying, thin extremities, chronic ulcerations of the ankles, atrophy of the skin and her right eye had been enucleated several years earlier due to acute glaucoma resulting from bilateral cataract extraction at the age of 27. She lived longer than many Werner patients and died as age 57. The gene responsible for Werner syndrome: The WRN gene encodes a DNA helicase of the RecQ family, in-vivo function unknown. In-vitro, WRN protein unwinds doublestranded DNA and has a high affinity for "G-DNA", a structure that may form at telomeres, ribosomal DNA (rDNA) and other GC-rich sequences. 3. Huntington’s Disease FIGURE 7: HUNTINGTON’ S DISEASE (5) It is a progressive neurodegenerative genetic disorder becoming noticeable in middle age The disease is caused by an autosomal dominant mutation on either of an individual's two copies of a gene called “Huntingtin” The Huntingtin gene normally provides the genetic information for a protein that is also called “huntingtin” The mutation of the Huntingtin gene codes for a different form of the protein, whose presence results in gradual damage to specific areas of the brain References: 1. http://www.nia.nih.gov/HealthInformation/Publications/AgingUndertheMicroscope/ 2. Looking for the fountain of youth, Scientists unlock the secrets of aging (Magazine) 3. http://www.ncbi.nlm.nih.gov/bookshelf/br.fcgi?book=gnd&part=wernersyndrome 4. http://monnatlab.gs.washington.edu/images/wrn_patient_pedigree.jpg 5. http://en.wikipedia.org/wiki/File:Neuron_with_mHtt_inclusion.jpg