Enzymes are macromolecular biological catalysts. Enzymes accelerate chemical reactions; the molecules upon which enzymes may act are called substrates and the enzyme converts the substrates into different molecules known as products. All metabolic processes in the cell need enzyme catalysis in order to occur at rates fast enough to sustain life. Metabolic pathways depend upon enzymes to catalyze individual steps; the study of enzymes is called enzymology and a new field of pseudoenzyme analysis has grown up, recognising that during evolution, some enzymes have lost the ability to carry out biological catalysis, reflected in their amino acid sequences and unusual'pseudocatalytic' properties. Enzymes are known to catalyze more than 5,000 biochemical reaction types. Most enzymes are proteins; the latter are called ribozymes. Enzymes' specificity comes from their unique three-dimensional structures. Like all catalysts, enzymes increase the reaction rate by lowering its activation energy; some enzymes can make their conversion of substrate to product occur many millions of times faster.
An extreme example is orotidine 5'-phosphate decarboxylase, which allows a reaction that would otherwise take millions of years to occur in milliseconds. Chemically, enzymes are like any catalyst and are not consumed in chemical reactions, nor do they alter the equilibrium of a reaction. Enzymes differ from most other catalysts by being much more specific. Enzyme activity can be affected by other molecules: inhibitors are molecules that decrease enzyme activity, activators are molecules that increase activity. Many therapeutic drugs and poisons are enzyme inhibitors. An enzyme's activity decreases markedly outside its optimal temperature and pH, many enzymes are denatured when exposed to excessive heat, losing their structure and catalytic properties; some enzymes are used commercially, in the synthesis of antibiotics. Some household products use enzymes to speed up chemical reactions: enzymes in biological washing powders break down protein, starch or fat stains on clothes, enzymes in meat tenderizer break down proteins into smaller molecules, making the meat easier to chew.
By the late 17th and early 18th centuries, the digestion of meat by stomach secretions and the conversion of starch to sugars by plant extracts and saliva were known but the mechanisms by which these occurred had not been identified. French chemist Anselme Payen was the first to discover an enzyme, diastase, in 1833. A few decades when studying the fermentation of sugar to alcohol by yeast, Louis Pasteur concluded that this fermentation was caused by a vital force contained within the yeast cells called "ferments", which were thought to function only within living organisms, he wrote that "alcoholic fermentation is an act correlated with the life and organization of the yeast cells, not with the death or putrefaction of the cells."In 1877, German physiologist Wilhelm Kühne first used the term enzyme, which comes from Greek ἔνζυμον, "leavened" or "in yeast", to describe this process. The word enzyme was used to refer to nonliving substances such as pepsin, the word ferment was used to refer to chemical activity produced by living organisms.
Eduard Buchner submitted his first paper on the study of yeast extracts in 1897. In a series of experiments at the University of Berlin, he found that sugar was fermented by yeast extracts when there were no living yeast cells in the mixture, he named the enzyme that brought about the fermentation of sucrose "zymase". In 1907, he received the Nobel Prize in Chemistry for "his discovery of cell-free fermentation". Following Buchner's example, enzymes are named according to the reaction they carry out: the suffix -ase is combined with the name of the substrate or to the type of reaction; the biochemical identity of enzymes was still unknown in the early 1900s. Many scientists observed that enzymatic activity was associated with proteins, but others argued that proteins were carriers for the true enzymes and that proteins per se were incapable of catalysis. In 1926, James B. Sumner crystallized it; the conclusion that pure proteins can be enzymes was definitively demonstrated by John Howard Northrop and Wendell Meredith Stanley, who worked on the digestive enzymes pepsin and chymotrypsin.
These three scientists were awarded the 1946 Nobel Prize in Chemistry. The discovery that enzymes could be crystallized allowed their structures to be solved by x-ray crystallography; this was first done for lysozyme, an enzyme found in tears and egg whites that digests the coating of some bacteria. This high-resolution structure of lysozyme marked the beginning of the field of structural biology and the effort to understand how enzymes work at an atomic level of detail. An enzyme's name is derived from its substrate or the chemical reaction it catalyzes, with the word ending in -ase. Examples are alcohol dehydrogenase and DNA polymerase. Different enzymes that catalyze the same chemical reaction are called isozymes; the International Union of Biochemistry and Molecular Biology have developed a nomenclature for enzymes, the EC numbers. The first number broadly classifies the enzyme based on its mechanism; the top-level classification is: EC 1, Oxidoreductases: catalyze oxidation/reducti
Actin is a family of globular multi-functional proteins that form microfilaments. It is found in all eukaryotic cells, where it may be present at a concentration of over 100 μM. An actin protein is the monomeric subunit of two types of filaments in cells: microfilaments, one of the three major components of the cytoskeleton, thin filaments, part of the contractile apparatus in muscle cells, it can be present as either a free monomer called G-actin or as part of a linear polymer microfilament called F-actin, both of which are essential for such important cellular functions as the mobility and contraction of cells during cell division. Actin participates in many important cellular processes, including muscle contraction, cell motility, cell division and cytokinesis and organelle movement, cell signaling, the establishment and maintenance of cell junctions and cell shape. Many of these processes are mediated by extensive and intimate interactions of actin with cellular membranes. In vertebrates, three main groups of actin isoforms, alpha and gamma have been identified.
The alpha actins, found in muscle tissues, are a major constituent of the contractile apparatus. The beta and gamma actins coexist in most cell types as components of the cytoskeleton, as mediators of internal cell motility, it is believed that the diverse range of structures formed by actin enabling it to fulfill such a large range of functions is regulated through the binding of tropomyosin along the filaments. A cell's ability to dynamically form microfilaments provides the scaffolding that allows it to remodel itself in response to its environment or to the organism's internal signals, for example, to increase cell membrane absorption or increase cell adhesion in order to form cell tissue. Other enzymes or organelles such as cilia can be anchored to this scaffolding in order to control the deformation of the external cell membrane, which allows endocytosis and cytokinesis, it can produce movement either by itself or with the help of molecular motors. Actin therefore contributes to processes such as the intracellular transport of vesicles and organelles as well as muscular contraction and cellular migration.
It therefore plays an important role in embryogenesis, the healing of wounds and the invasivity of cancer cells. The evolutionary origin of actin can be traced to prokaryotic cells. Actin homologs from prokaryotes and archaea polymerize into different helical or linear filaments consisting of one or multiple strands; however the in-strand contacts and nucleotide binding sites are preserved in prokaryotes and in archaea. Lastly, actin plays an important role in the control of gene expression. A large number of illnesses and diseases are caused by mutations in alleles of the genes that regulate the production of actin or of its associated proteins; the production of actin is key to the process of infection by some pathogenic microorganisms. Mutations in the different genes that regulate actin production in humans can cause muscular diseases, variations in the size and function of the heart as well as deafness; the make-up of the cytoskeleton is related to the pathogenicity of intracellular bacteria and viruses in the processes related to evading the actions of the immune system.
Actin was first observed experimentally in 1887 by W. D. Halliburton, who extracted a protein from muscle that'coagulated' preparations of myosin that he called "myosin-ferment". However, Halliburton was unable to further refine his findings, the discovery of actin is credited instead to Brunó Ferenc Straub, a young biochemist working in Albert Szent-Györgyi's laboratory at the Institute of Medical Chemistry at the University of Szeged, Hungary. In 1942, Straub developed a novel technique for extracting muscle protein that allowed him to isolate substantial amounts of pure actin. Straub's method is the same as that used in laboratories today. Szent-Gyorgyi had described the more viscous form of myosin produced by slow muscle extractions as'activated' myosin, since Straub's protein produced the activating effect, it was dubbed actin. Adding ATP to a mixture of both proteins causes a decrease in viscosity; the hostilities of World War II meant Szent-Gyorgyi and Straub were unable to publish the work in Western scientific journals.
Actin therefore only became well known in the West in 1945, when their paper was published as a supplement to the Acta Physiologica Scandinavica. Straub continued to work on actin, in 1950 reported that actin contains bound ATP and that, during polymerization of the protein into microfilaments, the nucleotide is hydrolyzed to ADP and inorganic phosphate. Straub suggested that the transformation of ATP-bound actin to ADP-bound actin played a role in muscular contraction. In fact, this is true only in smooth muscle, was not supported through experimentation until 2001; the amino acid sequencing of actin was completed by M. Elzinga and co-workers in 1973; the crystal structure of G-actin was solved in 1990 by colleagues. In the same year, a model for F-actin was proposed by Holmes and colleagues following experiments using co-crystallization with different proteins; the procedure of co-crystallization with different proteins was used during the following years, until in 2001 the isolated protein was crystallized along with ADP.
However, there is still no high-resolution X-ray structure of F-actin. The crystallization of F-actin was possible due to the use of a rhodamine conjugate that impedes polymerization by blocking the amino acid cys-374. Christine
Apoptosis is a form of programmed cell death that occurs in multicellular organisms. Biochemical events lead to death; these changes include blebbing, cell shrinkage, nuclear fragmentation, chromatin condensation, chromosomal DNA fragmentation, global mRNA decay. The average adult human loses between 70 billion cells each day due to apoptosis. For an average human child between the ages of 8 to 14 year old 20 to 30 billion cells die per day. In contrast to necrosis, a form of traumatic cell death that results from acute cellular injury, apoptosis is a regulated and controlled process that confers advantages during an organism's lifecycle. For example, the separation of fingers and toes in a developing human embryo occurs because cells between the digits undergo apoptosis. Unlike necrosis, apoptosis produces cell fragments called apoptotic bodies that phagocytic cells are able to engulf and remove before the contents of the cell can spill out onto surrounding cells and cause damage to them; because apoptosis cannot stop once it has begun, it is a regulated process.
Apoptosis can be initiated through one of two pathways. In the intrinsic pathway the cell kills itself because it senses cell stress, while in the extrinsic pathway the cell kills itself because of signals from other cells. Weak external signals may activate the intrinsic pathway of apoptosis. Both pathways induce cell death by activating caspases, which are proteases, or enzymes that degrade proteins; the two pathways both activate initiator caspases, which activate executioner caspases, which kill the cell by degrading proteins indiscriminately. Research on apoptosis has increased since the early 1990s. In addition to its importance as a biological phenomenon, defective apoptotic processes have been implicated in a wide variety of diseases. Excessive apoptosis causes atrophy, whereas an insufficient amount results in uncontrolled cell proliferation, such as cancer; some factors like Fas receptors and caspases promote apoptosis, while some members of the Bcl-2 family of proteins inhibit apoptosis.
German scientist Karl Vogt was first to describe the principle of apoptosis in 1842. In 1885, anatomist Walther Flemming delivered a more precise description of the process of programmed cell death. However, it was not until 1965. While studying tissues using electron microscopy, John Foxton Ross Kerr at the University of Queensland was able to distinguish apoptosis from traumatic cell death. Following the publication of a paper describing the phenomenon, Kerr was invited to join Alastair R. Currie, as well as Andrew Wyllie, Currie's graduate student, at University of Aberdeen. In 1972, the trio published a seminal article in the British Journal of Cancer. Kerr had used the term programmed cell necrosis, but in the article, the process of natural cell death was called apoptosis. Kerr and Currie credited James Cormack, a professor of Greek language at University of Aberdeen, with suggesting the term apoptosis. Kerr received the Paul Ehrlich and Ludwig Darmstaedter Prize on March 14, 2000, for his description of apoptosis.
He shared the prize with Boston biologist H. Robert Horvitz. For many years, neither "apoptosis" nor "programmed cell death" was a cited term. Two discoveries brought cell death from obscurity to a major field of research: identification of components of the cell death control and effector mechanisms, linkage of abnormalities in cell death to human disease, in particular cancer; the 2002 Nobel Prize in Medicine was awarded to Sydney Brenner and John E. Sulston for their work identifying genes that control apoptosis; the genes were identified by studies in the nematode C. elegans and homologues of these genes function in humans to regulate apoptosis. In Greek, apoptosis translates to the "falling off" of leaves from a tree. Cormack, professor of Greek language, reintroduced the term for medical use as it had a medical meaning for the Greeks over two thousand years before. Hippocrates used the term to mean "the falling off of the bones". Galen extended its meaning to "the dropping of the scabs".
Cormack was no doubt aware of this usage. Debate continues over the correct pronunciation, with opinion divided between a pronunciation with the second p silent and the second p pronounced, as in the original Greek. In English, the p of the Greek -pt- consonant cluster is silent at the beginning of a word, but articulated when used in combining forms preceded by a vowel, as in helicopter or the orders of insects: diptera, etc. In the original Kerr, Wyllie & Currie paper, there is a footnote regarding the pronunciation: "We are most grateful to Professor James Cormack of the Department of Greek, University of Aberdeen, for suggesting this term; the word "apoptosis" is used in Greek to describe the "dropping off" or "falling off" of petals from flowers, or leaves from trees. To show the derivation we propose that the stress should be on the penultimate syllable, the second half of the word being pronounced like "ptosis", which comes from the same root "to fall", is used to describe the drooping of the upper eyelid."
The initiation of apoptosis is regulated by activation mechanisms, because once apoptosis has begun, it leads to the death of the cell. The two best-understood activation mechanisms are the extrinsic pathway; the intrinsic pathway is activated by intracellular signals generated when cells are stressed and depends on the release of proteins from th
The western blot is a used analytical technique in molecular biology and other molecular biology disciplines to detect specific proteins in a sample of tissue homogenate or extract. In brief, the sample undergoes protein denaturation, followed by gel electrophoresis. A synthetic or animal-derived antibody is created that recognises and binds to a specific target protein; the electrophoresis membrane is washed in a solution containing the primary antibody, before excess antibody is washed off. A secondary antibody is added which binds to the primary antibody; the secondary antibody is visualised through various methods such as staining, immunofluorescence, radioactivity, allowing indirect detection of the specific target protein. Other related techniques include dot blot analysis, quantitative dot blot, immunohistochemistry, immunocytochemistry where antibodies are used to detect proteins in tissues and cells by immunostaining, enzyme-linked immunosorbent assay; the name western blot is a play on the eponymously-named Southern blot, a technique for DNA detection.
Detection of RNA is termed northern blot. The term "western blot" was given by W. Neal Burnette, although the method itself originated in the laboratory of Harry Towbin at the Friedrich Miescher Institute; the western blot is extensively used in biochemistry for the qualitative detection of single proteins and protein-modifications. It is used as a general method to identify the presence of a specific single protein within a complex mixture of proteins. A semi-quantitative estimation of a protein can be derived from the size and color intensity of a protein band on the blot membrane. In addition, applying a dilution series of a purified protein of known concentrations can be used to allow a more precise estimate of protein concentration; the western blot is used for verification of protein production after cloning. It is used in medical diagnostics, e. g. in the HIV test or BSE-Test. The confirmatory HIV test employs a western blot to detect anti-HIV antibody in a human serum sample. Proteins from known HIV-infected cells are blotted on a membrane as above.
The serum to be tested is applied in the primary antibody incubation step. The stained bands indicate the proteins to which the patient's serum contains antibody. A western blot is used as the definitive test for variant Creutzfeldt-Jakob Disease, a type of prion disease linked to the consumption of contaminated beef from cattle with Bovine spongiform encephalopathy. Another application is in the diagnosis of tularemia. An evaluation of the western blot’s ability to detect antibodies against F. tularensis revealed that its sensitivity is 100% and the specificity is 99.6%. Some forms of Lyme disease testing employ western blotting. A western blot can be used as a confirmatory test for Hepatitis B infection and HSV-2 infection. In veterinary medicine, a western blot is sometimes used to confirm FIV+ status in cats. Further applications of the western blot technique include its use by the World Anti-Doping Agency. Blood doping is the misuse of certain techniques and/or substances to increase one's red blood cell mass, which allows the body to transport more oxygen to muscles and therefore increase stamina and performance.
There are three known substances or methods used for blood doping, erythropoietin, synthetic oxygen carriers and blood transfusions. Each is prohibited under WADA's List of Prohibited Methods; the western blot technique was used during the 2014 FIFA World Cup in the anti-doping campaign for that event. In total, over 1000 samples were collected and analyzed by Reichel, et al. in the WADA accredited Laboratory of Lausanne, Switzerland. Recent research utilizing the western blot technique showed an improved detection of EPO in blood and urine based on novel Velum SAR precast horizontal gels optimized for routine analysis. With the adoption of the horizontal SAR-PAGE in combination with the precast film-supported Velum SAR gels the discriminatory capacity of micro-dose application of rEPO was enhanced; the western blot method is composed of a gel electrophoresis to separate native proteins by 3-D structure or denatured proteins by the length of the polypeptide, followed by an electrophoretic transfer onto a membrane and an immunostaining procedure to visualize a certain protein on the blot membrane.
SDS-PAGE is used for the denaturing electrophoretic separation of proteins. SDS is used as a buffer in order to give all proteins present a uniform negative charge, since proteins can be positively, negatively, or neutrally charged; this type of electrophoresis is known as SDS-PAGE. Prior to electrophoresis, protein samples are boiled to denature the proteins present; this ensures that proteins are separated based on size and prevents proteases from degrading samples. An alternative means of protein separation is the sonication using a Hielscher Ultrasonics VialTweeter placed in a 4 °C cold room using 10 cycles, each cycle consisting of 30 seconds sonication at 100% amplitude followed by a 30 second rest period. Following electrophoretic separation, the proteins are transferred to a membrane, where they are blocked with milk to prevent non-specific antibody binding, stained with antibodie
Bleb (cell biology)
In cell biology, a bleb is a bulge or protrusion of the plasma membrane of a cell, human bioparticulate or abscess with an internal environment similar to that of a simple cell, characterized by a spherical, bulky morphology. It is characterized by the decoupling of the cytoskeleton from the plasma membrane, degrading the internal structure of the cell, allowing the flexibility required to allow the cell to separate into individual bulges or pockets of the intercellular matrix. Most blebs are seen in apoptosis but are seen in other non-apoptotic functions. Blebbing, or zeiosis, is the formation of blebs. Bleb growth is driven by intracellular pressure generated in the cytoplasm when the actin cortex undergoes actomyosin contractions; the disruption of the membrane-actin cortex interactions are dependent on the activity of myosin-ATPaseBleb formation can be initiated in two ways: 1) through local rupture of the cortex or 2) through local detachment of the cortex from the plasma membrane. This generates a weak spot through which the cytoplasm flows, leading to the expansion of the bulge of membrane by increasing the surface area through tearing of the membrane from the cortex, during which time, actin levels decrease.
The cytoplasmic flow is driven by hydrostatic pressure inside the cell. Blebbing is one of the defined features of apoptosis. During apoptosis, the cell's cytoskeleton causes the membrane to bulge outward; these bulges may separate from the cell, taking a portion of cytoplasm with them, to become known as apoptotic blebs. Phagocytic cells consume these fragments and the components are recycled. Two types of blebs are recognized in apoptosis. Small surface blebs are formed. During stages, larger so-called dynamic blebs may appear, which may carry larger organelle fragments such as larger parts of the fragmented apoptotic cell nucleus. Blebbing has important functions in other cellular processes, including cell locomotion, cell division, physical or chemical stresses. Blebs have been seen in cultured cells in certain stages of the cell cycle; these blebs are used for cell locomotion in embryogenesis. The types of blebs vary including variations in bleb growth rates, size and actin content, it plays an important role in all five varieties of necrosis, a detrimental process.
However, cell organelles do not spread into necrotic blebs. In 2004, a chemical known as blebbistatin was shown to inhibit the formation of blebs; this agent was discovered in a screen for small molecule inhibitors of nonmuscle myosin IIA and was shown to lower the affinity of myosin with actin, thus altering the contractile forces that impinge on the cytoskeleton-membrane interface. Charras GT, Coughlin M, Mitchison TJ, Mahadevan L. "Life and times of a cellular bleb". Biophys. J. 94: 1836–53. Bibcode:2008BpJ....94.1836C. doi:10.1529/biophysj.107.113605. PMC 2242777. PMID 17921219. Charras GT, Hu CK, Coughlin M, Mitchison TJ. "Reassembly of contractile actin cortex in cell blebs". J. Cell Biol. 175: 477–90. Doi:10.1083/jcb.200602085. PMC 2064524. PMID 17088428. Dai J, Sheetz MP. "Membrane tether formation from blebbing cells". Biophys. J. 77: 3363–70. Bibcode:1999BpJ....77.3363D. Doi:10.1016/S0006-349577168-7. PMC 1300608. PMID 10585959. Drug Stops Motor Protein, Shines Light on Cell Division - FOCUS March 21, 2003.
Retrieved April 8, 2008. Hagmann J, Burger MM, Dagan D. "Regulation of plasma membrane blebbing by the cytoskeleton". J. Cell. Biochem. 73: 488–99. Doi:10.1002/1097-464473:4<488::AID-JCB7>3.0. CO. PMID 10733343. MBInfo - Bleb MBInfo - Bleb Assembly
Caenorhabditis elegans is a free-living, transparent nematode, about 1 mm in length, that lives in temperate soil environments. It is the type species of its genus; the name is rhabditis and Latin elegans. In 1900, Maupas named it Rhabditides elegans, Osche placed it in the subgenus Caenorhabditis in 1952, in 1955, Dougherty raised Caenorhabditis to the status of genus. C. Elegans lacks respiratory or circulatory systems. Most of these nematodes are hermaphrodites and a few are males. Males have specialised tails for mating. In 1963, Sydney Brenner proposed research into C. elegans in the area of neuronal development. In 1974, he began research into the molecular and developmental biology of C. elegans, which has since been extensively used as a model organism. It was the first multicellular organism to have its whole genome sequenced, as of 2012, is the only organism to have its connectome completed. C. elegans is unsegmented and bilaterally symmetrical. It has a cuticle, four main epidermal cords, a fluid-filled pseudocoelom.
It has some of the same organ systems as larger animals. About one in a thousand individuals is male and the rest are hermaphrodites; the basic anatomy of C. elegans includes a mouth, intestine and collagenous cuticle. Like all nematodes, they have neither a respiratory system; the four bands of muscles that run the length of the body are connected to a neural system that allows the muscles to move the animal's body only as dorsal bending or ventral bending, but not left or right, except for the head, where the four muscle quadrants are wired independently from one another. When a wave of dorsal/ventral muscle contractions proceeds from the back to the front of the animal, the animal is propelled backwards; when a wave of contractions is initiated at the front and proceeds posteriorly along the body, the animal is propelled forwards. Because of this dorsal/ventral bias in body bends, any normal living, moving individual tends to lie on either its left side or its right side when observed crossing a horizontal surface.
A set of ridges on the lateral sides of the body cuticle, the alae, is believed to give the animal added traction during these bending motions. In relation to lipid metabolism, C. elegans does not have any specialized adipose tissues, a pancreas, a liver, or blood to deliver nutrients compared to mammals. Neutral lipids are instead stored in the intestine and embryos; the epidermis corresponds to the mammalian adipocytes by being the main triglyceride depot. The pharynx is a muscular food pump in the head of C. elegans, triangular in cross-section. This transports it directly to the intestine. A set of "valve cells" connects the pharynx to the intestine, but how this valve operates is not understood. After digestion, the contents of the intestine are released via the rectum, as is the case with all other nematodes. No direct connection exists between the pharynx and the excretory canal, which functions in the release of liquid urine. Males have a single-lobed gonad, a vas deferens, a tail specialized for mating, which incorporates spicules.
Hermaphrodites have two ovaries and spermatheca, a single uterus. Numerous gut granules are present in the intestine of C. elegans, the functions of which are still not known, as are many other aspects of this nematode, despite the many years that it has been studied. These gut granules are found in all of the Rhabditida orders, they are similar to lysosomes in that they feature an acidic interior and the capacity for endocytosis, but they are larger, reinforcing the view of their being storage organelles. A remarkable feature of the granules is that when they are observed under ultraviolet light, they react by emitting an intense blue fluorescence. Another phenomenon seen is termed'death fluorescence'; as the worms die, a dramatic burst of blue fluorescence is emitted. This death fluorescence takes place in an anterior to posterior wave that moves along the intestine, is seen in both young and old worms, whether subjected to lethal injury or peacefully dying of old age. Many theories have been posited on the functions of the gut granules, with earlier ones being eliminated by findings.
They are thought to store zinc as one of their functions. Recent chemical analysis has identified the blue fluorescent material they contain as a glycosylated form of anthranilic acid; the need for the large amounts of AA the many gut granules contain is questioned. One possibility is. Another possibility is; this is seen a possible link to the melanin–containing melanosomes. The hermaphroditic worm is considered to be a specialized form of self-fertile female, as its soma is female; the hermaphroditic germline produces male gametes first, lays eggs through its uterus after internal fertilization. Hermaphrodites produce all their sperm in the L4 stage and produce only oocytes; the hermaphroditic gonad acts as an ovotestis with sperm cells being stored in the same area of the gonad as the oocytes until the first oocyte pushes the sperm into the spermatheca. The male can inseminate the hermaphrodite; the sperm of