Na+/K+-ATPase (also known as the Na+/K+ pump, sodium-potassium pump, or simply NAKA, for short) is an enzyme (EC 3.6.3.9) located in the plasma membrane (specifically an electrogenic transmembrane ATPase). It is found in the human cell and is common to all cellular life.
The Na+/K+-ATPase helps maintain resting potential, avail transport and regulate cellular volume.
Resting potential
In order to maintain the cell potential, cells must keep a low concentration of sodium ions and high levels of potassium ions within the cell (intracellular). Outside of the cells (extracellular), there are high concentrations of sodium and low concentrations of potassium, so diffusion occurs through ion channels in the plasma membrane. In order to keep the appropriate concentrations, the sodium-potassium pump pumps sodium out and potassium in through active transport. As the plasma membrane is far less permeable to sodium than it is to potassium ions, an electric potential (negative intracellularly) is the eventual result.
Transport
Export of sodium from the cell provides the driving force for several facilitated membrane transport proteins, which import glucose, amino acids and other nutrients into the cell. Translocation of sodium from one side of an epithelium to the other side creates an osmotic gradient that drives the absorption of water.[citation needed]
Another important task of the Na+-K+ pump is to provide a Na+ gradient that is used by certain carrier processes. In the gut, for example, sodium is transported out of the resorbing cell on the blood side via the Na+-K+ pump, whereas, on the resorbing side, the Na+-Glucose symporter uses the created Na+ gradient as a source of energy to import both Na+ and Glucose, which is far more efficient than simple diffusion. Similar processes are located in the renal tubular system.
Mechanism
The pump, with bound ATP, binds 3 intracellular Na+ ions.
ATP is hydrolyzed, leading to phosphorylation of the pump at a highly conserved aspartate residue and subsequent release of ADP.[citation needed]
A conformational change in the pump exposes the Na+ ions to the outside. The phosphorylated form of the pump has a low affinity for Na+ ions, so they are released.[citation needed]
The pump binds 2 extracellular K+ ions. This causes the dephosphorylation of the pump, reverting it to its previous conformational state, transporting the K+ ions into the cell.[citation needed]
The unphosphorylated form of the pump has a higher affinity for Na+ ions than K+ ions, so the two bound K+ ions are released. ATP binds, and the process starts again.
RegulationEndogenous The Na+/K+-ATPase is thought to be downregulated by cAMP.[1] Thus, substances causing an increase in cAMP downregulates Na+/K+-ATPase. These include the ligands of the Gs-coupled GPCRs.
In contrast, substances causing a decrease in cAMP upregulates Na+/K+-ATPase. These include the ligands of the Gi-coupled GPCRs. It should be noted that cAMP also acts as a second messenger causing an increase in protein abundance of Na-K-ATPase.
Exogenous The Na+-K+-ATPase can be pharmacologically modified by administrating drugs exogenously.
For instance, Na+-K+-ATPase found in the membrane of heart cells is an important target of cardiac glycosides (for example digoxin and ouabain), inotropic drugs used to improve heart performance by increasing its force of contraction.
Contraction of any muscle is dependent on a 100- to 10,000-times higher-than-resting intracellular Ca2+ concentration, which, as soon as it is put back again on its normal level by a carrier enzyme in the plasma membrane, and a calcium pump in sarcoplasmic reticulum, muscle relaxes.
Since this carrier enzyme (Na+-Ca2+ translocator) uses the Na gradient generated by the Na+-K+ pump to remove Ca2+ from the intracellular space, slowing down the Na+-K+ pump results in a permanently-higher Ca2+ level in the muscle, which will eventually lead to stronger contractions.
Northern blot is a technique used in molecular biology research to study gene expression. It takes its name from its similarity to the Southern blot technique, named for biologist Edwin Southern. The major difference is that RNA, rather than DNA, is analyzed in the northern blot. Both techniques use electrophoresis and detection with a hybridization probe. The northern blot technique was developed in 1977 by James Alwine, David Kemp, and George Stark at Stanford University.
A northern blot is very similar to a Southern blot except that it is RNA rather than DNA which is extracted, run on a gel and transferred to a filter membrane. There are 3 types of RNA: tRNA (transfer RNA - active in assembly of polypeptide chains), rRNA (ribosomal RNA - part of the structure of ribosomes) and mRNA (messenger RNA - the product of DNA transcription and used for translation of a gene into a protein). It is mRNA which is isolated and hybridized in northern blots.
mRNA is extracted from the cells grown in galactose and cells grown in glucodse and purified.
The mRNA is loaded onto a gel for electrophoresis. Lane 1 has gal mRNa Lane 2 has the Glucose mRNA.
An electric current is passed through the gel and the RNA moves away from the negative electrode. The distance moved depends on the size of the RNA fragment. Since genes are different sizes the size of the mRNAs varies also. This results in a smear on a gel. Standards are used to quantitate the size. The RNA can be visualized by staining first with a fluorescent dye and then lighting with UV.
RNA is single-stranded, so it can be transferred out of the gel and onto a membrane without any further treatment. The transfer can be done electrically or by capillary action with a high salt solution.
A GAL DNA probe is incubated with the blot.the single stranded GAL DNA probe binds with immobilized GAL mRNA The blot is washed to remove non-specifically bount probe and then a development step allows visualization of the probe that is bound.
Hereditary nonpolyposis colorectal cancer (HNPCC), also known as Lynch syndrome, is characterised by a risk of colorectal cancer and other cancers of the endometrium, ovary, stomach, small intestine, hepatobiliary tract, upper urinary tract, brain, and skin. HNPCC is subdivided into Lynch Syndrome I (familial colon cancer) and Lynch Syndrome II (other cancer of the gastrointestinal system or the reproductive system). The increased risk for these cancers is due to inherited mutations that degrade the self-repair capability of DNA.
Colon Cancer
Genes are related to Lynch syndrome
Variations in the MLH1, MSH2, MSH6, and PMS2 genes increase the risk of developing Lynch syndrome. All of these genes are involved in the repair of mistakes made when DNA is copied (DNA replication) in preparation for cell division. Mutations in any of these genes prevent the proper repair of DNA replication mistakes. As the abnormal cells continue to divide, the accumulated mistakes can lead to uncontrolled cell growth and possibly cancer. Although mutations in these genes predispose individuals to cancer, not all people who carry these mutations develop cancerous tumors.
Lynch syndrome cancer risk is inherited in an autosomal dominant pattern, which means one inherited copy of the altered gene in each cell is sufficient to increase cancer risk. It is important to note that people inherit an increased risk of cancer, not the disease itself. Not all people who inherit mutations in these genes will develop cancer.
Biomarker discovery is the process by which biomarkers are discovered. It is a medical term.
Many commonly used blood tests in medicine are biomarkers. The way that these tests have been found can be seen as biomarker discovery. However, their identification has mostly been a one-at-a time approach. Many of these well-known tests have been identified based on clear biological insight, from physiology or biochemistry. This means that only a few markers at a time have been considered. One example of this way of biomarker discovery is the use of injections of inulin for measuring kidney function. From this, one discovered a naturally occurring molecule, creatinine, that enabled the same measurements to be made easily without injections. This can be seen as a serial process.
The recent interest in biomarker discovery is because new molecular biologic techniques promise to find relevant markers rapidly, without detailed insight into mechanisms of disease. By screening many possible biomolecules at a time, a parallel approach can be tried. Genomics and proteomics are some technologies that are used in this process. Significant technical difficulties remain.
There is considerable interest in biomarker discovery from the pharmaceutical industry. Blood test or other biomarkers could serve as intermediate markers of disease in clinical trials, and also be possible drug targets.
Enzymes serve a wide variety of functions inside living organisms. They are indispensable for signal transduction and cell regulation, often via kinases and phosphatases. They also generate movement, with myosin hydrolysing ATP to generate muscle contraction and also moving cargo around the cell as part of the cytoskeleton. Other ATPases in the cell membrane are ion pumps involved in active transport. Enzymes are also involved in more exotic functions, such as luciferase generating light in fireflies. Viruses can also contain enzymes for infecting cells, such as the HIV integrase and reverse transcriptase, or for viral release from cells, like the influenza virus neuraminidase.
An important function of enzymes is in the digestive systems of animals. Enzymes such as amylases and proteases break down large molecules (starch or proteins, respectively) into smaller ones, so they can be absorbed by the intestines. Starch molecules, for example, are too large to be absorbed from the intestine, but enzymes hydrolyse the starch chains into smaller molecules such as maltose and eventually glucose, which can then be absorbed. Different enzymes digest different food substances. In ruminants which have a herbivorous diets, microorganisms in the gut produce another enzyme, cellulase to break down the cellulose cell walls of plant fiber.
Several enzymes can work together in a specific order, creating metabolic pathways. In a metabolic pathway, one enzyme takes the product of another enzyme as a substrate. After the catalytic reaction, the product is then passed on to another enzyme. Sometimes more than one enzyme can catalyze the same reaction in parallel, this can allow more complex regulation: with for example a low constant activity being provided by one enzyme but an inducible high activity from a second enzyme.
Enzymes determine what steps occur in these pathways. Without enzymes, metabolism would neither progress through the same steps, nor be fast enough to serve the needs of the cell. Indeed, a metabolic pathway such as glycolysis could not exist independently of enzymes. Glucose, for example, can react directly with ATP to become phosphorylated at one or more of its carbons. In the absence of enzymes, this occurs so slowly as to be insignificant. However, if hexokinase is added, these slow reactions continue to take place except that phosphorylation at carbon 6 occurs so rapidly that if the mixture is tested a short time later, glucose-6-phosphate is found to be the only significant product. Consequently, the network of metabolic pathways within each cell depends on the set of functional enzymes that are present.
Pharmacogenomics is the branch of pharmacology which deals with the influence of genetic variation on drug response in patients by correlating gene expression or single-nucleotide polymorphisms with a drug's efficacy or toxicity. By doing so, pharmacogenomics aims to develop rational means to optimise drug therapy, with respect to the patients' genotype, to ensure maximum efficacy with minimal adverse effects. Such approaches promise the advent of "personalized medicine"; in which drugs and drug combinations are optimised for each individual's unique genetic makeup.
In this lecture, Ogechi N. Ikediobi, UCSF School of Pharmacy, discusses pharmacogenomics (how an individual's genetic inheritance affects the body's response to drugs) and cancer.
According to the World Health Organization cancer is the second leading cause of death in developed countries.but is also among the leading causes of death and develop among adults in developing countries .as well as edit increasing proportion of elderly people in the world and that is estimated to result in 16 million new cases of cancer a by year 2020 so statistics paint a picture that cancer is going be a socio-economic and Health burden for not only developed countries but also for developing nations
cancer can be classifed into two parts
Inherited (germline)-5-10% of cancers
Acquired (somatic) 90-95 % cancers
Inherited cancer usually manifest themselves at earlier age whereas most cancers manifest the during living .
cancer is largely a disease of aging because most cancers at the 90% of them are due to acquired mutation of the cells which is correlated with living longer.
cancer occurs in a multistage process and this theory is based on the work of several scientists accumulated over many years and the theory is cancer arises from the transformational of the genetic material of the normal cell that transforms cell then acquires subsequent successive mutations in its Genome then lead to uncontrolled growth is define as tumor or cancers
Transfer RNA (abbreviated tRNA) is a small RNA molecule (usually about 74-95 nucleotides) that transfers a specific active amino acid to a growing polypeptide chain at the ribosomal site of protein synthesis during translation. It has a 3' terminal site for amino acid attachment. This covalent linkage is catalyzed by an aminoacyl tRNA synthetase. It also contains a three base region called the anticodon that can base pair to the corresponding three base codon region on mRNA. Each type of tRNA molecule can be attached to only one type of amino acid, but because the genetic code contains multiple codons that specify the same amino acid, tRNA molecules bearing different anticodons may also carry the same amino acid.
Structure
tRNA has primary structure, secondary structure (usually visualized as the cloverleaf structure), and tertiary structure (all tRNAs have a similar L-shaped 3D structure that allows them to fit into the P and A sites of the ribosome).
1. The 5'-terminal phosphate group.
2. The acceptor stem is a 7-bp stem made by the base pairing of the 5'-terminal nucleotide with the 3'-terminal nucleotide (which contains the CCA 3'-terminal group used to attach the amino acid). The acceptor stem may contain non-Watson-Crick base pairs.
3. The CCA tail is a CCA sequence at the 3' end of the tRNA molecule. This sequence is important for the recognition of tRNA by enzymes critical in translation. In prokaryotes, the CCA sequence is transcribed. In eukaryotes, the CCA sequence is added during processing and therefore does not appear in the tRNA gene.
4. The D arm is a 4 bp stem ending in a loop that often contains dihydrouridine.
5. The anticodon arm is a 5-bp stem whose loop contains the anticodon. It also contains a Y that stands for a modified purine nucleotide.
6. The T arm is a 5 bp stem containing the sequence TΨC where Ψ is a pseudouridine.
7. Bases that have been modified, especially by methylation, occur in several positions outside the anticodon. The first anticodon base is sometimes modified to inosine (derived from adenine) or pseudouridine (derived from uracil).
Anticodon
An anticodon is a unit made up of three nucleotides that correspond to the three bases of the codon on the mRNA. Each tRNA contains a specific anticodon triplet sequence that can base-pair to one or more codons for an amino acid. For example, one codon for lysine is AAA; the anticodon of a lysine tRNA might be UUU. Some anticodons can pair with more than one codon due to a phenomenon known as wobble base pairing. Frequently, the first nucleotide of the anticodon is one of two not found on mRNA: inosine and pseudouridine, which can hydrogen bond to more than one base in the corresponding codon position. In the genetic code, it is common for a single amino acid to be specified by all four third-position possibilities; for example, the amino acid glycine is coded for by the codon sequences GGU, GGC, GGA, and GGG.
To provide a one-to-one correspondence between tRNA molecules and codons that specify amino acids, 61 types of tRNA molecules would be required per cell. However, many cells contain fewer than 61 types of tRNAs because the wobble base is capable of binding to several, though not necessarily all, of the codons that specify a particular amino acid.
Aminoacylation
Aminoacylation is the process of adding an aminoacyl group to a compound. It produces tRNA molecules with their CCA 3' ends covalently linked to an amino acid.
Each tRNA is aminoacylated (or charged) with a specific amino acid by an aminoacyl tRNA synthetase. There is normally a single aminoacyl tRNA synthetase for each amino acid, despite the fact that there can be more than one tRNA, and more than one anticodon, for an amino acid. Recognition of the appropriate tRNA by the synthetases is not mediated solely by the anticodon, and the acceptor stem often plays a prominent role.
Reaction:
1. amino acid + ATP → aminoacyl-AMP + PPi
2. aminoacyl-AMP + tRNA → aminoacyl-tRNA + AMP
Sometimes, certain organisms can have one or more aminoacyl tRNA synthetases missing. This leads to mischarging of the tRNA by a chemically related amino acid. The correct amino acid is made by enzymes that modify the mischarged amino acid to the correct one.
For example, Helicobacter pylori has glutaminyl tRNA synthetase missing. Thus, glutamate tRNA synthetase mischarges tRNA-glutamine(tRNA-Gln) with glutamate. An amidotransferase then converts the acid side chain of the glutamate to the amide, forming the correctly charged gln-tRNA-Gln.
Binding to ribosome
The ribosome has three binding sites for tRNA molecules: the A (aminoacyl), P (peptidyl), and E (exit) sites. During translation the A site binds an incoming aminoacyl-tRNA as directed by the codon currently occupying this site. This codon specifies the next amino acid to be added to the growing peptide chain. The A site only works after the first aminoacyl-tRNA has attached to the P site. The P-site codon is occupied by peptidyl-tRNA that is a tRNA with multiple amino acids attached as a long chain. The P site is actually the first to bind to aminoacyl tRNA. This tRNA in the P site carries the chain of amino acids that has already been synthesized. The E site is occupied by the empty tRNA as it is about to exit the ribosome.
Atherosclerosis is a disease affecting arterial blood vessels. It is a chronic inflammatory response in the walls of arteries, in large part due to the accumulation of macrophage white blood cells and promoted by low density (especially small particle) lipoproteins (plasma proteins that carry cholesterol and triglycerides) without adequate removal of fats and cholesterol from the macrophages by functional high density lipoproteins(HDL), (see apoA-1 Milano). It is commonly referred to as a "hardening" or "furring" of the arteries. It is caused by the formation of multiple plaques within the arteries.
The atheromatous plaque is divided into three distinct components:
1. The atheroma ("lump of porridge", from Athera, porridge in Greek,), which is the nodular accumulation of a soft, flaky, yellowish material at the center of large plaques, composed of macrophages nearest the lumen of the artery
2. Underlying areas of cholesterol crystals
3. Calcification at the outer base of older/more advanced lesions.
The following terms are similar, yet distinct, in both spelling and meaning, and can be easily confused: arteriosclerosis, arteriolosclerosis, and atherosclerosis. Arteriosclerosis is a general term describing any hardening (and loss of elasticity) of medium or large arteries (from the Greek Arterio, meaning artery, and sclerosis, meaning hardening), arteriolosclerosis is any hardening (and loss of elasticity) of arterioles (small arteries), atherosclerosis is a hardening of an artery specifically due to an atheromatous plaque. Therefore, atherosclerosis is a form of arteriosclerosis.
Atherosclerosis causes two main problems. First, the atheromatous plaques, though long compensated for by artery enlargement (see IMT), eventually lead to plaque ruptures and stenosis (narrowing) of the artery and, therefore, an insufficient blood supply to the organ it feeds. If the compensating artery enlargement process is excessive, then a net aneurysm results.
These complications are chronic, slowly progressing and cumulative. Most commonly, soft plaque suddenly ruptures (see vulnerable plaque), causing the formation of a thrombus that will rapidly slow or stop blood flow, leading to death of the tissues fed by the artery in approximately 5 minutes. This catastrophic event is called an infarction. One of the most common recognized scenarios is called coronary thrombosis of a coronary artery, causing myocardial infarction (a heart attack). Another common scenario in very advanced disease is claudication from insufficient blood supply to the legs, typically due to a combination of both stenosis and aneurysmal segments narrowed with clots. Since atherosclerosis is a body-wide process, similar events occur also in the arteries to the brain, intestines, kidneys, legs, etc.
Causes
Atherosclerosis develops from low-density lipoprotein cholesterol (LDL), colloquially called "bad cholesterol". Many believe that, when this lipoprotein gets through the wall of an artery, oxygen free radicals react with it to form oxidized-LDL. The body's immune system responds by sending specialised white blood cells (macrophages and T-lymphocytes) to absorb the oxidised-LDL. Unfortunately, these white blood cells are not able to process the oxidised-LDL, and ultimately grow then rupture, depositing a greater amount of oxidised cholesterol into the artery wall. This triggers more white blood cells, continuing the cycle.
Eventually, the artery becomes inflamed. The cholesterol plaque causes the muscle cells to enlarge and form a hard cover over the affected area. This hard cover is what causes a narrowing of the artery, reduces the blood flow and increases blood pressure.
Some researchers believe that atherosclerosis may be caused by an infection of the vascular smooth muscle cells. Chickens, for example, develop atherosclerosis when infected with the Marek's disease herpesvirus. Herpesvirus infection of arterial smooth muscle cells has been shown to cause cholesteryl ester (CE) accumulation. ester accumulation is associated with atherosclerosis.
Yi Cui is an assistant professor in the Materials Science and Engineering Department at Stanford University. He is a recipient of the Technology Review World Top 100 Young Innovator Award. He received his PhD degree from Harvard University working with Prof. Charles Lieber. He received his B.S. degree from Univ of Science and Technology of China. ABSTRACT Nanowires and nanocrystals represent important nanomaterials with one-dimensional and zero-dimensional morphology, respectively. Here I will give an overview on the research about how these nanomaterials impact the critical applications in faster transistors, smaller nonvolatile memory devices, efficient solar energy conversion, high-energy battery and nanobiotechnology.
Adenovirus infects all the cells ,In normal cell to DNA from the virus moves towards to the nucleus which has a antidode p53 tumor suppressor gene, which prevents the viral DNA from replication. If same virus infects the cancer cell ,becuase the mutant P53 gene,it is unable to stop viral DNA replication.so viral DNA continues to multiply and eventually burst the cell release the viruses into the environment. it will infect both normal and cancer cells ,But the normal cells are kind of immune to the virus whereas the cancer cells will continue to be killed by Virus
Tumor cells have developed mechanism to avoid dectection by immune system,when Left undetected Tumor cells will continue to grow and progress.However the posibilty exisit to exploit inherit characteristic of tumor cells in fight against them
This may used to target tumor cells. The potential lies on harnessing the power of the bodies own immune response, when activated Antigen Presenting cells also known as APC are in turn responsible for T-cell activation. Scientist are currently studying the different ways to activating APC’s .so on exposure to particular antigen, APC will take-up and process that antigen in preparation for T-cell activation. The antigen peptides are presented on APC surface. The APC starts to mature and travels to lymph system, here it completes its maturation and encounters T-cells. It selects the T-cells that posses antigen receptors matching the antigen peptides on APC surface. The APC then presents the antigen to T-cells. Antigen presentation along with other signals results in T-cell activation .When Activated the T-cells multiplies and can now seek out and recognize that particular antigen as result activated T-cells attack target cells. Activated T-cells may be immune systems most potent defense against cancer.
Macrophages are cells within the tissues that originate from specific white blood cells called monocytes. Monocytes and macrophages are phagocytes, acting in both non-specific defense (or innate immunity) as well as specific defence (or cell-mediated immunity) of vertebrate animals. Their role is to phagocytose (engulf and then digest) cellular debris and pathogens either as stationary or mobile cells, and to stimulate lymphocytes and other immune cells to respond to the pathogen.
Macrophages are versatile cells that play many roles. As scavengers, they rid the body of worn-out cells and other debris. They are foremost among the cells that "present" antigen; a crucial role in initiating an immune response. As secretory cells, monocytes and macrophages are vital to the regulation of immune responses and the development of inflammation; they churn out an amazing array of powerful chemical substances (monokines) including enzymes, complement proteins, and regulatory factors such as interleukin-1. At the same time, they carry receptors for lymphokines that allow them to be "activated" into single-minded pursuit of microbes and tumour cells.
After digesting a pathogen, a macrophage will present the antigen (a molecule, most often a protein found on the surface of the pathogen, used by the immune system for identification) of the pathogen to a corresponding helper T cell. The presentation is done by integrating it into the cell membrane and displaying it attached to a MHC class II molecule, indicating to other white blood cells that the macrophage is not a pathogen, despite having antigens on its surface.
Eventually the antigen presentation results in the production of antibodies that attach to the antigens of pathogens, making them easier for macrophages to adhere to with their cell membrane and phagocytose. In some cases, pathogens are very resistant to adhesion by the macrophages. Coating an antigen with antibodies could be compared to coating something with Velcro to make it stick to fuzzy surfaces.
The antigen presentation on the surface of infected macrophages (in the context of MHC class II) in a lymph node stimulates TH1 (type 1 helper T cells) to proliferate (mainly due to IL-12 secretion from the macrophage). When a B-cell in the lymph node recognizes the same unprocessed surface antigen on the bacterium with its surface bound antibody, the antigen is endocytosed and processed. The processed antigen is then presented in MHCII on the surface of the B-cell. TH1 receptor that has proliferated recognizes the antigen-MHCII complex (with co-stimulatory factors- CD40 and CD40L) and causes the B-cell to produce antibodies that help opsonisation of the antigen so that the bacteria can be better cleared by phagocytes.
Macrophages provide yet another line of defense against tumor cells and body cells infected with fungus or parasites. Once a T cell has recognized its particular antigen on the surface of an aberrant cell, the T cell becomes an activated effector cell, releasing chemical mediators known as lymphokines that stimulate macrophages into a more aggressive form. These activated or angry macrophages, can then engulf and digest affected cells much more readily. The angry macrophage does not generate a response specific for an antigen, but attacks the cells present in the local area in which it was activated.
Describes in general terms the concepts of high-throughput protein expression coupled with immobilizations in functionalized nanoporous materials to carry out multiple kinds of diverse reactions. The animations also illustrate that immobilized enzymes potentially can refold inactive proteins.
My general research interests lie in elucidating the inner-workings of proteins, enzymes and biological motors, using instrumentation that combines optical tweezers, single molecule fluorescence and pulsed spectroscopy. This research will be directed towards developing a molecular level description of the motions associated with structural, mechanical, dynamic and energetic changes of these biological systems. Through these emerging technologies, significant advances in our ability to measure receptor/ligand interactions, the inner-workings of biological motors and interactions between multi-unit protein complexes will be achieved, thus providing a deeper understanding of these biological systems.
Lamellar keratoplasty involves replacement of your damaged or diseased anterior corneal stroma (middle layer of your cornea) and Bowman's membrane (second layer of your cornea) with donor material. Most of the bottom three layers of your cornea can be preserved. The donor corneal disc becomes repopulated with host cells, and the recipient epithelium usually covers the anterior corneal surface. This procedure is technically more difficult than penetrating keratoplasty. Lamellar keratoplasty has the advantage of being primarily outside the eye, making it a procedure that preserves your endothelium. The risk of rejection becomes less of an issue. The risks of wound leaks or flat anterior chambers associated with an intraocular procedure may be eliminated. Microsurgical techniques have vastly improved the technique of lamellar keratoplasty, but they have also substantially improved the results with penetrating keratoplasty. The use of conjunctival flaps and therapeutic soft contact lenses has reduced the indications for lamellar keratoplasty.
Meniscus is a crescent-shaped fibrocartilaginous structure present in the knee, acromioclavicular, sternoclavicular, and temporomandibular joints that, in contrast to articular disks, only partly divides a joint cavity. A small meniscus also occurs in the radio-carpal joint.
It usually refers to either of two specific parts of cartilage of the knee: The lateral and medial menisci. Both are cartilaginous tissues that provide structural integrity to the knee when it undergoes tension and torsion. The menisci are also known as 'semi-lunar' cartilages - referring to their half-moon "C" shape - a term which has been largely dropped by the medical profession, but which led to the menisci being called knee 'cartilages' by the lay public.
Menisci can be torn during innocuous activities such as walking or squatting. They can also be torn due to traumatic forces encountered in sports. The trauma mechanism is most often a twisting movement while the knee is bent. In older adults, the meniscus can be damaged following prolonged 'wear and tear'; this is called a degenerative tear.
Tears can lead to pain and/or swelling of the knee joint. Especially acute injuries (typically in younger, more active patients) can lead to displaced tears which can cause mechanical symptoms such as clicking, catching, or locking during motion of the knee joint. The joint will be in pain when in use, but when there is no load, the pain goes away.
Cancer (medical term: malignant neoplasm) is a class of diseases in which a group of cells display the traits of uncontrolled growth (growth and division beyond the normal limits), invasion (intrusion on and destruction of adjacent tissues), and sometimes metastasis (spread to other locations in the body via lymph or blood). These three malignant properties of cancers differentiate them from benign tumors, which are self-limited, do not invade or metastasize. Most cancers form a tumor but some, like leukemia, do not.
Cancer may affect people at all ages, even fetuses, but risk for the more common varieties tends to increase with age. Cancer causes about 13% of all deaths. According to the American Cancer Society, 7.6 million people died from cancer in the world during 2007. Cancers can affect other animals besides humans, and plants, too.
Crohns disease
Crohn's disease (also known as regional enteritis) is a chronic, episodic, inflammatory bowel disease (IBD) and is generally classified as an autoimmune disease. Crohn's disease can affect any part of the gastrointestinal tract from mouth to anus; as a result, the symptoms of Crohn's disease vary among afflicted individuals. The disease is characterized by areas of inflammation with areas of normal lining between in a symptom known as skip lesions. The main gastrointestinal symptoms are abdominal pain, diarrhea (which may be bloody, though this may not be visible to the naked eye), constipation, vomiting, weight loss or weight gain. Crohn's disease can also cause complications outside of the gastrointestinal tract such as skin rashes, arthritis, and inflammation of the eye.
ALS
Amyotrophic Lateral Sclerosis (ALS, sometimes called Maladie de Charcot, or Lou Gehrig's Disease (US)) is a progressive, usually fatal, neurodegenerative disease caused by the degeneration of motor neurons, the nerve cells in the central nervous system that control voluntary muscle movement. As a motor neuron disease, the disorder causes muscle weakness and atrophy throughout the body as both the upper and lower motor neurons degenerate, ceasing to send messages to muscles. Unable to function, the muscles gradually weaken, develop fasciculations (twitches) because of denervation, and eventually atrophy because of that denervation. The patient may ultimately lose the ability to initiate and control all voluntary movement except of the eyes.
Cognitive function is generally spared except in certain situations such as when ALS is associated with frontotemporal dementia. However, there are reports of more subtle cognitive changes of the frontotemporal type in many patients when detailed neuropsychological testing is employed. Sensory nerves and the autonomic nervous system, which controls functions like sweating, generally remain functional.
Lecture is presented by Sir John Bertrand Gurdon ,In 1962, Gurdon, then at Oxford University, announced that he had used the nucleus of fully differentiated adult intestinal cells to clone South African clawed frogs (Xenopus laevis).This was the first demonstration in animals that the nucleus of a differentiated somatic cell retains the potential to develop into all cell types (ie, is totipotent) and paved the way for future somatic cell nuclear transfer experiments, including the 1996 cloning of the sheep, Dolly.
Gurdon began cloning experiments using nonembryonic cells—specifically, cells from the intestinal lining of tadpoles. Gurdon believed that the tadpoles were old enough so that cells taken from them would be differentiated. Gurdon exposed a frog egg to ultraviolet light, which destroyed its nucleus. He then removed the nucleus from the tadpole intestinal cell and implanted it in the enucleated egg. The egg grew into a tadpole that was genetically identical to the DNA-donating tadpole. But the tadpoles cloned in Gurdon’s early experiments never survived to adulthood and scientists now believe that many of the cells used in these experiments may not have been differentiated cells after all. In later work, however, Gurdon successfully produced sexually mature adult frogs from eggs into which genetically marked nuclei had been transplanted from differentiated tadpole cells.
Gurdon’s experiments captured the attention of the scientific community and the tools and techniques he developed for nuclear transfer are still used today. The term clone (from the Greek word klōn, meaning “twig”) had already been in use since the beginning of the 20th century in reference to plants. In 1963 the British biologist J. B. S. Haldane, in describing Gurdon’s results, became one of the first to use the word clone in reference to animals.
In this Hitchcock lecture he explores the process of going from egg to organism.
Proteasomes are large protein complexes inside all eukaryotes and archaea, as well as in some bacteria. In eukaryotes, they are located in the nucleus and the cytoplasm.The main function of the proteasome is to degrade unneeded or damaged proteins by proteolysis, a chemical reaction that breaks peptide bonds. Enzymes that carry out such reactions are called proteases. Proteasomes are a major mechanism by which cells regulate the concentration of particular proteins and degrade misfolded proteins. The degradation process yields peptides of about seven to eight amino acids long, which can then be further degraded into amino acids and used in synthesizing new proteins. Proteins are tagged for degradation by a small protein called ubiquitin. The tagging reaction is catalyzed by enzymes called ubiquitin ligases. Once a protein is tagged with a single ubiquitin molecule, this is a signal to other ligases to attach additional ubiquitin molecules. The result is a polyubiquitin chain that is bound by the proteasome, allowing it to degrade the tagged protein.
Structurally, the proteasome is a large barrel-like complex containing a "core" of four stacked rings around a central pore. Each ring is composed of seven individual proteins. The inner two rings are made of seven β subunits that contain the six protease active sites. These sites are located on the interior surface of the rings, so that the target protein must enter the central pore before it is degraded. The outer two rings each contain seven α subunits whose function is to maintain a "gate" through which proteins enter the barrel. These α subunits are controlled by binding to "cap" structures or regulatory particles that recognize polyubiquitin tags attached to protein substrates and initiate the degradation process. The overall system of ubiquitination and proteasomal degradation is known as the ubiquitin-proteasome system.
The proteasomal degradation pathway is essential for many cellular processes, including the cell cycle, the regulation of gene expression, and responses to oxidative stress.
Discovery
Before the discovery of the ubiquitin proteasome system, protein degradation in cells was thought to rely mainly on lysosomes, membrane-bound organelles with acidic and protease-filled interiors that can degrade and then recycle exogenous proteins and aged or damaged organelles. However, work on ATP-dependent protein degradation in reticulocytes, which lack lysosomes, suggested the presence of a second intracellular degradation mechanism. This was shown in 1978 to be composed of several distinct protein chains, a novelty among proteases at the time. Later work on modification of histones led to the identification of an unexpected covalent modification of the histone protein by a branched bond between a histone lysine residue and the N-terminal glycine residue of the protein ubiquitin, which had no known function. It was then discovered that a previously-identified protein associated with proteolytic degradation, known as ATP-dependent proteolysis factor 1 (APF-1), was the same protein as ubiquitin.
Much of the early work leading up to the discovery of the ubiquitin proteasome system occurred in the late 1970s and early 1980s at the Technion in the laboratory of Avram Hershko, where Aaron Ciechanover worked as a graduate student. Hershko's year-long sabbatical in the laboratory of Irwin Rose at the Fox Chase Cancer Center provided key conceptual insights, though Rose later downplayed his role in the discovery.The three shared the 2004 Nobel Prize in Chemistry for their work in discovering this system.
Although electron microscopy data revealing the stacked-ring structure of the proteasome became available in the mid-1980s, the first structure of the proteasome core particle was not solved by X-ray crystallography until 1994. As of 2006, no structure has been solved of the core particle in complex with the most common form of regulatory cap.
Structure and organization
A schematic diagram of the proteasome 20S core particle viewed from one side. The α subunits that make up the outer two rings are shown in green, and the β subunits that make up the inner two rings are shown in blue.
Top view of the same schematic, illustrating the seven-fold symmetry of the rings.
The proteasome subcomponents are often referred to by their Svedberg sedimentation coefficient (denoted S). The most common form of the proteasome is known as the 26S proteasome, which is about 2000 kilodaltons (kDa) in molecular mass and contains one 20S core particle structure and two 19S regulatory caps. The core is hollow and provides an enclosed cavity in which proteins are degraded; openings at the two ends of the core allow the target protein to enter. Each end of the core particle associates with a 19S regulatory subunit that contains multiple ATPase active sites and ubiquitin binding sites; it is this structure that recognizes polyubiquitinated proteins and transfers them to the catalytic core. An alternative form of regulatory subunit called the 11S particle can associate with the core in essentially the same manner as the 19S particle; the 11S may play a role in degradation of foreign peptides such as those produced after infection by a virus.
20S core particle
The number and diversity of subunits contained in the 20S core particle depends on the organism; the number of distinct and specialized subunits is larger in multicellular than unicellular organisms and larger in eukaryotes than in prokaryotes. All 20S particles consist of four stacked heptameric ring structures that are themselves composed of two different types of subunits; α subunits are structural in nature, while β subunits are predominantly catalytic. The outer two rings in the stack consist of seven α subunits each, which serve as docking domains for the regulatory particles and form an exterior gate blocking unregulated access to the interior cavity. The inner two rings each consist of seven β subunits and contain the protease active sites that perform the proteolysis reactions. The size of the proteasome is relatively conserved and is about 150 angstroms (Å) by 115 Å. The interior chamber is at most 53 Å wide, though the entrance can be as narrow as 13 Å, suggesting that substrate proteins must be at least partially unfolded to enter.
In archaea such as Thermoplasma acidophilum, all the α and all the β subunits are identical, while eukaryotic proteasomes such as those in yeast contain seven distinct types of each subunit. In mammals, the β1, β2, and β5 subunits are catalytic; although they share a common mechanism, they have three distinct substrate specificities considered chymotrypsin-like, trypsin-like, and peptidyl-glutamyl peptide-hydrolyzing (PHGH).Alternative β forms denoted β1i, β2i, and β5i can be expressed in hematopoietic cells in response to exposure to pro-inflammatory signals such as cytokines, in particular, interferon gamma. The proteasome assembled with these alternative subunits is known as the immunoproteasome, whose substrate specificity is altered relative to the normal proteasome.
19S regulatory particle
The 19S particle in eukaryotes consists of 19 individual proteins and is divisible into two subassemblies, a 10-protein base that binds directly to the α ring of the 20S core particle, and a 9-protein lid where polyubiquitin is bound. Six of the ten base proteins have ATPase activity. The association of the 19S and 20S particles requires the binding of ATP to the 19S ATP-binding sites.ATP hydrolysis is required for the assembled complex to degrade a folded and ubiquitinated protein, although it is not yet clear whether that energy is used mainly for substrate unfolding, opening of the core channel, or some combination of processes. As of 2006, the structure of the 26S proteasome has not been solved, although the 19S and 11S particles are believed to bind in a similar way to the α rings of the 20S core particle.
Individual components of the 19S particle have their own regulatory roles. Gankyrin, a recently identified oncoprotein, is one of the 19S subcomponents that also tightly binds the cyclin-dependent kinase CDK4 and plays a key role in recognizing ubiquitinated p53, via its affinity for the ubiquitin ligase MDM2. Gankyrin is anti-apoptotic and has been shown to be overexpressed in some tumor cell types such as hepatocellular carcinoma.
11S regulatory particle
20S proteasomes can also associate with a second type of regulatory particle, a heptameric structure that does not contain any ATPases and can promote the degradation of short peptides, but not of complete proteins. Presumably this is because the complex cannot unfold larger substrates. This structure is also known as PA28 or REG. The mechanisms by which it binds to the core particle through the C-terminal tails of its subunits and induces α-ring conformational changes to open the 20S gate suggest a similar mechanism for the 19S particle. The expression of the 11S particle is induced by interferon gamma and is responsible, in conjunction with the immunoproteasome β subunits, for the generation of peptides that bind to the major histocompatibility complex.
Assembly
The assembly of the proteasome is a complex process due to the number of subunits that must associate to form an active complex. The β subunits are synthesized with N-terminal "propeptides" that are post-translationally modified during the assembly of the 20S particle to expose the proteolytic active site. The 20S particle is assembled from two half-proteasomes, each of which consists of a seven-membered pro-β ring attached to a seven-membered α ring. The association of the β rings of the two half-proteasomes triggers threonine-dependent autolysis of the propeptides to expose the active site. These β interactions are mediated mainly by salt bridges and hydrophobic interactions between conserved alpha helices whose disruption by mutation damages the proteasome's ability to assemble. The assembly of the half-proteasomes, in turn, is initiated by the assembly of the α subunits into their heptameric ring, forming a template for the association of the corresponding pro-β ring. The assembly of α subunits has not been characterized.
In general, less is known about the assembly and maturation of the 19S regulatory particles. They are believed to assemble as two distinct subcomponents, the ATPase-containing base and the ubiquitin-recognizing lid. The six ATPases in the base may assemble in a pairwise manner mediated by coiled-coil interactions. The order in which the nineteen subunits of the regulatory particle are bound is a likely regulatory mechanism that prevents exposure of the active site before assembly is complete.
The protein degradation process
Ribbon diagram of ubiquitin, the highly conserved protein that serves as a molecular tag targeting proteins for degradation by the proteasome.
Ubiquitination and targeting
Proteins are targeted for degradation by the proteasome by covalent modification of a lysine residue that requires the coordinated reactions of three enzymes. In the first step, a ubiquitin-activating enzyme (known as E1) hydrolyzes ATP and adenylates a ubiquitin molecule. This is then transferred to E1's active-site cysteine residue in concert with the adenylation of a second ubiquitin. This adenylated ubiquitin is then transferred to a cysteine of a second enzyme, ubiquitin-conjugating enzyme (E2). Lastly, a member of a highly-diverse class of enzymes known as ubiquitin ligases (E3) recognize the specific protein to be ubiquitinated and catalyze the transfer of ubiquitin from E2 to this target protein. A target protein must be labelled with at least four ubiquitin monomers (in the form of a polyubiquitin chain) before it is recognized by the proteasome lid. It is therefore the E3 that confers substrate specificity to this system. The number of E1, E2, and E3 proteins expressed depends on the organism and cell type, but there are many different E3 enzymes present in humans, indicating that there are a huge number of targets for the ubiquitin proteasome system.
The mechanism by which a polyubiquitinated protein is targeted to the proteasome is not fully understood. Ubiquitin-receptor proteins have an N-terminal ubiquitin-like (UBL) domain and one or more ubiquitin-associated (UBA) domains. The UBL domains are recognized by the 19S proteasome caps and the UBA domains bind ubiquitin via three-helix bundles. These receptor proteins may escort polyubiquitinated proteins to the proteasome, though the specifics of this interaction and its regulation are unclear.
The ubiquitin protein itself is 76 amino acids long and was named due to its ubiquitous nature, as it has a highly conserved sequence and is found in all known eukaryotic organisms. The genes encoding ubiquitin in eukaryotes are arranged in tandem repeats, possibly due to the heavy transcription demands on these genes to produce enough ubiquitin for the cell. It has been proposed that ubiquitin is the slowest-evolving protein identified to date.
The ubiquitination pathway.
Unfolding and translocation
After a protein has been ubiquitinated, it is recognized by the 19S regulatory particle in an ATP-dependent binding step. The substrate protein must then enter the interior of the 20S particle to come in contact with the proteolytic active sites. Because the 20S particle's central channel is narrow and gated by the N-terminal tails of the α ring subunits, the substrates must be at least partially unfolded before they enter the core. The passage of the unfolded substrate into the core is called translocation and necessarily occurs after deubiquitination. However, the order in which substrates are deubiquitinated and unfolded is not yet clear. Which of these processes is the rate-limiting step in the overall proteolysis reaction depends on the specific substrate; for some proteins, the unfolding process is rate-limiting, while deubiquitination is the slowest step for other proteins. The extent to which substrates must be unfolded before translocation is not known, but substantial tertiary structure, and in particular nonlocal interactions such as disulfide bonds, are sufficient to inhibit degradation.
The gate formed by the α subunits prevents peptides longer than about four residues from entering the interior of the 20S particle. The ATP molecules bound before the initial recognition step are hydrolyzed before translocation, although there is disagreement whether the energy is needed for substrate unfolding or for gate opening. The assembled 26S proteasome can degrade unfolded proteins in the presence of a non-hydrolyzable ATP analog, but cannot degrade folded proteins, indicating that energy from ATP hydrolysis is used for substrate unfolding. Passage of the unfolded substrate through the opened gate occurs via facilitated diffusion if the 19S cap is in the ATP-bound state.
The mechanism for unfolding of globular proteins is necessarily general, but somewhat dependent on the amino acid sequence. Long sequences of alternating glycine and alanine have been shown to inhibit substrate unfolding decreasing the efficiency of proteasomal degradation; this results in the release of partially degraded byproducts, possibly due to the decoupling of the ATP hydrolysis and unfolding steps. Such glycine-alanine repeats are also found in nature, for example in silk fibroin; in particular, certain Epstein-Barr virus gene products bearing this sequence can stall the proteasome, helping the virus propagate by preventing antigen presentation on the major histocompatibility complex.
Proteolysis
The mechanism of proteolysis by the β subunits of the 20S core particle is through a threonine-dependent nucleophilic attack. This mechanism may depend on an associated water molecule for deprotonation of the reactive threonine hydroxyl. Degradation occurs within the central chamber formed by the association of the two β rings and normally does not release partially degraded products, instead reducing the substrate to short polypeptides typically 7–9 residues long, though they can range from 4 to 25 residues depending on the organism and substrate. The biochemical mechanism that determines product length is not fully characterized. Although the three catalytic β subunits share a common mechanism, they have slightly different substrate specificities, which are considered chymotrypsin-like, trypsin-like, and peptidyl-glutamyl peptide-hydrolyzing (PHGH)-like. These variations in specificity are the result of interatomic contacts with local residues near the active sites of each subunit. Each catalytic β subunit also possesses a conserved lysine residue required for proteolysis.
Although the proteasome normally produces very short peptide fragments, in some cases these products are themselves biologically active and functional molecules. Certain transcription factors, including one component of the mammalian complex NF-κB, are synthesized as inactive precursors whose ubiquitination and subsequent proteasomal degradation converts them to an active form. Such activity requires the proteasome to cleave the substrate protein internally: rather than processively degrading it from one terminus. It has been suggested that long loops on these proteins' surfaces serve as the proteasomal substrates and enter the central cavity, while the majority of the protein remains outside. Similar effects have been observed in yeast proteins; this mechanism of selective degradation is known as regulated ubiquitin/proteasome dependent processing (RUP).
Ubiquitin-independent degradation
Although most proteasomal substrates must be ubiquitinated before being degraded, there are some exceptions to this general rule, especially when the proteasome plays a normal role in the post-translational processing of the protein. The proteasomal activation of NF-κB by processing p105 into p50 via internal proteolysis is one major example.Some proteins that are hypothesized to be unstable due to intrinsically unstructured regions, are degraded in a ubiquitin-independent manner. The most well-known example of a ubiquitin-independent proteasome substrate is the enzyme ornithine decarboxylase. Ubiquitin-independent mechanisms targeting key cell cycle regulators such as p53 have also been reported, although p53 is also subject to ubiquitin-dependent degradation. Finally, structurally-abnormal, misfolded, or highly oxidized proteins are also subject to ubiquitin-independent and 19S-independent degradation under conditions of cellular stress.
Evolution
The 20S proteasome is both ubiquitous and essential in eukaryotes. Some prokaryotes, including many archaea and the bacterial order Actinomycetales also share homologs of the 20S proteasome, while most bacteria possess heat shock genes hslV and hslU, whose gene products are a multimeric protease arranged in a two-layered ring and an ATPase. The hslV protein has been hypothesized to resemble the likely ancestor of the 20S proteasome.HslV is generally not essential in bacteria, and not all bacteria possess it, while some protists possess both the 20S and the hslV systems.
Sequence analysis suggests that the catalytic β subunits diverged earlier in evolution than the predominantly-structural α subunits. In bacteria that express a 20S proteasome, the β subunits have high sequence identity to archaeal and eukaryotic β subunits, while the α sequence identity is much lower. The presence of 20S proteasomes in bacteria may result from lateral gene transfer, while the diversification of subunits among eukaryotes is ascribed to multiple gene duplication events.
Cell cycle control
Cell cycle progression is controlled by ordered action of cyclin-dependent kinases (CDKs), activated by specific cyclins that demarcate phases of the cell cycle. Mitotic cyclins, which persist in the cell for only a few minutes, have one of the shortest life spans of all intracellular proteins. After a CDK-cyclin complex has performed its function, the associated cyclin is polyubiquitinated and destroyed by the proteasome, which provides directionality for the cell cycle. In particular, exit from mitosis requires the proteasome-dependent dissociation of the regulatory component cyclin B from the mitosis promoting factor complex. In vertebrate cells, "slippage" through the mitotic checkpoint leading to premature M phase exit can occur despite the delay of this exit by the spindle checkpoint.
Earlier cell cycle checkpoints such a post-restriction point check between G1 phase and S phase similarly involve proteasomal degradation of cyclin A, whose ubiquitination is promoted by the anaphase promoting complex (APC), an E3 ubiquitin ligase. The APC and the Skp1/Cul1/F-box protein complex (SCF complex) are the two key regulators of cyclin degradation and checkpoint control; the SCF itself is regulated by the APC via ubiquitination of the adaptor protein, Skp2, which prevents SCF activity before the G1-S transition.
Regulation of plant growth
In plants, signaling by auxins, or phytohormones that order the direction and tropism of plant growth, induces the targeting of a class of transcription factor repressors known as Aux/IAA proteins for proteasomal degradation. These proteins are ubiquitinated by SCFTIR1, or SCF in complex with the auxin receptor TIR1. Degradation of Aux/IAA proteins derepresses transcription factors in the auxin-response factor (ARF) family and induces ARF-directed gene expression. The cellular consequences of ARF activation depend on the plant type and developmental stage, but are involved in directing growth in roots and leaf veins. The specific response to ARF derepression is thought to be mediated by specificity in the pairing of individual ARF and Aux/IAA proteins.
Apoptosis
Both internal and external signals can lead to the induction of apoptosis, or programmed cell death. The resulting deconstruction of cellular components is primarily carried out by specialized proteases known as caspases, but the proteasome also plays important and diverse roles in the apoptotic process. The involvement of the proteasome in this process is indicated by both the increase in protein ubiquitination, and of E1, E2, and E3 enzymes that is observed well in advance of apoptosis, During apoptosis, proteasomes localized to the nucleus have also been observed to translocate to outer membrane blebs characteristic of apoptosis.
Proteasome inhibition has different effects on apoptosis induction in different cell types. The proteasome is not generally required for apoptosis, although inhibiting it is pro-apoptotic in most cell types that have been studied. However, some cell lines—in particular, primary cultures of quiescent and differentiated cells such as thymocytes and neurons—are prevented from undergoing apoptosis on exposure to proteasome inhibitors. The mechanism for this effect is not clear, but is hypothesized to be specific to cells in quiescent states, or to result from the differential activity of the pro-apoptotic kinase JNK. The ability of proteasome inhibitors to induce apoptosis in rapidly dividing cells has been exploited in several recently developed chemotherapy agents.
Response to cellular stress
In response to cellular stresses - such as infection, heat shock, or oxidative damage - heat shock proteins are expressed that identify misfolded or unfolded proteins and target them for proteasomal degradation. Both Hsp27 and Hsp90—chaperone proteins have been implicated in increasing the activity of the ubiquitin-proteasome system, though they are not direct participants in the process. Hsp70, on the other hand, binds exposed hydrophobic patches on the surface of misfolded proteins and recruits E3 ubiquitin ligases such as CHIP to tag the proteins for proteasomal degradation. The CHIP protein (carboxyl terminus of Hsp70-interacting protein) is itself regulated via inhibition of interactions between the E3 enzyme CHIP and its E2 binding partner.
Similar mechanisms exist to promote the degradation of oxidatively-damaged proteins via the proteasome system. In particular, proteasomes localized to the nucleus are regulated by PARP and actively degrade inappropriately oxidized histones. Oxidized proteins, which often form large amorphous aggregates in the cell, can be degraded directly by the 20S core particle without the 19S regulatory cap and do not require ATP hydrolysis or tagging with ubiquitin. However, high levels of oxidative damage increases the degree of cross-linking between protein fragments, rendering the aggregates resistant to proteolysis. Larger numbers and sizes of such highly oxidized aggregates are associated with aging.
Impaired proteasomal activity has been suggested as an explanation for some of the late-onset neurodegenerative diseases that share aggregation of misfolded proteins as a common feature, such as Parkinson's disease and Alzheimer's disease. In these diseases large insoluble aggregates of misfolded proteins can form and then result in neurotoxicity, through mechanisms that are not yet well understood. Decreased proteasome activity has been suggested as a cause of aggregation and Lewy body formation in Parkinson's. This hypothesis is supported by the observation that yeast models of Parkinson's are more susceptible to toxicity from alpha-synuclein, the major protein component of Lewy bodies, under conditions of low proteasome activity.
Role in the immune system
The proteasome plays a straightforward but critical role in the function of the adaptive immune system. Peptide antigens are displayed by the major histocompatibility complex class I (MHC) proteins on the surface of antigen-presenting cells. These peptides are products of proteasomal degradation of proteins originated by the invading pathogen. Although constitutively-expressed proteasomes can participate in this process, a specialized complex composed of proteins whose expression is induced by interferon gamma produces peptides of the optimal size and composition for MHC binding. These proteins whose expression increases during the immune response include the 11S regulatory particle, whose main known biological role is regulating the production of MHC ligands, and specialized β subunits called β1i, β2i, and β5i with altered substrate specificity. The complex formed with the specialized β subunits is known as the immunoproteasome.
The strength of MHC class I ligand binding is dependent on the composition of the ligand C-terminus, as peptides bind by hydrogen bonding and by close contacts with a region called the "B pocket" on the MHC surface. Optimal residues for the C-terminal end of these peptides are leucine and valine.The N-terminal residues, particularly the second residue in the peptide, also play a key role in determining binding affinity. The immunoproteasome complex generates the correct C-terminal tails; later processing of the products by interferon gamma-induced aminopeptidases trims the N-termini for optimal MHC ligand production.
Due to its role in generating the activated form of NF-κB, an anti-apoptotic and pro-inflammatory regulator of cytokine expression, proteasomal activity has been linked to inflammatory and autoimmune diseases. Increased levels of proteasome activity correlate with disease activity and have been implicated in autoimmune diseases including systemic lupus erythematosus and rheumatoid arthritis.
Proteasome inhibitors have effective anti-tumor activity in cell culture, inducing apoptosis by disrupting the regulated degradation of pro-growth cell cycle proteins.This approach of selectively-inducing apoptosis in tumor cells has proven effective in animal models and human trials. Bortezomib, a molecule developed by Millennium Pharmaceuticals and marketed as Velcade, is the first proteasome inhibitor to reach clinical use as a chemotherapy agent. Bortezomib is used in the treatment of multiple myeloma. Notably, multiple myeloma has been observed to result in increased proteasome levels in blood serum that decrease to normal levels in response to successful chemotherapy.Studies in animals have indicated that bortezomib may also have clinically-significant effects in pancreatic cancer. Preclinical and early clinical studies have been started to examine bortezomib's effectiveness in treating other B-cell-related cancers, particularly some types of non-Hodgkin's lymphoma.
The molecule ritonavir, marketed as Norvir, was developed as a protease inhibitor and used to target HIV infection. However, it has been shown to inhibit proteasomes as well as free proteases; specifically, the chymotrypsin-like activity of the proteasome is inhibited by ritonavir, while the trypsin-like activity is somewhat enhanced. Studies in animal models suggest that ritonavir may have inhibitory effects on the growth of glioma cells.
Proteasome inhibitors have also shown promise in treating autoimmune diseases in animal models. For example, studies in mice bearing human skin grafts found a reduction in the size of lesions from psoriasis after treatment with a proteasome inhibitor.Inhibitors also show positive effects in rodent models of asthma.
Labeling and inhibition of the proteasome is also of interest in laboratory settings for both in vitro and in vivo study of proteasomal activity in cells. The most commonly used laboratory inhibitor is lactacystin, a natural product synthesized by Streptomyces bacteria. Fluorescent inhibitors have also been developed to specifically label the active sites of the assembled proteasome.
The tocotrienol (or TCT) group—together with tocopherols—compose the vitamin E family. Natural tocotrienols exist in four different forms or isomers, named alpha-, beta-, gamma- and delta- tocotrienol, each which contain different number of methyl groups on the chromanol ring. The major structural difference from tocopherol is through its unsaturated side chain that has three double bonds in its farnesyl isoprenoid tail. All of the isomers have been demonstrated to have some level of antioxidant activity due to the donating hydrogen atom from the hydroxyl group on the chromanol ring that might reduce free radicals in the body. However, some of the isomers have been further investigated through a number of clinical and non-clinical studies for other biological activities. The investigations have shown promise in pre-clinical and clinical applications that include cancer treatment and cholestrol control, benefits that may be lesser or even lacking in the more commonly used synthetic alpha-tocopherol as discussed later.
Tocotrienols are named by analogy to tocopherols (from Greek words meaning to bear a pregnancy . but with this word changed to include the chemical difference that tocotrienols are trienes, meaning that they share identical structure with the tocopherols except for the addition of three double bonds to their side chains.
Tocotrienols and breast cancer
A study showed that tocotrienols are the components of vitamin E responsible for growth inhibition in human breast cancer cells in vitro as well as in vivo through estrogen-independent mechanisms. Tocotrienols can also affect cell homeostasis, possibly independently of their antioxidant activity. Anti-cancer effects of α- and γ-tocotrienol have been reported, although δ-tocotrienol was verified to be the most effective tocotrienol in inducing apoptosis (cell death) in estrogen-responsive and estrogen-nonresponsive human breast cancer cells. A daily dose of 30 - 50 mg mixture of α- and γ-tocotrienols can reduce breast cancer risk, and a treatment plan for breast cancer should use higher dosage.
Tocotrienols and prostate cancer
Investigation of the antiproliferative effect of tocotrienols in PC3 and LNCaP prostate cancer cells suggests that the transformation of vitamin E to CEHC is mostly a detoxification mechanism, useful to maintain the malignant properties of prostate cancer cells.[4] However, recent research suggested that γ-tocotrienol was most potent in suppressing prostate cancer cell proliferation, and that the antiproliferative effect of γ-tocotrienol act through multiple-signalling pathways (NF-B, EGF-R and Id family proteins). In addition, the same study demonstrated the anti-invasion and chemosensitisation effect of γ-tocotrienol against PCa cells.
The follicular phase (or proliferative phase) is the phase of the estrous cycle, (or, in humans and great apes, the menstrual cycle) during which follicles in the ovary mature. It ends with ovulation. The main hormone controlling this stage is estradiol.
Follicle recruitment
Follicle-stimulating hormone (FSH) is secreted by the anterior pituitary gland. FSH secretion begins to rise in the last few days of the previous menstrual cycle, and is highest and most important during the first week of the follicular phase. The rise in FSH levels recruits five to seven tertiary-stage ovarian follicles (this stage follicle is also known as a Graafian follicle or antral follicle) for entry into the menstrual cycle. These follicles, that have been growing for the better part of a year in a process known as folliculogenesis, compete with each other for dominance.
FSH induces the proliferation of granulosa cells in the developing follicles, and the expression of luteinizing hormone (LH) receptors on these granulosa cells. Under the influence of FSH, aromatase and p450 enzymes are activated, causing the granulosa cells to begin to secrete estrogen. This increased level of estrogen stimulates production of gonadotropin-releasing hormone (GnRH), which increases production of LH. LH induces androgen synthesis by thecal cells, stimulates proliferation, differentiation, and secretion of follicular thecal cells and increases LH receptor expression on granulosa cells.
Estrogen surge
Two or three days after LH levels begin to increase, usually by day seven of the cycle, one (or occasionally two) of the recruited follicles has emerged as dominant. Many endocrinologists believe that the estrogen secretion of the dominant follicle has increased to a level that GnRH production is suppressed, which lowers the levels of LH and FSH. This slowdown in LH and FSH production leads to the atresia (death) of most of the recruited follicles, though the dominant follicle continues to mature. Estrogen levels will continue to increase for several days (on average, six days, but this varies widely).
These high estrogen levels initiate the formation of a new layer of endometrium in the uterus, histologically identified as the proliferative endometrium. Crypts in the cervix are also stimulated to produce fertile cervical mucus.This mucus reduces the acidity of the vagina, creating a more hospitable environment for sperm. It also has a characteristic texture that helps guide sperm through the cervix and to the fallopian tubes, where they wait for ovulation.In addition, basal body temperature may lower slightly under the influence of high estrogen levels.
LH surge and ovulation
Estrogen levels are highest right before the LH surge begins. The short-term drop in steroid hormones between the beginning of the LH surge and the event of ovulation may cause mid-cycle spotting or bleeding.Under the influence of the preovulatory LH surge, the first meiotic division of the oocytes is completed. The surge also initiates luteinization of thecal and granulosa cells. Ovulation normally occurs 30 (± 2) hours after the beginning of the LH surge (when LH is first detectable in urine).
Follicular waves
The time from recruitment of tertiary stage follicles to ovulation is normally about two weeks, comprising days 1-14 of an idealized 28-day menstrual cycle. However, it is common for the follicular phase to last much longer, as sometimes no dominant follicle is selected. In this case, normally more tertiary stage follicles are recruited and the process begins again. One study of women with regular menstrual cycles found that none of them ovulated after the first wave of follicle development: 68% of cycles showed ovulation after the second wave, while 32% of cycles showed ovulation after the third wave. Fertility awareness sources may refer to multiple waves of follicular development as a split peak because of the characteristic pattern of cervical mucus changes that is sometimes observed.
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