cell biology 5 Flashcards
Epidemiology of cholera
Cholera is an acute intestinal infection caused by toxigenic Vibrio cholerae . The main areas of the world in which cholera is present are Africa, Asia and parts of the Middle East. There are more than 100 types of cholera. However, there are only two types of cholera that affect humans: Vibrio cholerae O1 and Vibrio cholerae O139. We are currently in the 7th Cholera Pandemic, caused by the El Tor biotype of V. cholerae O1. It was first identified in 1905 at a quarantine camp on the Siniai Peninsula in El-Tor, Egypt from a group of pilgrims returning from Mecca. El Tor reappeared in an outbreak in Indonesia in 1937, but the pandemic did not arise until the 1960’s when El Tor spread through Bangladesh and India. It then arose in parts of Africa and Italy in the 1970’s. Spreading through parts of Europe in the 1980’s, it then affected 21 countries in Latin America in the 1990’s. It is responsible now for over 1 million cases worldwide. In 1992, a second serotype was discovered in Bangladesh, designated O139 (“Bengal”), and is now endemic in the region. The strain currently in Haiti since 2010 is the O1 Ogawa serotype.
Vibrio cholerae O1
there are two subtypes (Classical and El Tor). There are 3 serotypes of O1: Inaba, Ogawa and Hikojima.
Vibrio cholerae O139
first described in Bangladesh in 1992, now considered endemic in the region.
Global trend (taken from WHO) for cholera
Globally for 2013, there was an estimated 1.4 to 4.3 million cases accounting for 28,000 to 142,000 deaths. The true number is not known due to limitations in surveillance systems and lack of diagnostic capacities in some areas, leading to both over- and under-reporting. “In 2013, 43% of cases were reported from Africa whereas between 2001–2009, 93% to 98% of total cases worldwide were reported from that continent. This proportion changed in 2010 with the outbreak in the island of Hispaniola. A higher proportion of cases started to be reported from Haiti and the Dominican Republic. Globally, cases reported from Africa have also decreased since 2012. However many people still die of the disease notably in Sub-Saharan Africa, Asia and in Hispaniola, clearly showing that cholera remains a significant public health problem.”
Cholera Symptoms
Voluminous (up 1 liter per hour) watery feces with bits of mucus - this is sometimes called ‘rice water’ stools since it looks like water in which rice has been washed, Vomiting, Severe and rapid dehydration. An infected individual could die within hours if left untreated from dehydration. If you lived in a developing country before the 1970’s and were infected with cholera, you had a 30-50% chance of dying. Due to oral rehydration therapy (ORT), the mortality is reduced to about 1%.
Pathophysiology of cholera
Vibrio cholerae is spread by the fecal-oral route. Therefore, it is most commonly spread contaminated food or water. Good sanitation is crucial to control cholera transmission.. The infectious dose of V. cholera is stated to be 108 cfu. The required dose is lower in the presence of reduced gastric acidity. On the other hand as few as 100,000 rods for Salmonella subspecies and 10 bacilli for Shigella subspecies are required for infection. Therefore, you need a good dose of contaminated food to be infected. Vibrio cholerae is a noninvasive species. The mechanism of diarrhea is through a “virulence cassette” composed of 3 genes encoding for the 3 toxins that result in diarrhea: ctx (cholera toxin), zot (zonulin occludens toxin), and ace (accessory cholera enterotoxin).
ctx (cholera toxin)
The primary mechanism of disease is through the actions of cholera toxin (Ctx). Ctx consists of an A subunit and B subunit. The A subunit is the active site while the B subunit is the transport molecule. Intestinal crypt cells, the primary secretory cells found in the small intestinal mucosa, respond to numerous secretagogues including acetylcholine, prostaglandins, and vasoactive intestinal peptide. The second messengers Ca2+ and cAMP lead to chloride secretion through the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR), located on the apical (luminal) side of the cells. Ctx binds to the GM1 ganglioside receptor on surface of the enterocyte via the B subunit. At the cell surface, the A subunit is then cleaved off and endocytosed. It subsequently binds to G protein intracellularly, and then stimulates adenylate cyclase to produce cAMP. cAMP leads to the continuous activation of CFTR, resulting in a massive efflux of chloride ions, followed by water, resulting in massive watery diarrhea high in electrolyte content.
zot (zonulin occludens toxin)
Zonulin occludens toxin (Zot) is located on the bacterial membrane and binds to a Zot receptor, resulting in an alteration of intestinal permeability through a cascade of intracellular events that lead to subsequent tight junction disassembly. It is believed to mimic Zonulin, the naturally occurring endogenous modulator of tight junctions. This results in lossening the tight junctions and an increased efflux of salt and water in the gut.
ace (accessory cholera enterotoxin)
Accessory cholera enterotoxin (Ace) affects the potential differences across cells, contributing to the diarrhea, but the precise mechanism is still unknown.
Cystic Fibrosis Transmembrane Conductance Regulator (CFTR)
CFTR functions as an ATP-gated anion channel, increasing the conductance for certain anions (e.g. Cl–) to flow down their electrochemical gradient. ATP-driven conformational changes in CFTR open and close a gate to allow transmembrane flow of anions down their electrochemical gradient. This in contrast to other ABC proteins, in which ATP-driven conformational changes fuel uphill substrate transport across cellular membranes. Essentially, CFTR is an ion channel that evolved as a ‘broken’ ABC transporter that leaks when in open conformation. The CFTR is found in the epithelial cells of many organs including the lung, liver, pancreas, digestive tract, reproductive tract, and skin. Normally, the protein moves chloride and thiocyanate[17] ions (with a negative charge) out of an epithelial cell to the covering mucus. Positively charged sodium ions follow passively, increasing the total electrolyte concentration in the mucus, resulting in the movement of water out of cell by osmosis.
Osmotic Diarrhea
Osmotic diarrhea is diarrhea driven by an osmotically active agent in the intestinal lumen which pulls water into the intestine. Lactose intolerance is one example where malabsorbed lactose causes water to be drawn into the colon leading to diarrhea. An osmotic diarrhea can also occur in the setting of malabsorptive conditions such as from celiac disease or following a particularly severe case of gastroenteritis, where malabsorbed carbohydrates can lead to an osmotic diarrhea. Medications such as polyethylene glycol (used to treat constipation) which is not absorbed, can result in more watery stools. This type of diarrhea will improve when you remove the osmotic source.
Secretory Diarrhea
If a patient has a secretory diarrhea, the watery stools will continue even they are fasting. This is because the intestine is actively secreting fluids and electrolytes into the lumen. Cholera is the prototypic secretory diarrhea. In the small intestines, you have villi (the long fingerlike projections in the musocsa) that serve to increase the absorptive capacity of the intestine, and you have crypts (located at the base of each villi) that serve to secrete fluids and electrolytes. In normal conditions, the villi outperform the crypts, so that you have a net fluid and electrolyte absorption. In the case of cholera, the crypts secrete so much that it overwhelms the absorptive capacity of the villi, even though there is no histological injury to the intestinal epithelium. Therefore, the fluid lost from a secretory diarrhea can have an electrolyte content that is close to that found in your serum!
How is cholera treated?
The treatment for cholera is rehydration, focusing on both volume repletion and replacing ongoing fluid losses. Antibiotics are not generally indicated in mild-moderate cases of cholera, but can be used to treat severely affected individuals. Antibiotics will shorten the duration of disease and reduce the risk of further infectivity by killing the organisms. Anti-diarrheal medications are not indicated for cholera, as while it may slow down intestinal motility, it will not affect the secretory component of the diarrhea (in fact, it may make you feel worse!). In the United States, it is convenient to deliver fluids intravenously. However, in regions where IV fluids are inaccessible, oral rehydration therapy (ORT) is cheap and easily accessible, either in the form of a package or homemade. More importantly, it is lifesaving. The fundamental principle of oral rehydration solutions is to take advantage of the sodium transporters in the apical surface of the intestinal epithelial cell. This is done by coupling glucose or starch with sodium in the intestinal lumen, to promote sodium absorption and hence chloride and water flow away from the lumen. Not only can oral rehydration replace lost fluids in an individual, it even has the potential to actually reduce the volume of diarrhea. For more than 25 years, UNICEF and WHO had recommended a single formulation of glucose-based ORS to prevent or treat dehydration from diarrhea. This product, which provides a solution containing 90 mEq/l of sodium with a total osmolarity of 311 mOsm/l, had proven to be effective. However, concerns about its use in non-cholera causes of diarrhea (although it has been shown to also be effective in rotavirus-induced diarrhea) and also about the high osmolarity (possibility of driving an osmotic-induced diarrhea) resulted in a new improved formulation. Therefore, in 2003, a “reduced osmolarity” formulation had been developed, with lower glucose and sodium concentrations. While there are some concerns about biochemical hyponatremia in individuals receiving the reduced osmolarity formula, it has not been associated with serious consequences and appears to be better tolerated by patients. There is now an additional recommendation of zinc supplementation for the management of diarrheal disease in addition to ORT, particularly for pediatric patients. There has been considerable debate over whether or not ORT should be given in prepackaged formulations or homemade. Even though the majority of mothers in developing countries affected by cholera are aware of ORT, only fewer than half will make the solution correctly. This could potentially lead to hypernatremia or continued dehydration. On the other hand, it is cheaper and more easily accessible than having to go to a local clinic to obtain prepackaged formulations, and it promotes self-sufficiency. More recently, rice-based ORT has been promoted as being able to even reduce the severity of diarrhea. From a homemade perspective, this has been made by essentially substituting glucose with rice cereal. Naturally, such a product has now been commercialized and produced as “Ceralyte.” Ceralyte-90 is designed for secretory diarrhea, and Ceralyte-70 and -50 have less sodium, and are intended for “less severe” forms of diarrhea. Nevertheless, there have been numerous studies suggesting that this rice-based ORT is actually superior to standard ORS for cholera (but not necessarily so for regular diarrhea). Rice-based and other cereal-based oral rehydration solutions are thought to reduce diarrhea by adding more substrate to the gut lumen without increasing osmolality, thus providing additional glucose molecules for glucose-mediated absorption. In addition, the amino acids in the solutions may also provide additional substrate for other cotransport mechanisms within the colon.
Mechanisms of fluid absorption by ORT
Remember, cholera results in the massive efflux of chloride out of the cell via the CFTR, in the form of salt, and is accompanied by water (it causes a secretory diarrhea). As you have likely already learned from Professor Betz’s lectures, there are several transporters that can bring sodium into a cell (and chloride will follow) from the apical side. These all rely on the sodium/potassium pump on the basolateral membrane, to create a sodium gradient favoring sodium entry into the cell. However, some will result in a net movement of a charge across the membrane (electrogenic transport) and some will not (electroneutral transport). Studies in animals and humans demonstrated that the maximum uptake of water and electrolytes occurs when the ratio of carbohydrate to sodium approaches one, and the WHO recommends a ratio of < 1.4 to 1. The WHO formulas take advantage of the sodium cotransporters on the apical side of the enterocyte, which are not affected during a cholera infection. These are in the form of sodium-glucose transporters, or sodium coupled with other substrates such as amino acids, which help in sodium reabsorption. Again, when sodium reenters a cell, chloride and water will then follow. The key to successful ORT is to start early and offer the solution continuously (small frequent sips if vomiting) in a patient with cholera. Likewise, as the patient is being rehydrated, early feeding is recommended.
Necrosis
premature death of cells. In necrosis, the organelle that suffers first seems to be the mitochondrion, which early on begins to swell. At the stage called “high-amplitude swelling” it can no longer maintain its ionic gradients or oxidative phosphorylation, and the cell runs out of energy. Starving for ATP, the plasma membrane’s ion pumps fail, water floods in, and the cell swells and bursts. Lysis releases the cell’s intracellular contents into the extracellular milieu, where they have no business being; these internal lipids, proteases, and small molecules are intensely proinflammatory. They attract white cells, primarily macrophages, from around the body. Given the extent of damage, this is usually desirable, as the local facilities for dealing with damage can be overwhelmed. The effect of the inflammatory process is debris removal, injury resolution, and, if the stroma has been damaged, scar formation.
COMMON FEATURES OF APOPTOSIS
The defining morphological feature of apoptosis is a collapse of the nucleus; chromatin, which is normally composed of mixed open and condensed regions (heterochromatin and euchromatin), becomes supercondensed, appearing as crescents around the nuclear envelope and, eventually, spherical featureless beads. The structural correlate of this morphological change is the fragmentation of DNA into units of one or several nucleosomes in length. (A nucleosome consists of a core of histone proteins wrapped by about 180 base pairs of DNA, and is the first stage of compaction of DNA.) This degradation reflects the action of an endonuclease on the DNA in the linkers between nucleosomes; this stretch of DNA is not very well protected by histones. Because a cell can only repair a few simultaneous double-stranded breaks in its DNA, the extensive DNA damage in apoptosis (up to 300,000 breaks/chromosome!) means that even if there were no other changes, the cell would certainly never divide again.
Early in apoptosis cells
shrink remarkably, losing about a third of their volume in a few seconds. This shrinkage is quite apparent in cell culture, and also in vivo, where apoptotic cells in tissue sections often pull away from their neighbors. As might be expected there are cytoskeletal changes that accompany shrinkage, and the result is a peculiar, vigorous “boiling” action of the plasma membrane, which has been called zeiosis.
zeiosis
a bleb is a protrusion, or bulge, of the plasma membrane of a cell, caused by localized decoupling of the cytoskeleton from the plasma membrane. During apoptosis (programmed cell death), the cell’s cytoskeleton breaks up and 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 bodies. Phagocytic cells eventually consume these fragments and the components are recycled.
apoptotic bodies
small sealed membrane vesicles that are produced from cells undergoing cell death by apoptosis. The formation of apoptotic bodies is a mechanism preventing leakage of potentially toxic or immunogenic cellular contents of dying cells and prevents inflammation or autoimmune reactions as well as tissue destruction. the apoptotic cell usually tears itself apart from other cells with zeiosis into apoptotic bodies, some of which contain chromatin. It is not known how, or even if, these changes lead to cell death. This is because early in apoptosis, while the cell is still fully “viable,” that is, still able to exclude vital dyes like trypan blue, it is recognized by another cell and phagocytosed; it dies within the phagocyte. So the goal of all the morphological changes is to ensure that the apoptotic cell gets taken up by a healthy cell, before it has had a chance to spill its dangerous contents.
PHAGOCYTOSIS OF APOPTOTIC CELLS
Apoptosis is also accompanied by changes in the plasma membrane, the most obvious of which involves the phospholipid phosphatidylserine (PS). All the PS in a normal plasma membrane is confined to the inner leaflet of the lipid bilayer; in fact, an enzyme (called “flippase”) ensures that any PS molecule that strays to the outer leaflet is quickly returned. Soon after apoptosis begins, the distribution of PS becomes equal on both sides of the membrane, by a “scrambling” mechanism involving “scramblase.” This means that PS is now exposed on the cell’s exterior surface. Phagocytic cells have receptors for PS, and recognize, bind to, and ingest cells that have committed to the apoptotic pathway, consuming them while they are still alive. In this way the apoptotic cell never has a chance to lyse and release inflammation-causing molecules to the extracellular space. Furthermore, a macrophage that recognizes a cell as apoptotic does not become activated. So the removal of apoptotic cells is physiological and silent, as would be appropriate for an event that occurs constantly in the normal human body. The correct removal of apoptotic cells is so vital that there are multiple mechanisms for their recognition, in addition to the PS system. The apoptotic cell dies inside the macrophage, before the membrane is permeable, preventing any inflammation.
morphogenetic death
A very important phenomenon during development that determines the final shape of body parts and organs. In limbs, the death by apoptosis of cells between the digits gives the final form to fingers and toes. In the nervous system, many more cells develop than the organism needs; those that form the correct contacts at the correct time are bathed in survival factors by the target they have innervated; if not, they are dispensable. Indeed, even the formation of as precise a structure as the brain depends on a Darwinian-style selection of cells that have chanced to make the best connections. Other local conditions could determine cell survival. For example, it has been shown very recently that cell shape, as influenced by the local tissue geometry, affects whether a cell will live or die.
Immune system and apoptosis
Apoptosis is very important in the immune system. In the thymus of the young rodent (and, we have pretty good evidence, human), 95-99% of the lymphocytes that develop there fail to be selected to mature as useful T cells, and die by apoptosis; the entire organ is replaced every 3 days. Clearly the process of generating effective, safe T cells is so exacting that most cells don’t make the cut.
cancer and apoptosis
it is thought that mutations that lead to cell growth are common, but tumors are rare. Perhaps as a small abnormal clone develops, it reaches a point where it exceeds the capacity of the microenvironment to provide growth and survival support, and involutes by apoptosis. But if, just before this critical period a second mutation or adaptation takes place, such that the cells are now more resistant to apoptosis, the clone may survive. There will be subsequent crises, and a new adaptation will be required each time; many experts estimate that it takes about 7 mutations for a cell to become fully, clinically, malignant. But this simple model stresses a key point: for cancer progression, mutations that inhibit death may be just as important as those that stimulate growth.
Bcl-2
Damage to the Bcl-2 gene has been identified as a cause of a number of cancers. Bcl-2 is specifically considered as an important anti-apoptotic protein and is thus classified as an oncogene.
cytochrome C role in apoptosis
Cytochrome c is also an intermediate in apoptosis, a controlled form of cell death used to kill cells in the process of development or in response to infection or DNA damage. Cytochrome c binds to cardiolipin in the inner mitochondrial membrane, thus anchoring its presence and keeping it from releasing out of the mitochondria and initiating apoptosis. While the initial attraction between cardiolipin and cytochrome c is electrostatic due to the extreme positive charge on cytochrome c, the final interaction is hydrophobic, where a hydrophobic tail from cardiolipin inserts itself into the hydrophobic portion of cytochrome c. During the early phase of apoptosis, mitochondrial ROS production is stimulated, and cardiolipin is oxidized by a peroxidase function of the cardiolipin–cytochrome c complex. The hemoprotein is then detached from the mitochondrial inner membrane and can be extruded into the soluble cytoplasm through pores in the outer membrane. The sustained elevation in calcium levels precedes cyt c release from the mitochondria. The release of small amounts of cyt c leads to an interaction with the IP3 receptor (IP3R) on the endoplasmic reticulum (ER), causing ER calcium release. The overall increase in calcium triggers a massive release of cyt c, which then acts in the positive feedback loop to maintain ER calcium release through the IP3Rs. This explains how the ER calcium release can reach cytotoxic levels. This release of cytochrome c in turn activates caspase 9, a cysteine protease. Caspase 9 can then go on to activate caspase 3 and caspase 7, which are responsible for destroying the cell from within.
Caspases
Caspases are essential in cells for apoptosis, or programmed cell death, in development and most other stages of adult life, and have been termed “executioner” proteins for their roles in the cell. Some caspases are also required in the immune system for the maturation of lymphocytes. Failure of apoptosis is one of the main contributions to tumour development and autoimmune diseases; this, coupled with the unwanted apoptosis that occurs with ischemia or Alzheimer’s disease, has stimulated interest in caspases as potential therapeutic targets since they were discovered in the mid-1990s.
THE INTRINSIC PATHWAY
involves perturbation of mitochondrial outer membrane function, either spontaneously or following withdrawal of growth factors or some other physiological or pathological signal. Normally the mitochondrial membrane is guarded by “anti-apoptotic” members of the Bcl-2 protein family, Bcl-2 and Bcl- XL. When the cell receives the suicide signal, “pro-apoptotic” members of the family such as Bim and PUMA are made; they move to the mitochondrion and replace Bcl-2 and Bcl-XL. This allows other members of the same family, Bax and Bax, to act on the membrane, making it permeable so it releases cytochrome C into the cytoplasm. That activates a cytoplasmic protein called Apaf-1. Finally, activated Apaf-1 activates the protease caspase-9, and it activates caspase-3, that eventually result in the classic appearance of apoptosis. caspase-9 is a signal caspase, caspase-3 an executioner. The details here are not for memorizing, though the signaling philosophy—death controlled by pro- and anti-apoptotic Bcl-2 factors—is very important.
THE EXTRINSIC PATHWAY
Cytotoxic (killer) T cells (CTL) are responsible for surveillance of the surfaces of all body cells. If a cytotoxic T cell recognizes that a cell is mutated or infected, it instructs the target cell to undergo apoptosis. Unneeded or undesirable lymphocytes are also eliminated by this mechanism. Different CTL seem to use one of two mechanisms to do this work. In one, the CTL upregulates expression of a surface molecule called Fas (or CD95) ligand (FasL, CD95L), which then engages and cross-links a corresponding molecule on the abnormal cell’s surface, Fas or CD95. CD95 transduces a signal into the cell’s interior, which recruits an adaptor molecule called FADD, which activates caspase-8. Like caspase-9 in the intrinsic pathway, caspase-8 then activates caspase-3. So the upstream process is different from that in the intrinsic pathway, but the downstream results are the same.
FAS receptor
Fas forms the death-inducing signaling complex (DISC) upon ligand binding. Membrane-anchored Fas ligand trimer on the surface of an adjacent cell causes oligomerization of Fas. Recent studies which suggested the trimerization of Fas could not be validated. Other models suggested the oligomerization up to 5-7 Fas molecules in the DISC.[6] This event is also mimicked by binding of an agonistic Fas antibody, though some evidence suggests that the apoptotic signal induced by the antibody is unreliable in the study of Fas signaling. To this end, several clever ways of trimerizing the antibody for in vitro research have been employed. Upon ensuing death domain (DD) aggregation, the receptor complex is internalized via the cellular endosomal machinery. This allows the adaptor molecule FADD to bind the death domain of Fas through its own death domain.
Fas-Associated protein with Death Domain (FADD)
an adaptor protein that bridges members of the tumor necrosis factor receptor superfamily, such as the Fas-receptor, to procaspases 8 and 10 to form the death-inducing signaling complex (DISC) during apoptosis. FADD also contains a death effector domain (DED) near its amino terminus,[8] which facilitates binding to the DED of caspase-8. Active caspase-8 is then released from the DISC into the cytosol, where it cleaves other effector caspases, eventually leading to DNA degradation, membrane blebbing, and other hallmarks of apoptosis.
Fas ligand (FasL)
It is expressed on cytotoxic T lymphocytes. Fas forms the death-inducing signaling complex (DISC) upon ligand binding. Membrane-anchored Fas ligand trimer on the surface of an adjacent cell causes trimerization of Fas receptor. Upon ensuing death domain (DD) aggregation, the receptor complex is internalized via the cellular endosomal machinery. This allows the adaptor molecule Fas-associated death domain (FADD) to bind the death domain of Fas through its own death domain. FADD also contains a death effector domain (DED) near its amino terminus, which facilitates binding to the DED of caspase-8. Active caspase-8 is then released from the DISC into the cytosol, where it cleaves other effector caspases, eventually leading to DNA degradation, membrane blebbing, and other hallmarks of apoptosis.
apoptosis mutations in ALPS
Recently, children with the condition named autoimmune lymphoproliferative syndrome (ALPS) have been identified. Their enormous lymphadenopathy suggests lymphoma or Hodgkin disease, although the pathogenesis is a failure of cells to die rather than uncontrolled proliferation; they have mutations in either Fas or FasL. Again it is worth considering that in every cell compartment of the adult at steady state, a cell must die for each one that divides. If proliferation exceeds death, the compartment grows; and this can happen because cells are dividing too fast, or not dying fast enough. This is a novel way of looking at malignancy.
FLIP
There is a protein related to caspase-8, called FLIP, which is however proteolytically-inactive. It competes with caspase-8 for binding to FADD, and thus inhibits apoptosis signaling (Fig. 4). Amazingly, there are viral FLIPs (v-FLIPs), known from herpes viruses such as HHV-8, the Kaposi’s sarcoma virus. Clever pathogens will develop (or, in the case of viruses, steal) anti- apoptotic genes to keep the cell alive until they can finish their replicative cycle. The cell becomes, for practical purposes, a zombie.
Cytotoxic T lymphocytes (CTLs)
a T lymphocyte (a type of white blood cell) that kills cancer cells, cells that are infected (particularly with viruses), or cells that are damaged in other ways. When exposed to infected/dysfunctional somatic cells, TC cells release the cytotoxins perforin, granzymes, and granulysin. Through the action of perforin, granzymes enter the cytoplasm of the target cell and their serine protease function triggers the caspase cascade, which is a series of cysteine proteases that eventually lead to apoptosis (programmed cell death). A second way to induce apoptosis is via cell-surface interaction between the TC and the infected cell. When a TC is activated it starts to express the surface protein FAS ligand (FasL)(Apo1L)(CD95L), which can bind to Fas (Apo1)(CD95) molecules expressed on the target cell. However, this Fas-Fas ligand interaction is thought to be more important to the disposal of unwanted T lymphocytes during their development or to the lytic activity of certain TH cells than it is to the cytolytic activity of TC effector cells. Engagement of Fas with FasL allows for recruitment of the death-induced signaling complex (DISC). The Fas-associated death domain (FADD) translocates with the DISC, allowing recruitment of procaspases 8 and 10. These caspases then activate the effector caspases 3, 6, and 7, leading to cleavage of death substrates such as lamin A, lamin B1, lamin B2, PARP (poly ADP ribose polymerase), and DNAPK (DNA-activated protein kinase). The final result is apoptosis of the cell that expressed Fas.
bacterial blockage of apoptosis
The nastiest E. coli bacteria induce terrible diarrhea by changing the physiology of colonic epithelial cells. To grow effectively the bacteria must keep the epithelial cells alive. They do so by making an enzyme that specifically glycosylates FADD, making it unable to activate caspase 8 and thus blocking apoptosis.
CELLULAR RESPONSES TO DAMAGE
As we noted, lymphocytes are more sensitive to radiation than any other cell. Why are lymphocytes so sensitive? It may be because they are so dangerous. A very minor change in the environment—the binding of antigen to the cell’s receptor—can drive a lymphocyte into rapid cycle, so that the one cell can become 64,000 cells within 4 days. If such a cell were to be damaged, perhaps mutated, the error would rapidly be locked into a substantial clone. This poses a risk of autoimmunity, or even lymphoma. So it seems reasonable that a damaged lymphocyte would respond not by repair, but by committing suicide. This is biologically sound, since the sole function of the body is to preserve and perpetuate gametes, and any single somatic cell may be sacrificed, if it presents a possible risk to the community of cells. We call this the “better dead than wrong” rule. Cells that are less risky, like fibroblasts, have the leisure to repair much more severe damage. So there is a continuum of response to injury: first, repair; if repair is impossible or unwise, apoptosis; if the damage is overwhelming, necrosis. For different cell types, the crossover to the next response will occur at different levels of damage. This could explain, for example, why certain toxins and chemicals are more harmful to specific tissues or cell types. At a more subtle level, if a population of cells were relatively resistant to apoptosis they might under some circumstances be more susceptible to malignant transformation—by their survival they would lock in mutations.
Endocytosis
There are two major routes of endocytosis: (1) phagocytosis and (2) pinocytosis or small vesicle formation.
phagocytosis
Phagocytosis in multicellular organisms is normally carried out by specialized cells in the blood, e.g., macrophages and neutrophils. These cells recognize foreign organisms like bacteria, engulf them, and deliver them to lysosomes for degradation. Macrophages and neutrophils also recognize apoptotic cells (one signal is negatively charged phosphatidylserine that moves from the inner to the outer leaflet) and aged cells (roughly 1011 of our red blood cells are phagocytosed every day).
Pinocytosis
Pinocytosis of vesicles involves small volumes, and usually is associated with specific uptake of ligands and receptors. Vesicles are typically formed by two mechanisms, either clathrin coat proteins or caveolae. Cargo molecules bind to a transmembrane receptor, which has a short motif on the cytoplasmic domain that is recognized and binds to an adaptor protein. An adaptor complex of proteins forms to enable a clathrin coat to assemble on a vesicle budding from the plasma membrane (or from the Golgi membrane in the secretory pathway). The vesicle remains attached to the membrane until dynamin pinches it off. The adaptor complex and clathrin rapidly dissociate from the endocytosed vesicle.
low density lipoprotein receptor (LDLR) as
cholesterol uptake is dependent on the LDL receptor. Before examining the details of the pathway, note the general scheme: LDLR is reutilized - it cycles between the surface and the lysosome and brings LDL particles to the lysosome, where they get degraded. LDLRs get clustered in membrane pits because an adaptor protein complex AP2 binds the receptor and also binds clathrin. Clathrin assembles over the surface of the nascent vesicle, dynamin pinches off the neck of membrane, creating the clathrin-coated vesicle. Shortly thereafter the clathrin coat disassembles, and this vesicle (the early endosome) moves to the next compartment (late endosome) in which the pH is low. The early endosome fuses with the late endosome and exposes the LDL particle to the lysosomal lumen, which is acidic. LDL dissociates from its receptor in the low pH. The LDLR is recycled to the plasma membrane and the LDL is broken down into cholesterol, fatty acids, and amino acids. The lifetime of an LDLR is about 20 hours and it goes through the cycle shown in the figure about once every ten minutes.
Caveolae
Caveolae (little cavities) are small endocytic vesicles that form without coat proteins. They are found in most cells and are thought to be especially important for membrane domains known as lipid rafts (regions high in cholesterol and signaling molecules). Some animal viruses and cholera toxin enter cells specifically through caveolae. Caveolin is the structural protein that is required for caveolae formation. Each vesicle contains 144 caveolins. Caveolin is also a scaffolding protein for coordinating protein complexes (see cartoon to right). There are three caveolin genes in humans (caveolin-1, -2, and -3). Caveolin-3 is expressed in skeletal and cardiac muscle; mutations in this gene cause Limb Girdle disease and Rippling Muscle disease.
Limb Girdle disease
an autosomal class of muscular dystrophy that is similar but distinct from Duchenne muscular dystrophy and Becker’s muscular dystrophy. Limb-girdle muscular dystrophy encompasses a large number of rare disorders. Currently, there is no cure and the disease inevitably worsens over time. LGMD is typically an inherited disorder, though it may be inherited as a dominant or recessive genetic defect. The result of the defect is that the muscles cannot properly form certain proteins needed for normal muscle function. Several different proteins can be affected, and the specific protein that is absent or defective identifies the specific type of muscular dystrophy. Among the proteins affected in LGMD are α, β, γ and δ sarcoglycans. The sarcoglycanopathies could be possibly amenable to gene therapy.
Rippling Muscle disease
Rippling muscle disease is a condition in which the muscles are unusually sensitive to movement or pressure (irritable). The muscles near the center of the body (proximal muscles) are most affected, especially the thighs. In most people with this condition, stretching the muscle causes visible ripples to spread across the muscle, lasting 5 to 20 seconds. A bump or other sudden impact on the muscle causes it to bunch up (percussion-induced muscle mounding) or exhibit repetitive tensing (percussion-induced rapid contraction). The rapid contractions can continue for up to 30 seconds and may be painful. Rippling muscle disease can be caused by mutations in the CAV3 gene. Muscle conditions caused by CAV3 gene mutations are called caveolinopathies. The CAV3 gene provides instructions for making a protein called caveolin-3, which is found in the membrane surrounding muscle cells. This protein is the main component of caveolae, which are small pouches in the muscle cell membrane. Within the caveolae, the caveolin-3 protein acts as a scaffold to organize other molecules that are important for cell signaling and maintenance of the cell structure. It may also help regulate calcium levels in muscle cells, which play a role in controlling muscle contraction and relaxation.
ways bacteria and viruses enter the cell
Viruses and bacteria are two environmental problems for humans that illustrate the usefulness of knowing different ways that cells take up materials from the extracellular space. Multiple portals exist for virus and bacterial entry into mammalian cells. Some examples are (1) Clathrin-mediated entry (e.g. vesicular stomatitis virus). (2) Fusion-entry (e.g. HIV). (3) Macropinocytosis-mediated entry (e.g. vaccinia virus). (4) Phagocytosis-like-mediated entry (e.g. herpes simplex virus). (5) Phagocytosis-mediated entry (e.g. bacteria). (6) Caveolin-mediated entry (e.g. simian virus 40).
Degradation
in the steady state, production and elimination are equal. All proteins have a characteristic lifetime, which for some proteins is minutes and for others can be days and weeks (even a lifetime for the lens of the eye). Moreover, even organelles have lifetimes and are being degraded and regenerated. Thus, protein and organelle degradation is vital for all cells. Two more examples emphasize the importance of degradation. A large fraction of proteins synthesized in the ER do not get properly folded and must be degraded to keep the cell healthy. Endocytosis brings both good and bad molecules and microbes into the cell and both need to be degraded (to utilize some components and destroy others).
cholera toxin (CTX or CT)
an oligomeric complex made up of six protein subunits: a single copy of the A subunit (part A), and five copies of the B subunit (part B), connected by a disulfide bond. The five B subunits form a five-membered ring that binds to GM1 gangliosides on the surface of the intestinal epithelium cells. The A1 portion of the A subunit is an enzyme that ADP-ribosylates G proteins, while the A2 chain fits into the central pore of the B subunit ring. Upon binding, the complex is taken into the cell via receptor-mediated endocytosis. Once inside the cell, the disulfide bond is reduced, and the A1 subunit is freed to bind with a human partner protein called ADP-ribosylation factor 6 (Arf6).[13] Binding exposes its active site, allowing it to permanently ribosylate the Gs alpha subunit of the heterotrimeric G protein. This results in constitutive cAMP production, which in turn leads to secretion of H2O, Na+, K+, Cl−, and HCO3− into the lumen of the small intestine and rapid dehydration.
