Structure and function of cholera toxin and the related Escherichia coli heat-labile enterotoxin.

Cholera and the related Escherichia coli-associated diarrheal disease are important problems confronting Third World nations and any area where water supplies can become contaminated. The disease is extremely debilitating and may be fatal in the absence of treatment. Symptoms are caused by the action of cholera toxin, secreted by the bacterium Vibrio cholerae, or by a closely related heat-labile enterotoxin, produced by Escherichia coli, that causes a milder, more common traveler's diarrhea. Both toxins bind receptors in intestinal epithelial cells and insert an enzymatic subunit that modifies a G protein associated with the adenylate cyclase complex. The consequent stimulated production of cyclic AMP, or other factors such as increased synthesis of prostaglandins by intoxicated cells, initiates a metabolic cascade that results in the excessive secretion of fluid and electrolytes characteristic of the disease. The toxins have a very high degree of structural and functional homology and may be evolutionarily related. Several effective new vaccine formulations have been developed and tested, and a growing family of endogenous cofactors is being discovered in eukaryotic cells. The recent elucidation of the three-dimensional structure of the heat-labile enterotoxin has provided an opportunity to examine and compare the correlations between structure and function of the two toxins. This information may improve our understanding of the disease process itself, as well as illuminate the role of the toxin in studies of signal transduction and G-protein function.     

Nitric oxide and ABA in the control of plant function

Abscisic acid (ABA) and nitric oxide (NO) are both extremely important signalling molecules employed by plants to control many aspects of physiology. ABA has been extensively studied in the mechanisms which control stomatal movement as well as in seed dormancy and germination and plant development. The addition of either ABA or NO to plant cells is known to instigate the actions of many signal transduction components. Both may have an influence on the phosphorylation of proteins in cells mediated by effects on protein kinases and phosphatases, as well as recruiting a wide range of other signal transduction molecules to mediate the final effects. Both ABA and NO may also lead to the regulation of gene expression. However, it is becoming more apparent that NO may be acting downstream of ABA, with such action being mediated by reactive oxygen species such as hydrogen peroxide in some cases. However not all ABA responses require the action of NO. Here, examples of where ABA and NO have been put together into the same signal transduction pathways are discussed.     

Vibrio cholerae infection of Drosophila melanogaster mimics the human disease cholera

Cholera, the pandemic diarrheal disease caused by the gram-negative bacterium Vibrio cholerae, continues to be a major public health challenge in the developing world. Cholera toxin, which is responsible for the voluminous stools of cholera, causes constitutive activation of adenylyl cyclase, resulting in the export of ions into the intestinal lumen. Environmental studies have demonstrated a close association between V. cholerae and many species of arthropods including insects. Here we report the susceptibility of the fruit fly, Drosophila melanogaster, to oral V. cholerae infection through a process that exhibits many of the hallmarks of human disease: (i) death of the fly is dependent on the presence of cholera toxin and is preceded by rapid weight loss; (ii) flies harboring mutant alleles of either adenylyl cyclase, Gsalpha, or the Gardos K channel homolog SK are resistant to V. cholerae infection; and (iii) ingestion of a K channel blocker along with V. cholerae protects wild-type flies against death. In mammals, ingestion of as little as 25 mug of cholera toxin results in massive diarrhea. In contrast, we found that ingestion of cholera toxin was not lethal to the fly. However, when cholera toxin was co-administered with a pathogenic strain of V. cholerae carrying a chromosomal deletion of the genes encoding cholera toxin, death of the fly ensued. These findings suggest that additional virulence factors are required for intoxication of the fly that may not be essential for intoxication of mammals. Furthermore, we demonstrate for the first time the mechanism of action of cholera toxin in a whole organism and the utility of D. melanogaster as an accurate, inexpensive model for elucidation of host susceptibility to cholera.     

cAMP-independent effects of cholera toxin on B cell activation. I. A possible role for cell surface ganglioside GM1 in B cell activation

Cholera toxin has been used as a tool to study the effects of cAMP on the activation of B cells but may have effects independent of its ability to elevate cAMP. We found five lines of evidence which suggested that cholera toxin suppressed mitogen-stimulated B cell activation through a cAMP-independent pathway. 1) Cholera toxin (1 microgram/ml) was consistently more suppressive than forskolin (100 microM) despite the induction of higher intracellular cAMP levels by forskolin. 2) Cholera toxin was more suppressive at 1 microgram/ml than at 0.1 microgram/ml despite equivalent elevations of cAMP. 3) Washing B cells following their incubation with cholera toxin reversed much of the inhibition without altering intracellular cAMP levels. 4) The A subunit of cholera toxin, which at high concentrations (10 micrograms/ml) induced levels of cAMP comparable to those induced by cholera toxin (1 and 0.1 microgram/ml), did not inhibit B cell activation. 5) cAMP derivatives at high concentrations were much less effective than was cholera toxin in suppressing B cell activation. Although the elevation of cAMP may cause a mild inhibition of B cell proliferation, we found that even a marked elevation of cAMP did not suppress B cell proliferation, unless the elevation was persistent. We did, however, observe that the degree of toxin inhibition more closely paralleled binding of the toxin to B cells than toxin stimulation of cAMP. This result raised the possibility that binding of cholera toxin to its ganglioside GM1 receptor mediated an inhibitory signal which suppressed B cell proliferation.     

Cholera Threat to Humans in Ghana Is Influenced by Both Global and Regional Climatic Variability

Cholera, an acute diarrheal illness, is caused by infection of the intestine with the bacterium Vibrio cholerae after ingestion of contaminated water or food. The disease had disappeared from most of the developed countries in the last 50 years, but cholera epidemics remain a major public health problem in many developing countries, most often localized in tropical areas. Cholera is an infectious disease for which a relationship between disease temporal patterns and climate has been demonstrated, but only in an endemic context and for local areas of Asia and South America. Until now, similar studies have not been done in an epidemic context, on the African continent, although the largest number of cholera cases has been reported for those countries by the World Health Organization. The wavelet method was used in order to explore periodicity in (i) a long-time monthly cholera incidence in Ghana, West Africa, (ii) proxy environmental variables, and (iii) climatic indices time series, from 1975 to 1995. Cross-analysis were done to explore links between these time series, i.e., between cholera and climate. Results showed strong statistical association (coherency) from the end of the 1980s, between cholera outbreak resurgences in Ghana and the climatic/environmental parameters under scrutiny. Further examination of the existence of common spatial and temporal patterns in infectious diseases on the continent of Africa will permit development of more effective treatment of disease.     

Crystal structure of cholera toxin B-pentamer bound to receptor GM1 pentasaccharide.

Cholera toxin (CT) is an AB5 hexameric protein responsible for the symptoms produced by Vibrio cholerae infection. In the first step of cell intoxication, the B-pentamer of the toxin binds specifically to the branched pentasaccharide moiety of ganglioside GM1 on the surface of target human intestinal epithelial cells. We present here the crystal structure of the cholera toxin B-pentamer complexed with the GM1 pentasaccharide. Each receptor binding site on the toxin is found to lie primarily within a single B-subunit, with a single solvent-mediated hydrogen bond from residue Gly 33 of an adjacent subunit. The large majority of interactions between the receptor and the toxin involve the 2 terminal sugars of GM1, galactose and sialic acid, with a smaller contribution from the N-acetyl galactosamine residue. The binding of GM1 to cholera toxin thus resembles a 2-fingered grip: the Gal(beta 1-3)GalNAc moiety representing the "forefinger" and the sialic acid representing the "thumb." The residues forming the binding site are conserved between cholera toxin and the homologous heat-labile enterotoxin from Escherichia coli, with the sole exception of His 13. Some reported differences in the binding affinity of the 2 toxins for gangliosides other than GM1 may be rationalized by sequence differences at this residue. The CTB5:GM1 pentasaccharide complex described here provides a detailed view of a protein:ganglioside specific binding interaction, and as such is of interest not only for understanding cholera pathogenesis and for the design of drugs and development of vaccines but also for modeling other protein:ganglioside interactions such as those involved in GM1-mediated signal transduction.     

Modulation of meiotic arrest in mouse oocytes by guanyl nucleotides and modifiers of G-proteins.

Guanyl nucleotide binding-proteins, or G-proteins, are ubiquitous molecules that are involved in cellular signal transduction mechanisms. Because a role has been established for cAMP in meiosis and G-proteins participate in cAMP-generating systems by stimulating or inhibiting adenylate cyclase, the present study was conducted to examine the possible involvement of G-proteins in the resumption of meiotic maturation. Cumulus cell-free mouse oocytes (denuded oocytes) were maintained in meiotic arrest in a transient and dose-dependent manner when microinjected with the nonhydrolyzable GTP analog, GTP gamma S. This effect was specific for GTP gamma S, because GppNHp, GTP, and ATP gamma S were without effect. Three compounds, known to interact with G-proteins, were tested for their ability to modulate meiotic maturation: pertussis toxin, cholera toxin, and aluminum fluoride (AlF4-). Pertussis toxin had little effect on maturation in either cumulus cell-enclosed oocytes or denuded oocytes when meiotic arrest was maintained with dibutyryl cAMP (dbcAMP) or hypoxanthine. Cholera toxin stimulated germinal vesicle breakdown (GVB) in cumulus cell-enclosed oocytes during long-term culture, but its action was inhibitory in denuded oocytes. AlF4- stimulated GVB in both cumulus cell-enclosed oocytes and denuded oocytes when meiotic arrest was maintained with hypoxanthine but was much less effective in dbcAMP-arrested oocytes. In addition, AlF4- abrogated the inhibitory action of cholera toxin in denuded oocytes and also that of follicle-stimulating hormone (FSH) in cumulus cell-enclosed oocytes. Cholera toxin or FSH alone each stimulated the synthesis of cAMP in oocyte-cumulus cell complexes, whereas pertussis toxin or AlF4- alone were without effect. Both cholera toxin and AlF4- augmented the stimulatory action of FSH on cAMP. These data suggest the involvement of guanyl nucleotides and G-proteins in the regulation of GVB, although different G-proteins and mediators may be involved at the oocyte and cumulus cell levels. Cholera toxin most likely acts by ADP ribosylation of the alpha subunit of Gs and increased generation of cAMP, whereas AlF4- appears to act by antagonizing a cAMP-dependent step.     

Meddling Vibrio cholerae Murmurs: A Neoteric Advancement in Cholera Research

Cholera, a known diarrheal disease is associated with various risk factors like hypovolemic shock, rice watery stools, and death in developing countries. The overuse of antibiotics to treat cholera imposed a selective pressure for the emergence and spread of multi-drug resistant Vibrio cholerae strains. The failure of conventional antimicrobial therapy urged the researchers to find an alternative therapy that could meddle the cholera murmurs (Quorum Sensing). It seems to effectively overcome the conventional cholera therapies in parallel to decrease the morbidity and mortality rate in the developing countries. The paramount objective of this review essentially focuses on the different Quorum Sensing (QS) regulatory switches governing virulence and pathogenicity of Vibrio cholerae. This review also provides an insight into the plausible QS targets that could be exploited to bring about a breakthrough to the prevailing cholera therapy.     

Hormone replacement therapy.

Hormone replacement therapy is increasingly being used for purposes unrelated to the alleviation of menopausal symptoms, such as the prevention of osteoporosis and cardiovascular disease. Clinical trials, however, suggest that the one drug/many purposes concept may be too optimistic. The availability of new estrogen-like compounds and the discovery of a second estrogen receptor have opened new possibilities for more specific drug development.     

Transduction of membrane tension by the ion channel alamethicin.

Mechanoelectrical transduction in biological cells is generally attributed to tension-sensitive ion channels, but their mechanisms and physiology remain controversial due to the elusiveness of the channel proteins and potential cytoskeletal interactions. Our discovery of membrane tension sensitivity in ion channels formed by the protein alamethicin reconstituted into pure lipid membranes has demonstrated two simple physical mechanisms of cytoskeleton-independent transduction. Single channel analysis has shown that membrane tension energizes mechanical work for changes of conductance state equal to tension times the associated increase in membrane area. Results show a approximately 40 A2 increase in pore area and transfer of an 80-A2 polypeptide into the membrane. Both mechanisms may be implicated in mechanical signal transduction by cells.     

The Role of eNSCs in Neurodegenerative Disease

Recent progress in biology has shown that many if not all adult tissues contain a population of stem cells. It is believed that these cells are involved in the regeneration of the tissue or organ in which they reside as a response to the natural turnover of differentiated cells or to injury. In the adult mammalian brain, stem cells in the subventricular zone and the dentate gyrus may also play a role in the replacement of neurons. A positive beneficial response to injury does not necessarily require cell replacement. New findings suggest that some populations of endogenous neural stem cells in the central nervous system may have adopted a function different from cell replacement and are involved in the protection of neurons in diverse paradigms of disease and injury. In this article, we will focus on the immature cell populations of the central nervous system and the signal transduction pathways that regulate them which suggest new possibilities for their manipulation in injury and disease.     

Jak2 tyrosine kinase

Discovered roughly 10 yr ago, Jak2 tyrosine kinase has emerged as a critical molecule in mammalian development, physiology, and disease. Here, we review the early history of Jak2 and its role in health and disease. We will also review, its critical role in mediating cytokine-dependent signal transduction. Additionally, more recent work demonstrating the importance of Jak2 in G protein-coupled receptor and tyrosine kinase growth factor receptor signal transduction will be discussed. The cellular and biochemical mechanisms by which Jak2 tyrosine kinase is activated and regulated within the cell also will be reviewed. Finally, structure-function and pharmacological-based studies that identified structural motifs and amino acids within Jak2 that are critical for its function will be examined. By reviewing the biology of Jak2 tyrosine kinase at the molecular. cellular, and physiological levels, we hope to advance the understanding of how a single gene can have such a profound impact on development, physiology, and disease.     

The unfolded protein response: no longer just a special teams player

The endoplasmic reticulum stress pathway known as the unfolded protein response is currently the best understood model of interorganellar signal transduction. Bridging a physical separation, the pathway provides a direct line of communication between the endoplasmic reticulum lumen and the nucleus. With the unfolded protein response, the cell has the means to monitor and respond to the changing needs of the endoplasmic reticulum. Beginning with the discovery of its remarkable signaling mechanism in yeast, the unfolded protein response has not ceased to reveal more of its many secrets. By applying powerful biochemical, genetic, genomic, and cytological approaches, the recent efforts of many groups have buried the long-held notion that the unfolded protein response is simply a regulatory platform for endoplasmic reticulum chaperones. We now know that the unfolded protein response regulates many genes that affect diverse aspects of cellular physiology. In addition, studies in mammals have revealed novel unfolded protein response signaling factors that may contribute to the specialized needs of multicellular organisms. This article focuses on these and other recent developments in the field.     

A protocol to assess cell cycle and apoptosis in human and mouse pluripotent cells

Embryonic stem cells (ESC) and induced pluripotent stem cells (iPSCs) present a great opportunity to treat and model human disease as a cell replacement therapy. There is a growing pressure to understand better the signal transduction pathways regulating pluripotency and self-renewal of these special cells in order to deliver a safe and reliable cell based therapy in the near future. Many signal transduction pathways converge on two major cell functions associated with self-renewal and pluripotency: control of the cell cycle and apoptosis, although a standard method is lacking across the field. Here we present a detailed protocol to assess the cell cycle and apoptosis of ESC and iPSCs as a single reference point offering an easy to use standard approach across the field.     

Jak2 tyrosine kinase: a true jak of all trades?

Discovered roughly 10 yr ago, Jak2 tyrosine kinase has emerged as a critical molecule in mammalian development, physiology, and disease. Here, we review the early history of Jak2 and its role in health and disease. We will also review its critical role in mediating cytokine-dependent signal transduction. Additionally, more recent work demonstrating the importance of Jak2 in G protein-coupled receptor and tyrosine kinase growth factor receptor signal transduction will be discussed. The cellular and biochemical mechanisms by which Jak2 tyrosine kinase is activated and regulated within the cell also will be reviewed. Finally, structure-function and pharmacological-based studies that identified structural motifs and amino acids within Jak2 that are critical for its function will be examined. By reviewing the biology of Jak2 tyrosine kinase at the molecular, cellular, and physiological levels, we hope to advance the understanding of how a single gene can have such a profound impact on development, physiology, and disease.     

A protein switch with tunable steepness reconstructed in Escherichia coli cells with eukaryotic signaling proteins

Cholera is a disease which shows a clear blood group profile, with blood group O individuals experiencing the most severe symptoms. For a long time, the cholera toxin has been suspected to be the main culprit of this blood group dependence. Here, we show that both El Tor and classical cholera toxin B-pentamers do indeed bind blood group determinants (with equal affinities), using Surface Plasmon Resonance and NMR spectroscopy. Together with previous structural data, this confirms our earlier hypothesis as to the molecular basis of cholera blood group dependence, with an interesting twist: the shorter blood group H-determinant characteristic of blood group O individuals binds with similar binding affinity compared to the A-determinant, however, with different kinetics.     

Glucose- but Not Rice-Based Oral Rehydration Therapy Enhances the Production of Virulence Determinants in the Human Pathogen Vibrio cholerae

Despite major attempts to prevent cholera transmission, millions of people worldwide still must address this devastating disease. Cholera research has so far mainly focused on the causative agent, the bacterium Vibrio cholerae, or on disease treatment, but rarely were results from both fields interconnected. Indeed, the treatment of this severe diarrheal disease is mostly accomplished by oral rehydration therapy (ORT), whereby water and electrolytes are replenished. Commonly distributed oral rehydration salts also contain glucose. Here, we analyzed the effects of glucose and alternative carbon sources on the production of virulence determinants in the causative agent of cholera, the bacterium Vibrio cholerae during in vitro experimentation. We demonstrate that virulence gene expression and the production of cholera toxin are enhanced in the presence of glucose or similarly transported sugars in a ToxR-, TcpP- and ToxT-dependent manner. The virulence genes were significantly less expressed if alternative non-PTS carbon sources, including rice-based starch, were utilized. Notably, even though glucose-based ORT is commonly used, field studies indicated that rice-based ORT performs better. We therefore used a spatially explicit epidemiological model to demonstrate that the better performing rice-based ORT could have a significant impact on epidemic progression based on the recent outbreak of cholera in Haiti. Our results strongly support a change of carbon source for the treatment of cholera, especially in epidemic settings.     

Adhesion of Vibrio cholerae to granular starches

Cholera is a severe diarrheal disease caused by specific serogroups of Vibrio cholerae that are pathogenic to humans. Cholera can become epidemic and deadly without adequate medical care. Appropriate rehydration therapy can reduce the mortality rate from as much as 50% of the affected individuals to <1%. Thus, oral rehydration therapy (ORT) is an important measure in the treatment of this disease. To further reduce the symptoms associated with cholera, improvements in oral rehydration solution (ORS) by starch incorporation were suggested. Here, we report that V. cholerae adheres to starch granules incorporated in ORS. Adhesion of 98% of the cells was observed within 2 min when cornstarch granules were used. Other starches showed varied adhesion rates, indicating that starch source and composition play an important role in the interaction of V. cholerae and starch granules. Sugars metabolized by V. cholerae showed a repressive effect on the adhesion process. The possible mechanisms involved are discussed. Comparing V. cholerae adhesion with the adhesion of other pathogens suggests the involvement of starch degradation capabilities. This adhesion to granular starch can be used to improve ORT.     

Sphingolipids and cell death

Sphingolipids (SLs) have been considered for many years as predominant building blocks of biological membranes with key structural functions and little relevance in cellular signaling. However, this view has changed dramatically in recent years with the recognition that certain SLs such as ceramide, sphingosine 1-phosphate and gangliosides, participate actively in signal transduction pathways, regulating many different cell functions such as proliferation, differentiation, adhesion and cell death. In particular, ceramide has attracted considerable attention in cell biology and biophysics due to its key role in the modulation of membrane physical properties, signaling and cell death regulation. This latter function is largely exerted by the ability of ceramide to activate the major pathways governing cell death such as the endoplasmic reticulum and mitochondria. Overall, the evidence so far indicates a key function of SLs in disease pathogenesis and hence their regulation may be of potential therapeutic relevance in different pathologies including liver diseases, neurodegeneration and cancer biology and therapy.     

Illuminating the complexity of GPCR pathway selectivity – advances in biosensor development

It should come as no surprise that G protein-coupled receptors (GPCRs) continue to occupy the focus of drug discovery efforts. Their widespread expression and broad role in signal transduction underline their importance in human physiology. Despite more than 800 GPCRs sharing a common architecture, unique differences govern ligand specificity and pathway selectivity. From the relatively simplified view offered by classical radioligand binding assays and contractility responses in organ baths, the road from ligand binding to biological action has become more and more complex as we learn about the molecular mediators that underly GPCR activation and translate it to physiological outcomes. In particular, the development of biosensors has evolved over the years to dissect the capacity of a given receptor to activate individual pathways. Here, we discuss how recent biosensor development has reinforced the idea that biased signaling may become mainstream in drug discovery programs.