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Immunol Commun, 1976, 5(5), 387 - 400 Immunological probes into the mechanism of cholera toxin action; Craig SW et al.; The use of antibodies to specific cell surface proteins or to ligands which interact with cell surface receptors is a powerful tool for analyzing the properties of membrane proteins and the consequences of specific cell surface ligand-receptor interactions . Two central observations concerning membrane structure and function, - the diffusibility of membrane proteins (1) and ligand-triggered modulation of specific receptors (2), have derived from the use of antibodies to analyze the properties of membrane proteins . In our study of the mechanism of action of cholera toxin, a protein which binds to a specific cell surface receptor and results in the activation of adenyl cyclase, considerable information has been gained through the use of immunological techniques . This review will briefly summarize the data underlying our current concept of cholera toxin action at the cell membrane and will emphasize those observations made through the use of immunological approaches. Immunol Commun, 1976, 5(9), 737 - 56 Cholera toxin, ganglioside receptors and the immune response; Holmgren J et al.; Cholera toxin activates plasma membrane adenylate cyclase in all mammalian cell types . The structure-function relationship of the toxin has recently been clarified, and the cell membrane receptor identified . This information has made cholera toxin the "agent of choice" for studies in many biological systems of the possible regulatory role of adenylate cyclase/cyclic AMP . This article describes briefly our current knowledge about the toxin and its receptor . It then reviews recent research which has revealed that cholera toxin has strong modulating influences on the proliferation of normal and malignant lympocytes as well as on the initiation and expression of immune responses . The toxin has been found to inhibit DNA synthesis of B and T lymphocytes in vitro without inducing cell death and also to inhibit seems to decrease antibody secretion from plasma cells in vitro, and might also interfere with the release of other soluble immunological mediator subtances . In vivo cholera toxin induces a transient involution of the spleen and a more prolonged lymphocyte depletion of the thymus in mice; these effects appear to be mediated through the adrenal glands . The toxin inhibitors allograft rejection, and either stimulates or suppresses antibody formation depending on the timing of the toxin in relation to the antigen administration . It increases the capacity of the spleen cells to induce graft-vs-host reactions and the "allogeneic effect" on antibody production . An inhibitory effect on a normal suppressor population among the spleen cells has been identified . The findings illustrate the complex effects induced on the immune system by the probably most discriminative investigative tool available for stimulation of the adenylate cyclase/cyclic AMP system. Adv Prostaglandin Thromboxane Res, 1976, 1, 331 - 5 Differential effects of prostaglandin synthetase inhibitors on prostaglandin E2 binding and on prostaglandin- or cholera toxin-induced cyclic AMP accumulation in the rabbit uterus; Zor U et al.; Cyclic 3',5'-nucleotide phosphodiesterase (PDE) activity in rabbit uterine homogenate was inhibited by indomethacin (10 mug/ml; 66% inhibition) or flufenamic and (10 mug/ml; 60%) . Indomethacin (100 mug/ml) reduced uterine prostaglandin E2 (PGE2) content by 80%, but potentiated the stimulatory action of purified cholera toxin (choleragen; 800%) and of exogenous PGE2 (140%) on cyclic AMP accumulation, probably through its inhibitory effect on cyclic AMP destruction . These findings suggest that endogenous PGE2 is not an essential mediator of choleragen action . By contrast, flufenamic acid abolished choleragen and PGE2 action on cyclic AMP production . Unlabeled PGE2 (10 mug/ml), flufenamic acid, indomethacin, and aspirin (100 mug/ml each) inhibited {3H}PGE2 binding to uterine slices by 78, 73, 62, and 20% respectively . It is concluded that while indomethacin and flufenamic acid have similar effects on prostaglandin biosynthesis and PDE activity, only fenamates have an inhibitory effect on the biological action of exogenous PGE2 and choleragen on the stimulation of cyclic AMP production, probably through the inhibition of the binding of PGE2 and choleragen to its specific receptor sites . The diverse biochemical actions of the above drugs indicate that care has to be taken when using these drugs in analyzing the physiopathological roles of prostaglandins. Ciba Found Symp, 1976, (42), 89 - 108 The activation of adenylate cyclase by cholera toxin: possible interaction with the nucleotide regulatory site; Flores J et al.; The application of cholera toxin to intact cells causes a stimulation of adenylate cyclase activity . The effect is characterized by a lag period followed by a progressive rise in enzyme activity over several hours . Only a few minutes' exposure to the toxin is required to produce effects lasting over several days . Stimulation of adenylate cyclase by cholera toxin in broken cell preparations requires the presence of nicotinamide-adenine dinucleotide (NAD) and an unidentified component of the cytosol . Guanyl nucleotides and certain non-hydrolysable analogues of guanosine triphosphate also stimulate adenylate cyclase . Stimulation by the analogues results in a highly activated enzyme which has characterisitcs similar to those of adenylate cyclase after stimulation by cholera toxin . Thus the stimulation is irreversible, the enzyme may be "solubilized" by non-ionic detergents in the activated state, and responses to certain hormones are enhanced . Therefore the possibility exists that cholera toxin acts on the guanyl nucleotide regulatory protein of the adenylate cyclase complex . In exploring this possibility it was found pretreatment with cholera toxin not only blocked the stimulatory effect of subsequently added guanylylimidodi-phosphate (GppNHp) but that the latter reduced the stimulation by toxin . Similarly, pretreatment with GppNHp blocked the effect of cholera toxin . The similarities in the effects of cholera toxin and GppNHp, together with the mutual interference of their activities, suggests that cholera toxin acts at the same regulatory site at which guanyl nucleotides exert their effects on adenylate cyclase. Int Arch Allergy Appl Immunol, 1976, 50(5), 555 - 73 Interaction of cholera toxin and toxin derivatives with lymphocytes . II . Modulating effects of cholera toxin on in vivo humoral and cellular immune responses; Lindholm L et al.; The in vivo effects of cholera toxin on lymphoid organ structure and function in mice were investigated . It was found that within a day following intravenous injection of 1 mug of toxin, thymus as well as spleen weight decreased but the animals remained healthy . Histological studies suggested that the involution of lymphoid organs was due to cell death . Injection of cholera toxin into adrenalectomized mice was lethal within 36 h . In these animals no decrease in lymphoid organ weight was noted . Thymus cells from toxin-treated mice were found to be much inferior to thymocytes of untreated animals in their in vitro response to Concanavalin A, whereas the response of spleen cells from toxin-treated animals to mitogens was slightly increased . 1 mug of cholera toxin increased primary antibody formation when given to mice together with antigen (sheep erythrocytes) and decreased primary antibody formation when given before or after the antigen . The toxin also increased secondary antibody formation when injected simultaneously with or after the booster antigen dose, and decreased the antibody formation when given a few days before the booster injection . Treatment of mice with toxin was found to increase the capacity of spleen cells from these animals to induce the parental effect on antibody formation and to induce graft-versus-host reactions . The mechanisms behind the observed effects are discussed . It is suggested that cholera toxin affects different types of cells involved in immune responses primarily by a direct inhibitory action on cellular proliferation but also indirectly by causing release of adrenal gland hormones. J Exp Med, 1975 Dec 1, 142(6), 1550 - 63 Cellular kinetics of the intestinal immune response to cholera toxoid in rats; Pierce NF et al.; The aims of this study were (a) to find a regime of immunization with cholera toxoid in rats which would establish a high density of antitoxin containing cells (ACC) in the lamina propria of the intestine and (b) to determine the origin of the ACC . The best cellular response was achieved by a single i.p . dose of toxoid in FCA followed by an intraintestinal boost 2 wk later . ACC appeared in the thoracic duct lymph 2 days after boosting, reaching a peak of about 200,000 ACC/h at 3--4 days . This was followed by the appearance of large numbers of ACC in the intestine . The i.p . dose of toxoid by itself gave rise to very few ACC in the gut or thoracic duct lymph, but it had clearly primed the gut immune system for a secondary response . Priming was also achieved by the prolonged oral intake of toxoid . The importance of the intestinal route for boosting was shown by the failure of i.p . challenge to give an ACC response in the intestine after i.p . priming and the small response it provoked after oral priming . ACC among thoracic duct lymphocytes (TDL) and in the lamina propria contained predominantly IgA . Two observations indicated that the major source of the lamina propria ACC was from cells that emerged in the thoracic duct lymph after intraintestinal challenge . Firstly, the establishment of a thoracic duct fistula immediately before challenge prevented the appearance of ACC in the intestine . Secondly, many ACC appeared in the intestine of normal rats after the injection of TDL rich in ACC . Although homing of ACC precursors to the gut was not antigen-dependent, the distribution of ACC in the lamina propria was considerably influenced by the site of the intestinal challenge, the density of ACC being greatest at or distal to the site of injection of toxoid into the lumen of the gut. J Gen Microbiol, 1975 Dec, 91(2), 263 - 77 Protein reagent modification of cholera toxin: characterization of effects on antigenic, receptor-binding and toxic properties; Lonnroth I et al.; The effects of protein modification procedures on the biologically most important properties of cholera toxin, i.e . the toxic activity, the GM1 receptor-binding capacity and the antigenic (antibody-fixing) properties, have been studied quantitatively using microgram amounts or less of toxin protein . Most of the 24 group-specific reagents used had either no inhibitory effect on the toxic or the combination of GM1-binding and antibody-fixing properties of cholera toxin, or they had a concomitant inhibitory effect on these activities . Separate testing of GM1- and antibody-binding revealed a close, but not absolute, structural association between these properties, Amino group reactive substances were particularly effective in decreasing the GM1-binding activity, while leucine aminopeptidase had no effect . This suggests that lysine residues may be involved in binding toxin to the acidic GM1 receptor . Sodium dodecylsulphate and mercaptoethanol, which caused dissociation of the subunits of cholera toxin as indicated by polyacrylamide gel electrophoresis, abolished toxicity without inhibiting the concomitant GM1- and antibody-binding properties of the toxin . Similar differential effects were also obtained with three reagents which did not seem to change the aggregation state of the toxin . These substances all had specificity for arginine, suggesting that arginyl residues of the toxin molecule may be involved in a 'toxic site' distinct from the receptor-binding site(s) . A selective effect on the toxic site was also found by treating the toxin with carboxypeptidase or trypsin in the presence of urea; in the absence of urea no enzymic effect on any toxin property was noted. Ann Intern Med, 1975 Dec, 83(6), 782 - 5 Islet cell carcinoma, pancreatic cholera, and vasoactive intestinal peptide; Graham DY et al.; Three patients with profuse diarrhea, hypokalemia, metastatic nonbeta islet cell carcinoma, and the absence of gastric hypersecretion were found to have elevated levels of vasoactive intestinal peptide . One patient also had elevated serum gastrin levels . Two patients experienced prolonged remissions of diarrhea after operations in which only tumor biopsies were done . These cases further implicate vasoactive intestinal peptide as the agent mediating the diarrhea in the syndrome of pancreatic cholera. J Clin Invest, 1975 Nov, 56(5), 1345 - 9 Effects of cholera toxin on adenylate cyclase . Studies with guanylylimidodiphosphate; Flores J et al.; Similarities exist between the properties of adenylate cyclase after stimulation by cholera toxin and after stimulation by guanylylimidodiphosphate (Gpp-(NH)p) . Thus a strong stimulation is achieved by both agents, the stimulation is essentially irreversible, the action of certain hormones is enhanced and the enzyme can be solublized with Lubrol PX in the activated state . Because of these similarities the interaction of cholera toxin and Gpp(NH)p on adenylate cyclase was examined . It was found that prior activation of rat liver adenylate cyclase by cholera toxin in vivo, or by cholera toxin and NAD in homogenates, blocked the stimulatory effect of Gpp(NH)p . Furthermore under conditions in which the effect of Gpp(NH)p was less than that of cholera toxin, inhibition of stimulation by cholera toxin was seen . Stimulation of adenylate cyclase by maximal concentrations of Gpp(NH)p, but not by submaximal concentrations, blocked the stimulatory effect of cholera toxin . The mutant interference of the actions of these two agents suggests a common target in the regulatory mechanism of the adenylate cyclase complex. Cancer Res, 1975 Nov, 35(11 Pt 1), 3009 - 13 Cholera toxin effects on cell growth accompanied by selective alterations in metabolite uptake and modification of cell surface proteins; Rieber M et al.; Exposure of Chinese hamster ovary cells to cholera toxin at or below mug levels causes a marked morphological changes and increased adhesion and orientation of the cells . Such changes are paralleled by alterations in surface proteins as indicated by the cholera toxin-mediated modifications detectable by lactoperoxidase-catalyzed radioiodination of outer proteins . Mild tryptic treatment of cells prelabeled with {3H}glucosamine revealed a different kinetics of release of external glycoproteins in cells exposed to the toxin . An alteration in a specific glycoprotein species in cholera toxin-treated cells became evident by polyacrylamide gel electrophoresis followed by fluorography of 3H-labeled cellular glycoproteins . The effects of cholera toxin on surface proteins and growth of the cells occurred in the absence of a modification in amino acid uptake or incorporation of precursors into protein . However, thymidine uptake and glucosamine incorporation were inversely affected to toxin treatment . Some of the effects of the toxin appeared to be antagonized by colchicine. Am J Dig Dis, 1975 Nov, 20(11), 1035 - 9 Prostaglandin E in cholera toxin-induced intestinal secretion . Lack of an intermediary role; Hudson N et al.; Prostaglandin E1 (PGE1) and cholera enterotoxin stimulate small-intestine mucosal adenylate cyclase and intestinal secretion of water and electrolytes . The previous suggestion that PGE may mediate cholera-toxin effects was explored in these studies . Closed rabbit jejunal loops were injected in vivo with cholera toxin and compared to similar loops in the same animal injected with buffer . Loop mucosal homogenates and intestinal secretions were analyzed by radioimmunoassay for cAMP and PGE concentrations . Cholera toxin produced significant increases in mucosal and intestinal fluid cAMP; however, there were no significant increases in PGE in the toxin-treated loops when compared to the control loops . In addition, there was no correlation between cAMP and PGE in the same samples . These studies indicate that cholera toxin stimulates intestinal cAMP anc secretion independent of PGE synthesis and provide evidence against a specific role for PGE in mediating cholera-toxin effects. Biochim Biophys Acta, 1975 Oct 9, 404(2), 221 - 30 On the mechanism of action of cholera toxin on isolated rat adrenocortical cells . Comparison with the effects of adrenocorticotropin on steroidogenesis and cyclic AMP output; Palfreyman JW et al.; The effects of cholera toxin on isolated rat adrenocortical cells have been investigated . Both steroid and cyclic AMP output from adrenal cells were increased by the toxin in a dose dependent fashion . The concentration of toxin for half maximal stimulation for both of these responses was about 40 ng/ml . Maximal steroidogenesis and cyclic AMP output was obtained with similar concentrations of the toxin . A correlation was observed between the low amounts of cyclic AMP produced in response to all doses of cholera toxin and to physiologically significant concentrations of adrenocorticotropin (ACTH) (less than 0.1 munit/ml; i.e . submaximal for steroidogenesis in this system) . This was in direct contrast to the much higher levels of cyclic AMP generated by concentrations of ACTH greater than 1 munits/ml . Time course studies demonstrated a time-lag between toxin addition and steroid response of at least 40 min . Binding of cholera toxin to adrenal cells was rapid and was 90% complete within 15 min at both 37 and 0 degrees C . These data indicate that most of the delay in response to cholera toxin is due to processes subsequent to the initial binding interaction . Following the initial delay the subsequent maximal rate of steroidogenesis brought about by cholera toxin was very similar to that obtained with a concentration of ACTH that was maximal for steroidogenesis . Significant increases in cyclic AMP levels were detected about 20 min before increased steroidogenesis was apparent . Possible explanations for this result are considered . The results presented indicate great potential use for cholera toxin in the study of adrenal steroidogenic control mechanisms, particularly at the level of receptor mechanisms and the role of cyclic AMP. Can J Comp Med, 1975 Oct, 39(4), 472 - 3 Stabilization of hog cholera virus by dimethyl sulfoxide; Tessler J et al.; The stability of hog cholera virus through five freeze-thaw cycles in the presence and absence of dimethyl sulfoxide was studied . In the absence of dimethyl sulfoxide the hog cholera virus titer was reduced 52% to 91% following successive freezing and thawing cycles . However, when dimethyl sulfoxide was added to the viral suspension the virus titer appeared to remain the same after the same number of freezing and thawing cycles. Proc Natl Acad Sci U S A, 1975 Oct, 72(10), 3844 - 8 Mobility of cholera toxin receptors on rat lymphocyte membranes; Craig SW et al.; Fluorescein-labeled cholera toxin binds detectably to 40-60% of rat mesenteric lymph node cells and induces a temperature-dependent redistribution (patch and cap formation) of cell surface toxin receptors . The redistribution is inhibited by several "metabolic," "microtubule," and "microfilament" inhibitors, by concanavalin A, and by anticholera toxin IgG . Various studies indicate that cholera toxin is at least bivalent, and that this property may be related to both the induction of receptor redistribution and to the activation of adenylate cyclase . Membrane components which are probably identical to the sialo-glycolipid, GM1 ganglioside, appear to be mobile in the plane of the membrane . The possible role of toxin multivalency and receptor mobility in the mechanism of toxin action is considered. Bangladesh Med Res Counc Bull, 1975 Oct, 1(2), 65 - 71 Thermal inactivation of cholera phages; Monsur KA et al.; Thermal inactivation of seven cholera phages have been tested over the temperature range between 50 degrees to 70 degrees C . It was found that the phages vary widely in their heat sensitivity, Mukerjee's phages III being the most sensitive of the whole group . With all the phages over the temperature range studies, the inactivation curve seem to follow the pattern of virus thermal inactivation in general, the inactivation proceeding initially at a rapid rate, which in about 15 minutes time, gradually changes to a slower rate, each component tending to follow kinetics of the first order . The difficulty of explaining this phenomenon on the basis of population heterogeneity has been discussed. Prostaglandins, 1975 Oct, 10(4), 581 - 7 Effects of indomethacin on intestinal secretion, prostaglandin E and cyclic AMP: evidence against a role for prostaglandins in cholera toxin-induced secretion; Wilson DE et al.; The effects of indomethacin on intestine mucosal cAMP, intestinal fluid secretion, and mucosal and fluid PGE were studied in rabbits in vivo following challenge with cholera toxin . Indomethacin had no effect on cholera toxin-induced fluid secretion or cAMP accumulation . Inhibition of PGE synthesis was achieved by the administration of two but not one injection of indomethacin . These studies provide evidence against a role for PGE in mediating cholera toxin-induced secretion and point out the need to measure prostaglandin levels when using prostaglandin synthetase inhibitors in vivo. Infect Immun, 1975 Oct, 12(4), 768 - 71 Interaction of cholera enterotoxin with cultured adrenal tumor cells; Wishnow RM et al.; In the adrenal tumor cell system ganglioside Gm1 inhibited cholera enterotoxin (CT)-induced steroidogenesis if it was preincubated with the toxin or added to adrenal cells 10 min before CT . In the preincubation studies a molar ratio of Gm1 to toxin of 3:1 was necessary for half-maximal inhibition of steroidogenesis . On the other hand, horse serum anticholeragenoid neutralized the steroidogenic response to cell-bound CT by 50% if it was added to adrenal monolayer cultures 15 min after the toxin . Specific antiserum was able to neutralized 20% of the toxin-induced activity even if it was added to adrenal cultures 2 h after CT . Phase contrast microscopy demonstrated that partial neutralization of the biochemical effect of CT by horse serum anticholeragenoid was accompanied by partial prevention of toxin-induced rounding of adrenal cells . Further studies showed that pretreatment of cultured adrenal cells with a maximal dose of CT increased cyclic adenosine 3'-5'-monophosphate formation in response to a maximal stimulating dose of adrenocorticotropin . This result suggested potentiation of hormonal activation of adenylate cyclase in intact adrenal tumor cells in response to CT. J Immunol, 1975 Oct, 115(4), 1126 - 34 The role of cyclic AMP in the chemotactic responsiveness and spontaneous motility of rabbit peritoneal neutrophils . The inhibition of neutrophil movement and the elevation of cyclic AMP levels by catecholamines, prostaglandins, theophylline and cholera toxin; Rivkin I et al.; Agents known to affect intracellular levels of cyclic AMP in many diverse systems have been tested for their effect on the chemotaxis induced by Escherichia coli culture filtrates, spontaneous motility and cyclic AMP levels of rabbit peritoneal neutrophils . Prostaglandin E1 and A1 but not prostaglandin F2alpha increased neutrophil cyclic AMP levels and, correspondingly, only the former two prostaglandins inhibited chemotaxis . Nevertheless, a quantitative relationship between prostaglandin stimulation of cyclic AMP and inhibition of chemotaxis could not be found . Epinephrine, isoproterenol, and, to a much lesser extent, norepinephrine increased neutrophil cyclic AMP through beta adrenergic stimulation . Only epinephrine and isoproterenol inhibited chemotaxis, but the inhibition was variable and not related to the ability of these catecholamines to increase intracellular cyclic AMP . Cholera toxin increased neutrophil cyclic AMP after a 30-min lag period which paralled its inhibitory effect on chemotaxis and spontaneous motility . However, the effect on chemotaxis require 50 ng/ml of toxin whereas the effect on cyclic AMP was manifested at 2 ng/ml of toxin . Prior to 30-min preincubation there was no effect of even 1250 ng/ml of toxin on either cyclic AMP or chemotaxis . Choleragenoid prevented the effects of toxin on both cyclic AMP and chemotaxis . The bacterial chemotactic factor obtained from E . coli culture filtrates did not effect a measurable change in levels of neutrophil cyclic AMP . The data indicate that even though cyclic AMP is not, in the main sequence of events, triggering the chemotactic response, increases in neutrophil cyclic AMP may modulate the movement and thus the chemotactic responsiveness of the neutrophil. Virchows Arch B Cell Pathol, 1975 Sep 11, 18(4), 287 - 96 On the possible role of intestinal hormones as the diarrhoeagenic messenger in cholera; Osaka M et al.; Cholera enterotoxin introduced into the duodenum of young rabbits causes severe degranulation of the enterochromaffin (EC) cells as revealed by electron microscopy . In the mucosal epithelium fixed mainly 1 hr after toxin administration, many of the basal granules of the EC cells are swollen up and open to the basal and lateral cell surface . The EC cells of the rabbit, as it is the rule for intestinal endocrine cells in mammals, are open to the lumen with an apical process covered by microvilli . A hypothesis is proposed that cholera toxin stimulates this apical receptor of the EC cells and that the aminic (serotonin) and polypeptide (motilin?) products of the cells released by the stimulus may mediate the diarrhoeagenic action of cholera enterotoxin. Infect Immun, 1975 Sep, 12(3), 466 - 9 Binding of cholera toxin by various tissues; Gascoyne N et al.; Under certain conditions, it is possible to confirm the observation by Peterson (1974) that the cholera toxin-binding capacities of tissues from brain and colon mucosa, and from liver and small intestine mucosa, are comparable . Binding of toxin by all tissues except brain is very variable, but is roughtly proportional to their content of the toxin-binding ganglioside galactosyl-N-acetylgalactosaminyl (sialosyl) lactosyl ceramide . It appears that some toxin-binding sites of the mucosa of the small intestin and colon may be masked . It has also been confirmed that there may be some solubilization of toxin-binding material from brain on standing a few days at 4 C, but this is comparatively slight . Some disadvantages of measuring toxin binding by adding small amounts of radioactive toxin to compartively large amounts of tissue are discussed. J Immunol, 1975 Sep, 115(3), 871 - 5 Studies on nonidet P40 lysis of murine lymphoid cells . I . Use of cholera toxin and cell surface Ig to determine degree of dissociation of the plasma membrane; Hart DA; Lymphoid cells from A/J mice were iodinated (125I) by the lactoperoxidase lysed with the non-ionic detergent NP-40 . The plasma membrane glycolipid receptor for cholera toxin and cell surface immunoglobulin were utilized in immune precipitation systems to characterize the degree of dissociation of the plasma membrane under various conditions . It was found that at 0.1% NP-40 and at cell concentration from 5 to 10 times 10(7) cells/ml, lipid-protein and protein-lipid-protein complexes formed in NP-40 which were soluble after centrifugation at 10(5) times G . Column chromatography of 125I-cell lysates on agarose A-0.5 M in 0.1% or 0.5% NP-40/PBS indicated that the majority of iodinated cell surface material existed as aggregates in detergent micelles . The availability of the oligosaccharide moiety of the glycolipid to interact with the cholera toxin was dependent on both the detergent concentration and the cell concentration used for cell lysis . However, the cell surface immunoglobulin was immunoprecipitable under all conditions of lysis tested. Zh Mikrobiol Epidemiol Immunobiol, 1975 Sep, 0(9), 36 - 40 {Epidemiology of El Tor cholera in Indonesia}; Grizhebovskii GM et al.; The authors analyze data on El Tor cholera morbidity in Indonesia in the past and at present . It was shown that before the year of 1961 the infection was limited to the Sulavesi island and was epidemic in character, but it differed from classic endemic cholera by a number of signs . In 1961 and 1970 the spread of El Tor cholera along Indonesia was practically synchronous with its spread along the vast territories of the world, this apparently pointing to the effect of some general factor which caused the activization of El Tor cholera in its various foci. Proc Natl Acad Sci U S A, 1975 Sep, 72(9), 3561 - 4 Stimulation of epinephrine-sensitive fat cell adenylate cyclase by cytosol: effect of cholera toxin; Ganguly U et al.; Cytosol prepared from rat epididymal fat cells by centrifugation at 100,000 X g for 1 hr was found to enhance the basal and epinephrine-sensitive adenylate cyclase {EC 4.6.1.1; ATP pyrophosphate-lyase (cyclizing)} of fat cell ghosts . Cholera toxin also stimulated adenylate cyclase and increased the response to epinephrine in fat cells . A possible relationship between the adenylate cyclase modifying activities of cytosol and the effects of cholera toxin was sought . Cytosol from freshly prepared fat cells added to ghosts prepared from cells that had been exposed to toxin for varying periods showed a progressive loss of responsiveness to cytosol epinephrine-enhancing activity . The effect appeared within 15 min after toxin exposure, a full 30 min before any direct effect of toxin on adenylate cyclase was seen . Since exposure to toxin decreased membrane response to cytosol epinephrine-enhancing activity, the possibility that epinephrine-enhancing activity in cytosol might be altered by toxin was explored . Cytosol from cells exposed to toxin for varying periods lost epinephrine-enhancing activity to an appreciable degree within 15 min . Examination of these early events after exposure to toxin should clarify the way in which this bacterial substance affects mammalian cells . The cytosol epinephrine-enhancing activity was destroyed by boiling for 3 min and was partially inactivated by trypsin . It was nondialyzable and stable at -70 degrees. Proc Natl Acad Sci U S A, 1975 Sep, 72(9), 3438 - 42 Mechanism of activation of adenylate cyclase by cholera toxin; Sahyoun N et al.; Cholera toxin (choleragen) can stimulate adenylate cyclase {EC 4.6.1.1; ATP pyrophosphate-lyase (cyclizing)} activity in whole particulate fractions or purified plasma membranes of homogenates of isolated fat cells provided special precautions are taken to stabilize the enzyme during the required preincubation period . As observed with intact cells, the activation exhibits a protracted (about 25 min) lag phase, and it is blocked by ganglioside GM1 and choleragenoid ("binding" subunit of toxin) . The 36,000 molecular weight subunit ("active" subunit), a hydrophobic polypeptide which does not block choleragen binding or action, can directly activate the enzyme in intact cells without a lag phase . Its effects are not blocked by ganglioside GM1 or choleragenoid, yet the stimulated activity exhibits reduced fluoride and enhanced isoproterenol sensitivity, properties characteristic of the choleragen-activated enzyme . Binding of the 125I-labeled 36,000 molecular weight subunit to cells is not saturable and is unaffected by gangliosides, choleragen, or choleragenoid, and the bound material behaves as an integral membrane protein; this protein may simply partition into the membrane matrix . With increasing time of incubation cell-bound choleragen may dissociate into its component subunits, but these remain in the membrane . Using a double antibody immunoprecipitin system, substantial precipitation of cyclase activity occurs with antisera against the 36,000 molecular weight subunit provided toxin activation has occurred . The normal process of activation may involve an initially inactive toxin--ganglioside complex which, as a result of lateral mobility and multivalent binding (lag phase), results in destabilization of the molecule with release of the "active" subunit into the membrane core where it can spontaneously associate with and perturb the cyclase complex. Infect Immun, 1975 Sep, 12(3), 621 - 4 Increased adhesion of Chinese hamster ovary cells to substratum by cholera enterotoxin; Nozawa R et al.; A new, simple assay method using Chinese hamster ovary cells was devised for cholera enterotoxin . The effect of the cholera toxin was measured by an increase in cell adhesion to the substratum . The increase was dependent on the concentration of the toxin used and was effective for a longer period of time than that of adhesion increased by dibutyryl cyclic adenosine monophosphate and theophyline . There was a delay of about 60 min before the increase in adhesion, using the toxin, appeared, whereas the increase caused by dibutyryl cyclic adenosine monophosphate appeared almost at once. J Biol Chem, 1975 Aug 25, 250(16), 6424 - 32 The mechanism of action of cholera toxin in pigeon erythrocyte lysates; Gill DM et al.; The adenylate cyclase activity of intact pigeon erythrocytes begins to rise after about 20 min of exposure to cholera toxin . The maximum rate at which the cyclase activity increases appears to be limited by the number of toxin molecules which can reach an intracellular target . If the erythrocytes are made permeable to the toxin by a bacterial hemolysin, no such limit exists, and adenylate cyclase activity starts to rise immediately upon the addition of toxin, and continues to rise to a maximum at an initially constant rate which is dependent upon the concentration of toxin . On lysed erythrocytes, the addition of cholera antitoxin immediately prevents any further rise in adenylate cyclase activity, but does not reverse any activation already achieved . Erythrocyte lysates may also be activated by isolated peptide A1 of cholera toxin, although activation of adenylate cyclase of intact erythrocytes requires the complete toxin molecule . In the intact cells, toxin first attaches by its Component B to surface receptors of which there are about 30 per erythrocyte . Subsequently, peptide A1 but not Component B is inserted into the erythrocyte . It takes only about 1 min at 37 degrees for peptide A1 to be sufficiently deep within the cell membrane to be inaccessible to extracellular antitoxin, but its complete transit through the membrane appears to take longer . The surface receptors are used only once, for they remain blocked by Component B . The number of receptors available on the surface may be increased by soaking cells in ganglioside GM1 . Cholera toxin also decreases the rate of apparently spontaneous loss of adenylate cyclase activity and increases the response to epinephrine . Theophylline inhibits the action of cholera toxin. Biochim Biophys Acta, 1975 Aug 13, 399(2), 277 - 90 Effects of cholera toxin on cyclic adenosine 3',5'-monophosphate concentration and secretory processes in the exocrine pancreas; Smith PA et al.; 1 . The effect of purified cholera toxin on secretory processes of exocrine pancreas has been studied in the isolated, saline-perfused cat pancreas and in incubated pieces of rat pancreas . 2 . The toxin evoked a biphasic secretory response from the perfused cat pancreas . An initial small phase, which began within minutes of toxin application, was an artefact due to the presence of NaN3 in the cholera toxin preparation as supplied; it could be entirely reproduced by NaN3 at the concentration expected during toxin stimulation . A second, sustained phase of secretion, due to the action of the toxin proper, began within 30-60 min, increasing in magnitude for many hours and persisting in the absence of toxin . It was accompanied by a parellel rise in tissue cyclic AMP concentration, and could be potentiated by theophylline . 3 . The composition of the secretion stimulated by cholera toxin resembled that evoked by secretin; e.g . it contained a high concentration of bicarbonate and only basal amounts of digestive enzymes . 4 . Similarly, cholera toxin did not stimulate enzyme secretion by incubated rat pancreas, despite large rises in tissue cyclic AMP concentration . 5 . Because cholera toxin has thus far been shown to have no other effect than that of stimulating adenylate cyclase, these observations support the conclusion that cyclic AMP does mediate the electrolyte secretory response of the pancreas to secretin, but offers no evidence that cyclic AMP plays a similar role in the regulation of pancreatic enzyme secretion stimulated by cholecystokinin-pancreozymin or acetylcholine. Gastroenterology, 1975 Aug, 69(2), 479 - 82 Intestinal villus and crypt cell responses to cholera toxin; Weiser MM et al.; Adenylate cyclase activity was measured in rat small intestinal villus and crypt cells after in vivo and in vitro exposure to cholera toxin . The increase in intestinal adenylate cyclase induced by cholera toxin in vivo appeared to be largely confined to the villus cell with the largest increase observed for upper villus cells . Crypt cell adenylate cyclase was not responsive to cholera toxin . No response could be demonstrated for isolated villus or crypt cells incubated with cholera toxin in vitro . In vivo incubation with 125I-cholera toxin demonstrated binding to only villus cells . These results suggest that the major effect of cholera toxin was on villus cells rather than crypt cells and this was due to the greater accessibility or binding capacity of the villus cell to luminal cholera toxin. Immunology, 1975 Aug, 29(2), 275 - 82 The differential effect of cholera toxin on the lymphocyte stimulation induced by various mitogens; Vischer TL et al.; BALB/c spleen cells (5 x 10(6)) were cultured in 1 ml of serum-free RPMI 1640 medium for 3 days in order to examine the effect of cholera enterotoxin (CN) and its spontaneously formed toxoid (CD) on lymphocyte stimulation . Stimulation was assessed after addition of {3H} thymidine for the last 16 hours of culture . One microgram of CN per culture markedly reduced the baseline of {3H} thymidine incorporation and the stimulation due to phytohaemagglutinin (PHA), concanavalin A (con A) and bacterial lipopolysaccharide (LPS) . One microgram of CD diminished the base-line to half, abolished the response to PHA, reduced the response to con A and had very little effect on the LPS-induced stimulation . One-tenth the amount (0-1 mug) of both CN and CD affected only the PHA reaction . A secondary response to haemocyanin in vitro was not decreased by this lower dose . The effect of 1 mug on CN on the LPS response could be reduced by pretreatment of the cells with CD, whereas the PHA reaction remained markedly diminished . Dibutyryl-cAMP added to culture tubes had a similar effect ot 1 mug of CN, affecting the PHA response much more than the response to LPS . Spleen cells of mice immunized with CD gave a significant proliferative response to both 1 mug of CD and CN . The results are interpreted as indicating a strong inhibitory effect of CN mediated by accumulation of intracellular cAMP . CD-immunized cells contain specific receptors for both CD and CN which probably compete with the sites responsible for adenylate cyclase stimulation by CN. Prostaglandins, 1975 Jul, 10(1), 117 - 27 Effects of crude and pure cholera toxin on prostaglandin; Bedwani JR et al.; Investigations were made into the effects of crude and pure preparations of cholera toxin on the release of prostaglandin-like substances (PLS) from rabbit ileum . Perfusion of ileal loops in vivo with buffer containing crude toxin was followed by a release of PLS into the perfusate, in amounts up to 37.5 ng/30 min (PGE2 equivalents) . In contrast, no detectable PLS was released when ileal loops were perfused with pure toxin . Similarly, pieces of ileum opened longitudinally released PLS in amounts up to 107 ng PGE2/g tissue when incubated with crude toxin for 1-4 hr, but no release of PLS was detected in the presence of pure toxin under comparable conditions . Treatment of rabbits with indomethacin, 1.6 mg/kg p.o., had no effect on the accumulation of fluid in ileal sacs injected with crude or pure cholera toxin . These results support the view that prostaglandins do not play an essential role in the action of cholera toxin. Gastroenterology, 1975 Jul, 69(1), 110 - 22 Pancreatic cholera . Sudies on tumoral secretions and pathophysiology of diarrhea; Rambaud JC et al.; Tumoral secretions and pathophysiology of diarrhea were studied in 1 patient with pancreatic cholera . High concentrations of vasoactive intestinal peptide were found in both systemic blood and tumoral extracts, together with increased plasma levels of calcitonin and protaglandins E and Falpha . Gastric inhibitory peptide and gastrointestinal and pancreatic hormones were absent from the tumor, except for small amounts of glucagon, and their blood levels were normal . Decreased basal but normal pentagastrin-stimulated gastric acid secretion, normal basal and secretin-stimulated pancreatic secretion, increased volume of gallbladder bile with high bicarbonate, and low bile salt concentrations were observed, but the electrolyte content and flow rate of fluid passing the duodenojejunal junction were within normal limits . Small intestine was found to be the origin of the water and electrolyte fasting losses . Jejunum was the site of bicarbonate secretion . Jejunal glucose and leucine-stimulated water and sodium transports were also strikingly decreased, whereas the absorption rates of the sugar and amino acid were normal . Colon reabsorbed high amounts of water and sodium but increased potassium losses . Biological effects of vasoactive intestinal peptide may explain most of the patient's upper digestive secretion abnormalities and small intestinal function impairments, whereas secondary aldosteronism might explain the modified colonic function. Proc Natl Acad Sci U S A, 1975 Jul, 72(7), 2572 - 6 Cholera toxin activation of adenylate cyclase in cancer cell membrane fragments; Bitensky MW et al.; Activation of adenylate {ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1} by cholera toxin (84,000 daltons, 5.5 S) is demonstrated in plasma membrane fragments of mouse ascites cancer cells . The activation of adenylate cyclase is mediated by a macromolecular cyclase activating factor (MCAF), which has a sedimentation constant of 2.7 S and a molecular weight of about 26,000 . MCAF is derived from, and may be identical to the "A fragment" of cholera toxin . Generation of MCAF depends on prior interaction of cholera toxin with either dithiothreitol, NADH, NAD, or a low-molecular-weight component (less than 700 daltons) present in cytoplasm . Subsequent exposure of this pretreated cholera toxin to cell membranes from a variety of mouse ascites cancer cells is followed rapidly by the appearance of MCAF, which no longer requires dithiothreitol, NADH, or NAD for the activation of adenylate cyclase . Activation of adenylate cyclase by MCAF in ascites cancer cell membrane fragments is not reversed by repeated washing of these membrane fragments . Adenylate cyclase in normal cell membrane fragments fails to respond either to cholera toxin or MCAF in the presence of dithiothreitol . In striking contrast, the adenylate cyclase in membrane fragments from five ascites cancer cells responds to either MCAF or native cholera toxin preincubated with dithiothreitol, NADH, or NAD.
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