MMWR Morb Mortal Wkly Rep, 1991 Aug 16, 40(32), 562 - 5 Update: cholera--Western Hemisphere, and recommendations for treatment of cholera; Cholera--New York et al.; Through June 26, 1991, cholera has been reported from seven countries in the Western Hemisphere: Brazil, Chile, Colombia, Ecuador, Mexico, Peru, and the United States . In the United States, a total of 14 confirmed cases of epidemic-associated cholera have been reported among persons in Florida (one) (1), Georgia (one) (2), New Jersey (eight) (1), and New York (four) . This report summarizes information regarding the four cases reported in New York and describes a new laboratory procedure used to confirm the vehicle of transmission in this outbreak. Zh Mikrobiol Epidemiol Immunobiol, 1991 Aug, (8), 33 - 5 {The types of the epidemic manifestations of cholera on the territory of the USSR}; Norkevich MI et al.; On the basis of the analysis of cholera cases for the period of 1965-1989 three main main types of epidemic manifestations of this infection on the territory of the USSR were determined with due attention to the complex of data, characterizing the intensity and types of the epidemic process, the danger of the outbreak and spread of cholera . This made it possible to differentiate and decrease the complex of prophylactic measures, depending on the type of the territory. Naunyn Schmiedebergs Arch Pharmacol, 1991 Aug, 344(2), 160 - 6 Terminal serotonin autoreceptor function in the rat hippocampus is not modified by pertussis and cholera toxins; Blier P; The possibility that the terminal serotonin (5-HT) autoreceptor in the rat hippocampus is coupled to Gi, Go or Gs regulatory proteins was investigated using the electrically evoked overflow of {3H}5-HT from preloaded slices . Pertussis toxin, which inactivates Gi/o or cholera toxin, which stimulates Gs, was injected directly in the hippocampus 3 to 11 days prior to the experiments . Hippocampus slices were prepared, loaded with {3H}5-HT, superfused continuously, and stimulated electrically 72 min (S1) and 116 min (S2) after the beginning of superfusion . In the absence of any drug, the evoked overflow of {3H}5-HT in S1 was not altered by either toxin . The enhancing effect of the 5-HT reuptake blocker paroxetine (1 mumol/l) on the evoked {3H}5-HT overflow was also unaltered by these toxins . 5-Carboxyamidotryptamine, a 5-HT autoreceptor agonist, inhibited in a concentration-dependent manner the stimulation-evoked release of {3H}5-HT . The concentration-effect curve (0.001-0.1 mumol/l) for this drug was not altered by pretreatment with either pertussis or cholera toxin . Similarly, the effect of another 5-HT autoreceptor agonist, 5-methoxytryptamine (0.1 and 1 mumol/l), was not altered in the pretreated rats . In addition, the reduction of {3H}5-HT overflow obtained by increasing the stimulation frequency from 1 Hz to 5 Hz, which is due to an increase in terminal 5-HT autoreceptor activation at the higher frequency, was not altered by either toxin . The enhancing effect of the 5-HT autoreceptor antagonist methiothepin (1 mumol/l) on stimulation-evoked {3H}5-HT overflow was not changed by either pretreatment . N-Ethylmaleimide inactivates Gi/o proteins by alkylation.(ABSTRACT TRUNCATED AT 250 WORDS) J Electron Microsc Tech, 1991 Aug, 18(4), 387 - 94 Structural analysis of two-dimensional arrays of cholera toxin B-subunit; Mosser G et al.; Two-dimensional arrays of cholera toxin B-subunit (CTB) have been obtained by specific interaction with lipid films, as described by Ludwig et al . (1986) . The relationship between two types of array, of either rectangular or hexagonal geometry, was analyzed using crystallographic methods of electron image analysis . Our results showed that the type of array obtained was highly dependent on the negative stain used and that both arrays presented related lattice parameters, indicating that they originated from a common unstained structure . Image analysis of hexagonal arrays at 17 A resolution revealed variable CTB projected structures, ranging from annularly symmetric particles to highly asymmetric particles, very distinct from the pentameric structure resolved from rectangular crystals . The present data suggest that hexagonal arrays result from an imperfect staining of CTB rectangular crystals . The staining distortion is such that the stain layer does not match faithfully the pentameric protein distribution whereas the regular organization of the specimen is maintained. Acta Paediatr Jpn, 1991 Aug, 33(4), 428 - 33 Neuronal differentiation of Ewing's sarcoma induced by cholera toxin B and bromodeoxyuridine--establishment of Ewing's sarcoma cell line and histochemical study; Ohta S et al.; An Ewing's sarcoma (ES) cell line was established from a metastatic bone marrow specimen in a patient with advanced disease, and some histochemical characteristics were investigated by neuronal differentiation induced with cholera toxin B (CTB) and bromodeoxyuridine (BrdU) . Neuronal differentiation was investigated by the expression of neurofilament and Leu-7, and glial differentiation was observed by expression of S-100 protein . Neurofilament (NF) and Leu-7 were positive in ES cells and these were expressed more intensively by induction with CTB than with BrdU . There was no expression of S-100 protein in untreated or differentiated ES cells . ES cells became differentiated to neuronal cells with CTB and BrdU, but it was not observed, that ES cells had the potential to differentiate to glial cells . It appears that ES is of more primitive neural origin than neuroblastoma, primitive neuroectodermal tumors and other related neural tumors. Res Commun Chem Pathol Pharmacol, 1991 Aug, 73(2), 145 - 52 Modulation of epidermal growth factor binding to receptor by isoproterenol and cholera toxin in primary cultured hepatocytes; Katoh S et al.; The treatment of rat hepatocytes with isoproterenol induced the increase in the affinity of high affinity binding sites and the decrease in the number of low affinity binding sites of epidermal growth factor (EGF) . Similar results were obtained from the pretreatment of hepatocytes with cholera toxin . These results suggest that adenylate cyclase affects the affinity and number of EGF-receptor in hepatocytes. Mol Immunol, 1991 Aug, 28(8), 865 - 76 Mapping epitopic regions of cholera toxin B-subunit protein; Kazemi M et al.; Continuous overlapping synthetic hexapeptides representing the entire 103 amino acid sequence of the immunodominant B-subunit protein of cholera enterotoxin were used to examine reactivities of a variety of antisera in attempts to detect and define sequence-related (continuous) antigenic regions . The validity of the methods was established by the reactions of polyclonal antisera raised against longer synthetic peptides with appropriate synthetic hexapeptides . An unexpected cross-reaction is attributed to the presence of three identical amino acids (Gln16-Ile17-His18)--although in different order (Gln56-His57-Ile58)--in two parts of the B-subunit chain . Adsorption studies using polyclonal rabbit antisera revealed that, in many instances, denatured B-subunit protein more effectively removed reactivity with hexapeptides than did the native protein . Native holotoxin was more effective than native B-subunit . Sera from human cholera convalescents gave diffuse patterns of reactivity with synthetic hexapeptides--primarily against regions of reactive hexapeptides rather than with clearly defined continuous epitopes . Among many epitopic regions encountered, a strongly reactive tetramer, Ser-Gln-His-Ile (SQHI), was discovered in a highly conserved region, residues 55-58, of the B-subunit amino acid sequence . Adsorption studies revealed that this epitope is apparently exposed on the surface of the native protein . Amino acid substitution revealed the essentiality of Gln and His residues to this epitope . Gly54 was not part of the epitope but substitution of acidic residues Glu and Asp for Gly eliminated reactivity with antibody . The results suggest that continuous epitopes may contribute to the antigenicity of the native toxin protein and may be potentially useful for development of a peptide vaccine. J Biol Chem, 1991 Jul 15, 266(20), 12858 - 65 Activation of rat liver adenylate cyclase by cholera toxin requires toxin internalization and processing in endosomes; Janicot M et al.; Involvement of acidic cell compartments in processing and action of cholera toxin (CT) in rat liver has been examined using subcellular fractionation . Liver cell fractions prepared various times after CT injection display, after a lag phase, a progressive increase in adenylate cyclase activity, detectable earlier in Golgi-endosomal fractions (20 min) than in plasma membrane fractions (30 min), with a maximum (3-fold basal activity) achieved by 60-90 min . Endosomes containing in vivo internalized CT display a time-dependent increase in their ability to bind anti-A-subunit antibodies and to stimulate exogenous adenylate cyclase, which kinetically parallels the generation of A1 peptide, suggesting a translocation of A-subunit (or A1 peptide) across the endosomal membrane . In vivo chloroquine treatment inhibits endocytosis of CT taken up into the liver, lengthens the lag phase for adenylate cyclase activation by CT, and reduces by 3- to 10-fold the apparent affinity of the toxin for the enzyme . Incubation of endosomes containing internalized toxin at 37 degrees C under isotonic conditions results in a pH-dependent increase in generation of A1 peptide, membrane translocation of A-subunit (or A1 peptide), and degradation of toxin, with a maximum at pH 5 . Addition of ATP, by decreasing the internal endosomal pH, stimulates both generation of the A1 peptide and degradation of toxin at pH 6-8 . It is concluded that activation of adenylate cyclase by CT in intact liver requires association and subsequent processing of toxin in an acidic cell compartment, presumably endosomal. Mol Microbiol, 1991 Jul, 5(7), 1755 - 67 Analysis of structure and function of the B subunit of cholera toxin by the use of site-directed mutagenesis; Jobling MG et al.; Oligonucleotide-directed mutagenesis of ctxB was used to produce mutants of cholera toxin B subunit (CT-B) altered at residues Cys-9, Gly-33, Lys-34, Arg-35, Cys-86 and Trp-88 . Mutants were identified phenotypically by radial passive immune haemolysis assays and genotypically by colony hybridization with specific oligonucleotide probes . Mutant CT-B polypeptides were characterized for immunoreactivity, binding to ganglioside GM1, ability to associate with the A subunit, ability to form holotoxin, and biological activity . Amino acid substitutions that caused decreased binding of mutant CT-B to ganglioside GM1 and abolished toxicity included negatively charged or large hydrophobic residues for Gly-33 and negatively or positively charged residues for Trp-88 . Substitution of lysine or arginine for Gly-33 did not affect immunoreactivity or GM1-binding activity of CT-B but abolished or reduced toxicity of the mutant holotoxins, respectively . Substitutions of Glu or Asp for Arg-35 interfered with formation of holotoxin, but none of the observed substitutions for Lys-34 or Arg-35 affected binding of CT-B to GM1 . The Cys-9, Cys-86 and Trp-88 residues were important for establishing or maintaining the native conformation of CT-B or protecting the CT-B polypeptide from rapid degradation in vivo. Microbiologica, 1991 Jul, 14(3), 213 - 7 Protection tests in pigs vaccinated with the lapinized Chinese strain of hog cholera virus (HCV) previously adapted in a minipig kidney (MPK) cell line, to challenge infection with virulent HCV; Gualandi GL et al.; Pigs which had been vaccinated with the Lapinized Chinese strain of Hog Cholera Virus previously adapted in a minipig cell line cultures (MPK-LC-HCV), resultet to be protected when they were subjected to challenge infection with virulent Hog Cholera Virus (HCV) 6 or 11 months later . The challenge virus was never isolated from any of the vaccinated pigs . The MPK-LC-HCV vaccine induced a significant rise of the antibody titer to the HCV in pigs kept under field conditions. FEMS Microbiol Lett, 1991 Jul 1, 65(3), 265 - 71 Amplification of the secretory IgA response to Toxoplasma gondii using cholera toxin; Bourguin I et al.; Our study demonstrates that cholera toxin (CT) markedly enhances the intestinal anti-T . gondii antibody response following oral immunisation of mice with a T . gondii sonicate (TSo) and CT . The antibodies induced were mostly IgA and secretory IgA but a small quantity of IgG was also produced . In contrast, no intestinal anti-T . gondii IgM antibodies were detected . Anti-CT IgA antibodies were also present in intestinal secretions but in much lower quantities than the T . gondii-specific IgA . No anti-CT IgG nor IgM antibodies were detected . Western blot analysis showed that CT induced not only an increase of the intensity of the intestinal IgA antibody response to the 30-kDa band but also induced intestinal IgA antibodies against other major T . gondii proteins (p22, and the 28-kDa antigen) as recognised by specific monoclonal antibodies . The amplification of the anti-T . gondii secretory IgA response by means of an appropriate adjuvant may be one major step leading towards an orally induced immune protection against toxoplasmosis. J Clin Invest, 1991 Jul, 88(1), 143 - 8 Intestinal immune responses in humans . Oral cholera vaccination induces strong intestinal antibody responses and interferon-gamma production and evokes local immunological memory; Quiding M et al.; We have examined secretory antibody and cell-mediated immune responses to oral cholera vaccine in the human gastrointestinal mucosa . Freshly isolated peripheral blood lymphocytes and intestinal lymphocytes obtained by enzymatic dispersion of duodenal biopsies were assayed for numbers of total and vaccine specific immunoglobulin-secreting cells by enzyme-linked immunospot assay (ELISPOT) techniques; the frequency of cells secreting interferon-gamma (IFN-gamma) was also examined by a new modification of the ELISPOT technique . After booster immunizations with oral cholera vaccine, large numbers of cholera toxin-specific antibody-secreting cells (ASC) appeared in the small intestine . The responses were dominated by IgA ASC . A single immunization, performed 5 mo after the initial vaccinations, gave rise to an ASC response similar to that seen after the first booster immunization, with respect to both magnitude and isotype distribution . Each of the immunizations also evoked an ASC response in blood which was of lower magnitude than that seen in the small intestine, and comprised similar proportions of IgA and IgG ASC . A booster immunization also resulted in increased frequencies of IFN-gamma-secreting cells, but this increase was confined to the duodenal mucosa . This study establishes the feasibility of studying, at the single-cell level, intestinal immune reactivity in humans . Furthermore, it indicates that the small intestinal mucosa is an enriched source of IFN-gamma . It also demonstrates marked differences between intestinal and peripheral blood immune responses after enteric immunization, and confirms the notion that the mucosal immune system in humans displays immunological memory. Zh Mikrobiol Epidemiol Immunobiol, 1991 Jun, (6), 40 - 3 {The practical performance of cholera control measures in the USSR during the 7th pandemic}; Narkevich MI et al.; The critical analysis of anticholera measures carried out in the USSR since 1965 till the present time is presented . The grounds for the abolition or considerable reduction of a number of measures are considered from the viewpoint of their scientific substantiation, the adequacy of means and efforts spent for their realization and their antiepidemic effectiveness . Special attention is paid to the necessity of differentiated approach to these measures, depending on concrete local climatic and geographical, sanitary, hygienic and other factors which determine the epidemic potential of a given administrative territory. Exp Cell Res, 1991 Jun, 194(2), 210 - 7 Differences in induction of c-fos transcription by cholera toxin-derived cyclic AMP and Ca2+ signals in astrocytes and 3T3 fibroblasts; Gabellini N et al.; The B subunit of cholera toxin, a protein which binds specifically to membrane ganglioside GM1, is known to affect cell growth and differentiation . To investigate the mechanism of these cellular responses at the nuclear level, we used the induction of c-fos in astrocytes and 3T3 fibroblasts as a model . Northern blot analysis showed that treatment with B subunit provokes a rapid and transient expression of c-fos mRNA, independent of a measurable increase in cyclic AMP . The B subunit signal, which is mediated by Ca2+, was compared to cholera toxin and other agents which increase intracellular cyclic AMP levels . In transient transfection assays of astrocytes and fibroblasts, functional analysis of c-fos promoter deletions was used to identify the elements involved in transcriptional activation by B subunit . In astrocytes, the DNA region including the serum response element and the cyclic AMP response element (CRE) are equally required, whereas 3T3 cells require only the CRE for maximal induction . A synergistic effect of signal transduction was mediated by calcium and cyclic AMP on the CRE, being positive in 3T3 cells and negative in astrocytes . Diverse regulatory elements may be thus involved in responses of different cell types to the same extracellular signal . Furthermore, a single regulatory element (CRE) can integrate both calcium and cyclic AMP signals in the control of gene expression. J Med Assoc Thai, 1991 Jun, 74(6), 306 - 10 An outbreak of El Tor cholera in an institution for the mentally retarded in Nonthaburi, June-July 1987; Swaddiwudhipong W et al.; In June and July 1987, an outbreak of cholera caused by V.cholerae O1, biotype El Tor, serotype Inaba, occurred in an institution for the mentally retarded in Nonthaburi . Of the 447 retarded inmates, 74 were found to be infected and one died . Epidemiological investigation revealed that the inmates with severe mental retardation who ate food in their own sleeping-room were significantly (p less than 0.001) more likely to be infected than those taking food in the dining-room . We hypothesize that the liquid diet commonly served to the more severely mentally retarded may have increased the risk of infection by more rapid gastric emptying . The long average period of time for meal consumption among these individuals may have allowed the organisms to multiply to a level capable of causing disease . Contamination of food with cholera might have occurred during food handling in the kitchen or within the sleeping-room where overcrowded conditions and poor personal hygiene facilitated person-to-person spread of infection . Prompt implementation of control measures effectively terminated cholera transmission in the outbreak. Scand J Immunol, 1991 Jun, 33(6), 691 - 8 The adjuvant action of cholera toxin is associated with an increased intestinal permeability for luminal antigens; Lycke N et al.; This study addresses the question of whether cholera toxin (CT) increases gut permeability for molecules greater than 3000 Da and whether such an effect is associated with an adjuvant function by CT on the gut immune response . We found that CT after oral administration gives rise to strikingly increased gut permeability for Dextran (Mw 3000) concomitantly with a strong enhancing effect on the anti-keyhole limpet hemocyanin (KLH) specific immune response in the lamina propria after oral immunization with KLH plus Dextran and CT . In contrast, the B-subunit of the holotoxin, which lacks the adenylate cyclase/cAMP-activating property of CT, failed to increase gut permeability as well as local anti-KLH immune responses . These results might suggest a causal linkage between the ability of CT to increase gut permeability and its adjuvant property on gut mucosal immune responses . In addition this finding supports the notion that the adenylate cyclase/cAMP system plays a regulatory role in gut permeability and is important in enhancing mucosal immune responses . Based on previous studies and the present data we propose that the mechanism for CT's adjuvant function on mucosal immune responses is by affecting antigen-presenting cells, T and B cells in the gut to give a net enhancing effect on the stimulation of local immunity, and that the CT-induced increase in gut permeability might be part of the adjuvant mechanism by facilitating luminal antigens to access the gut mucosal immune system. Immunobiology, 1991 Jun, 182(3-4), 266 - 76 Cholera toxin-mediated inhibition of signalling in Jurkat cells is followed by, but not due to a loss of T cell receptor complex; Sommermeyer H et al.; Cholera toxin treatment of the human T cell lymphoma Jurkat resulted in inhibition of signalling via the T cell antigen receptor complex (TcR/CD3-complex) . Cholera toxin specifically ADP-ribosylated the alpha-subunit of the stimulatory G-protein of the adenylate cyclase (Gs alpha), no other proteins were modified in the intact cells . ADP-ribosylation of Gs alpha and its subsequent activation led to an increase of the cyclic AMP level and in addition, to a drastic reduction of the cell-surface density of the TcR/CD3-complex . Recently, we demonstrated that the effect of cholera toxin at the receptor level is not due to an increased cAMP level (4) . As inhibition of signalling is also not cAMP-mediated (8), we examined whether the modulation of the TcR/CD3-complex could be the reason for the interruption of the signalling cascade . Analyzing the time courses of the multiple cholera toxin effects in Jurkat cells at 37 degrees C, the following sequence was found: ADP-ribosylation of Gs alpha--increase of cyclic AMP level--inhibition of signalling via the TcR/CD3-complex--decrease of cell-surface density of the TcR/CD3-complex . Treatment of Jurkat cells at 20 degrees C with cholera toxin resulted in an increase of cyclic AMP and inhibition of signal transduction, while no decrease of TcR/CD3-complex density could be observed . These data imply that receptor loss from the cell-surface is not causative for the inhibition of signalling . More likely, activation of Gs uncouples signal transduction in Jurkat cells via the TcR, which by a so far unknown mechanism is followed by a loss of the receptor from the cell surface. J Bone Miner Res, 1991 Jun, 6(6), 551 - 60 On the role of cyclic AMP as a mediator of bone resorption: gamma-interferon completely inhibits cholera toxin- and forskolin-induced but only partially inhibits parathyroid hormone-stimulated 45Ca release from mouse calvarial bones; Lerner UH et al.; The effects of gamma-interferon (gamma-IFN) on bone resorption and cyclic AMP formation stimulated by parathyroid hormone (PTH), forskolin, and cholera toxin have been studied in cultured neonatal mouse calvarial bones . Bone resorption was assessed by the release of 45Ca from prelabeled mouse calvarial bone fragments . Cyclic AMP formation was quantified by analyzing the amount of the nucleotide in calvarial bone tissue . gamma-IFN completely blocked the 45Ca release response to forskolin and cholera toxin in 96 h cultures . In contrast, the 45Ca release response to PTH was only partially inhibited, an effect that was seen over a wide range of PTH concentrations . The inhibitory effect of gamma-IFN was dose dependent, with a threshold for action at 10 U/ml . Forskolin-stimulated 45Ca release could only be inhibited when gamma-IFN was added simultaneously with forskolin; gamma-IFN added to bones prestimulated with forskolin had no effect . The inhibitory effect of gamma-IFN on PTH-stimulated 45Ca release was seen first after a time lag of 48 h . In contrast calcitonin caused an inhibition after only 3 h . PTH and cholera toxin stimulation of radioactive calcium release was also inhibited by gamma-IFN in bones treated with indomethacin . gamma-IFN inhibited forskolin-induced 45Ca release in bones treated with the mitotic inhibitor hydroxyurea . No effect of gamma-IFN on cyclic AMP formation induced by PTH, cholera toxin, or forskolin could be seen . These data show that gamma-IFN inhibits forskolin- and cholera toxin-induced bone resorption by a mechanism unrelated to prostaglandin production or mitotic activity.(ABSTRACT TRUNCATED AT 250 WORDS) Br J Pharmacol, 1991 Jun, 103(2), 1347 - 50 Regulation of bradykinin receptor level by cholera toxin, pertussis toxin and forskolin in cultured human fibroblasts; Etscheid BG et al.; 1 . The effect of bacterial toxins on bradykinin-triggered release of arachidonic acid was studied in serum-deprived human foreskin (HSWP) fibroblasts prelabelled with {3H}-arachidonic acid . An 18-h exposure of HSWP cells to cholera toxin, pertussis toxin, or forskolin enhanced the bradykinin-stimulated release of arachidonic acid and metabolites . 2 . Prolonged treatment of HSWP cells with these agents also caused a 3 to 4 fold rise in cell surface {3H}-bradykinin binding . The rise was inhibited by concurrent incubation with cycloheximide or actinomycin D . In addition, cholera toxin and foreskolin increased {3H}-bradykinin binding in wildtype PC12 cells, but not in mutant PC12 cells with reduced cyclic AMP-dependent protein kinase type II activity . 3 . In conclusion, cholera toxin, pertussis toxin and forskolin enhanced arachidonic acid release in response to bradykinin, and increased the number of bradykinin receptors in HSWP fibroblasts . A cyclic AMP-dependent mechanism appears to mediate the actions of the toxins and forskolin. Eur J Immunol, 1991 Jun, 21(6), 1439 - 44 Interleukin 2 counteracts the inhibition of cytotoxic T lymphocytes by cholera toxin in vitro and in vivo; Moscovitch-Lopatin M et al.; Cholera toxin irreversibly activates a 43-kDa guanosine triphosphate (GTP)-binding protein by adenosine diphosphate ribosylation, resulting in activation of adenylate cyclase and increased intracellular levels of cyclic adenosine monophosphate (cAMP) . Because increases in intracellular cAMP inhibit interleukin 2 (IL 2) expression and cytotoxic T lymphocyte (CTL) generation and function in vitro and in vivo, we hypothesized that IL 2 may counteract the inhibition of CTL by cholera toxin . Activated CTL treated with IL 2 were protected from the inhibitory effects of cholera toxin . IL 2 also counteracted the inhibitory effect of cholera toxin on steady-state levels of CTL-specific serine esterase mRNA . Given the putative role of serine esterase for in vitro generated CTL effector activity, these results may account for recovery of CTL activity . Although IL 2 restored CTL function and serine esterase transcription, it did not block cholera toxin-catalyzed ribosylation of the 43-kDa GTP-binding protein, nor did it prevent the accumulation of intracellular levels of cAMP . In vivo, C57BL/6 mice challenged with the allogeneic tumor P815 had suppressed CTL function when cholera toxin was administered . These cholera toxin-treated mice died of tumor overgrowth, whereas untreated mice rejected the allogeneic tumor . Co-treatment of alloimmunized mice with cholera toxin and IL 2 prevented death from tumor overgrowth and restored CTL function; 67% of these mice survived . These data provide evidence that IL 2 acts in CTL through a mechanism independent of cholera toxin-sensitive GTP-binding protein in vitro and in vivo, despite elevated intracellular cAMP levels. Nature, 1991 May 30, 351(6325), 371 - 7 Crystal structure of a cholera toxin-related heat-labile enterotoxin from E . coli; Sixma TK et al.; Examination of the structure of Escherichia coli heat-labile enterotoxin in the AB5 complex at a resolution of 2.3A reveals that the doughnut-shaped B pentamer binds the enzymatic A subunit using a hairpin of the A2 fragment, through a highly charged central pore . Putative ganglioside GM1-binding sites on the B subunits are more than 20A removed from the membrane-crossing A1 subunit . This ADP-ribosylating (A1) fragment of the toxin has structural homology with the catalytic region of exotoxin A and hence also to diphtheria toxin. Neurosci Lett, 1991 May 27, 126(2), 199 - 202 Does androgen affect axonal transport of cholera toxin HRP in spinal motoneurons? Leslie M, Forger NG, Breedlove SM. We examined the effect of systemic androgen levels upon the rate at which lumbosacral motoneurons are labeled with cholera toxin-conjugated horseradish peroxidase (CT-HRP) injected into target muscles . CT-HRP first reaches the spinal nucleus of the bulbocavernosus between 8 and 10 h after injection into the bulbocavernosus muscle of adult male rats, but the number of motoneurons filled with CT-HRP does not differ between androgen-treated and control castrates at any of the time points examined . Thus, contrary to current speculation, we found no evidence that androgen can affect retrograde transport of CT-HRP by rat motoneurons. Biochemistry, 1991 May 21, 30(20), 5055 - 60 Thermodynamic identification of stable folding intermediates in the B-subunit of cholera toxin; Bhakuni V et al.; The structural stability and domain structure of the pentameric B-subunit of cholera toxin have been measured as a function of different perturbants in order to assess the magnitude of the interactions within the B-subunits . For these studies, temperature, guanidine hydrochloride (GuHCl), and pH were used as perturbants, and the effects were measured by high-sensitivity differential scanning calorimetry, isothermal reaction calorimetry, fluorescence spectroscopy, and partial protease digestion . At pH 7.5 and in the absence of any additional perturbants, the thermal unfolding of the B-subunit pentamer is characterized by a single peak in the heat capacity function centered at 77 degrees C and characterized by a delta Hcal of 328 kcal/mol of B-subunit pentamer and delta Hvh/delta Hcal of 0.3 . Lowering the pH down to 4 or adding GuHCl up to 2 M results in a decrease of the calorimetric enthalpy with no significant effect on the van't Hoff enthalpy . The transition enthalpy decreases in a sigmoidal fashion with pH, with an inflection point centered at pH 5.3 . Isothermal titration calorimetric studies as a function of pH also report a transition centered at pH 5.3 and characterized by an enthalpy change of 27 kcal/mol of B-subunit pentamer at 27 degrees C . Below this pH, the enthalpy change for the unfolding transition is reduced to approximately 100 kcal/mol of B-subunit pentamer . Similar behavior is obtained with GuHCl . In this case, a first transition is observed at 0.5 M GuHCl and a second one at 3 M GuHCl.(ABSTRACT TRUNCATED AT 250 WORDS) FEBS Lett, 1991 May 6, 282(2), 347 - 50 Protein phosphorylation in isolated nuclei from etiolated Avena seedlings . Effects of red/far-red light and cholera toxin; Romero LC et al.; We have studied the phosphorylation/dephosphorylation of several nuclear proteins in isolated nuclei from etiolated Avena seedlings as a function of red/far-red light . The effect of stimulatory (ADP-ribosylation by cholera toxin) or inhibitory (GDP beta S) conditions for GTP-binding proteins was also studied . Red or far-red light enhanced the phosphorylation level of 2 nuclear proteins with molecular masses of 75 and 60 kDa . The phosphorylation pattern was affected by the addition of cholera toxin or GDP beta S to the isolated nuclei . At least 2 proteins with molecular masses of 24 and 75 kDa cross-reacted by Western blot with GTP-binding protein antibodies. J Biol Chem, 1991 May 5, 266(13), 8213 - 9 Differential expression during development of ADP-ribosylation factors, 20-kDa guanine nucleotide-binding protein activators of cholera toxin; Tsai SC et al.; Cholera toxin exerts its effects on cells in large part through the ADP-ribosylation of guanine nucleotide-binding proteins . Toxin-catalyzed ADP-ribosylation is enhanced by approximately 20-kDa guanine nucleotide-binding proteins termed ADP-ribosylation factors (ARFs), which are allosteric activators of the toxin catalytic unit . Rabbit antiserum against a purified bovine brain ARF (sARF II) reacted on immunoblots with two approximately 20-kDa ARF-like proteins (sARF I and II) in tissue extracts from bovine, rat, frog, and chicken . Levels of ARF were higher in brain than in non-neural tissues . In rat brain, on the second postnatal day, amounts of sARF I and II were similar . By the 10th postnatal day and thereafter, sARF II predominated . Relative levels of ARF determined by immunoreactivity were in agreement with levels assessed in functional assays of cholera toxin-catalyzed ADP-ribosylation . Based on nucleotide and deduced amino acid sequences of human and bovine cDNAs, there appear to be at least six different ARF-like genes . Northern blots of rat brain poly(A)+ RNA were hybridized with cDNA and oligonucleotide probes specific for each of the human and bovine ARF genes . From the second to the 27th postnatal day, ARF 3 mRNA increased, whereas mRNAs for ARFs 2 and 4 decreased; and those for ARFs 1, 5, and 6 were apparently unchanged . Partial amino acid sequence of sARF II is consistent with it being either the ARF 1 or 3 gene product . The developmental changes in rat brain ARF parallel neuronal maturation and synapse formation. MMWR Morb Mortal Wkly Rep, 1991 May 3, 40(17), 287 - 9 Cholera--New Jersey and Florida; Cholera toxin and Gs protein modulation of synaptic transmission in guinea pig mesenteric artery; Department of Physiology and Biophysics, University of Cincinnati, College of Medicine, OH 45267-0576Cholera toxin (CTX) was used to test whether the presynaptic beta-adrenoceptors of guinea-pig mesenteric artery are coupled via stimulatory GTP-binding proteins . The vascular smooth muscle cells were electrically quiescent unless stimulated and had a mean resting potential of -68.7 +/- 2.8 mV (n = 16) and input resistance of 12.1 +/- 0.5 M omega (n = 4) . Perivascular nerve stimulation with brief square pulses evoked excitatory junction potentials (EJPs) in the muscle cells . Isoproterenol (0.1 microM) enhanced the EJP amplitude without modifying the passive membrane properties of the muscle cells . The beta-blocker, propranolol (0.5 microM), prevented the effects of isoproterenol on EJP amplitude . The permeant analogue of cyclic AMP, 8-bromocAMP, also potentiated EJP amplitude . EJP amplitude was markedly enhanced by treatment of the isolated blood vessels with CTX (10 micrograms/ml for 1 h) . The muscle cells became hyperpolarized (-74.6 +/- 2.1 mV, n = 5), and their input resistances were significantly reduced (8.2 +/- 0.5 M omega, n = 4) . These effects of CTX persisted after washout . Addition of GM1 ganglioside (5 micrograms/ml) prevented the CTX effects . The CTX enhancement of EJP amplitude was not prevented by application of depolarizing current (ca . 0.5 nA) the muscle cells (to counter the hyperpolarization) . These results suggest that CTX increases the neurotransmitter release from the nerve terminals; the hyperpolarization may be due to an increase in K+ conductance . These effects of CTX may be mainly due to elevation of cAMP in the nerve terminal and in the muscle cell. J Immunol, 1991 May 1, 146(9), 2908 - 14 Cholera toxin stimulates IL-1 production and enhances antigen presentation by macrophages in vitro; Bromander A et al.; Cholera toxin (CT) is a strong systemic and mucosal adjuvant that greatly enhances IgG and IgA immune responses . We investigated whether CT potentiates Ag presentation by macrophages as a possible mechanism underlying its adjuvant function . This was tested by preculturing APC in CT and analyzing the effect of CT treatment on the capacity to trigger 1) an allogeneic proliferative response of normal mesenteric lymph node T cells (H-2b) to the macrophage cell line P388D1 (H-2d) or 2) an Ag-specific proliferative response of D10.G4.1 clonal T cells in co-culture with normal macrophages and Ag . Pretreatment of APC, normal peritoneal macrophages or the P388D1 cells, with CT strongly enhanced Ag- and allogen-specific T cell proliferation . Also P388D1 APC treated with CT and then formalin-fixed demonstrated enhanced ability to stimulate T cell proliferation as compared to cells not exposed to CT, suggesting that the effect of CT on APC might be to enhance expression of a cell-associated factor . Flow microfluorimetry analysis of P388D1 cells cultured in CT-containing medium failed to detect an increase in class II MHC-Ag expression as compared to that found on cells not cultured in CT . In contrast, both soluble and cell-associated IL-1 formation was increased several-fold by CT, but with different CT dose requirements . A total of 10 to 100 times more CT were required for elevating the soluble IL-1 as compared to the cell associated IL-1, which was increased by as little as 1 ng/ml of CT . The soluble and cell-associated IL-1 activity induced by CT was abrogated by a polyclonal antiserum to IL-1-alpha . Similarly, the potentiating effect of CT on the ability of P388D1 APC to trigger alloreactive T cell proliferation was also blocked completely by the addition of the anti-IL-1-alpha antibody to the test system . This is the first study to demonstrate that CT potentiates Ag presentation . The mechanism for this effect probably involves induction of IL-1 production and in particular of a cell-associated form of IL-1 (IL-1-alpha) . Potentiation of APC function might be important for the adjuvant action of CT on the immune response in vivo. J Virol, 1991 May, 65(5), 2761 - 5 Live attenuated pseudorabies virus expressing envelope glycoprotein E1 of hog cholera virus protects swine against both pseudorabies and hog cholera; van Zijl M et al.; To investigate whether live attenuated pseudorabies virus (PRV) can be used as a vaccine vector, PRV recombinants that expressed envelope glycoprotein E1 of hog cholera virus (HCV) were generated . Pigs inoculated with these recombinants developed high levels of neutralizing antibodies against PRV and HCV and were protected against both pseudorabies and hog cholera (classical swine fever). Rev Med Chil, 1991 May, 119(5), 601 - 3 {Andrés Bello and cholera}; Costa-Casaretto C; Andres Bello, an intellectual and humanist and the first Rector of the University of Chile, published several articles about cholera in the Araucano, a newspaper of Santiago . Basically, they were translations and comments of articles about the epidemics affecting Europe and the British Isles between 1830 and 1846 . Cholera affected Chile in 1886. Arch Biochem Biophys, 1991 May 1, 286(2), 579 - 85 Multiple changes induced by cholera toxin contribute to the stimulation of aerobic lactate production in rat-1 fibroblasts; Resnick RJ et al.; Exposure of rat-1 fibroblasts to cholera toxin increased aerobic lactate production 3- to 8-fold with maximal stimulation observed between 1 and 2 h at a concentration of 1-2 micrograms/ml . Concomitant with this change was a 10- to 40-fold elevation in the intracellular concentration of cAMP . The cell permeable cAMP analogue, N6,2'-O-dibutyryl cAMP and the cyclic nucleotide phosphodiesterase inhibitor RO-20-1724 also increased lactate production and intracellular cAMP levels, although less effectively . Cholera toxin and dibutyryl cAMP induced a 2- to 3-fold elevation of intracellular fructose 2,6-bisphosphate and 2- to 3-fold increases in both 3-O-methylglucose and inorganic phosphate transport . A survey of five additional cell lines revealed striking variabilities in their individual responses to cholera toxin and dibutyryl cAMP . All were observed to be considerably less sensitive to either agent than rat-1 cells . These data suggest that a cooperative effect involving multiple parameters may be responsible for the observed increases in aerobic lactate production in response to cAMP and that these parameters may vary significantly among cell lines. Biochemistry, 1991 Apr 16, 30(15), 3697 - 703 Guanine nucleotide dependent formation of a complex between choleragen (cholera toxin) a subunit and bovine brain ADP-ribosylation factor; Tsai SC et al.; Cholera toxin activates adenylyl cyclase by catalyzing the ADP-ribosylation of Gs alpha, the stimulatory guanine nucleotide binding protein of the cyclase system . This toxin-catalyzed reaction, as well as the ADP-ribosylation of guanidino compounds and auto-ADP-ribosylation of the toxin A1 protein (CTA1), is stimulated, in the presence of GTP (or GTP analogue), by 19-21-kDa proteins, termed ADP-ribosylation factors or ARFs . These proteins directly activate CTA1 in a reaction enhanced by sodium dodecyl sulfate (SDS) or dimyristoylphosphatidylcholine (DMPC)/cholate . To determine whether ARF stimulation of ADP-ribosylation is associated with formation of a toxin-ARF complex, these proteins were incubated with guanine nucleotides and/or detergents and then subjected to gel permeation chromatography . An active ARF-toxin complex was observed in the presence of SDS and GTP gamma S {guanosine 5'-O-(3-thiotriphosphate)} but not GDP beta S {guanosine 5'-O-(2-thiodiphosphate)} . Only a fraction of the ARF was capable of complex formation . The substrate specificities of complexed and noncomplexed CTA differed; complexed CTA exhibited markedly enhanced auto-ADP-ribosylation . In the presence of GTP gamma S and DMPC/cholate, an ARF-CTA complex was not detected . A GTP gamma S-dependent ARF aggregate was observed, however, exhibiting a different substrate specificity from monomeric ARF . These studies support the hypothesis that in the presence of guanine nucleotide and either SDS or DMPC/cholate, ARF and toxin exist as multiple species which exhibit different substrate specificities. J Comp Neurol, 1991 Apr 8, 306(2), 344 - 60 Retinohypothalamic tract in the female albino rat: a study using horseradish peroxidase conjugated to cholera toxin; Levine JD et al.; There are several anatomically and functionally distinct retinofugal pathways, one of which is the retinohypothalamic tract (RHT) . In this study, horseradish peroxidase conjugated to cholera toxin (CT-HRP), a sensitive neural tracer, was employed to describe the RHT in the female albino rat . Following uniocular injection of CT-HRP, both medial and lateral components of the RHT were evident . The medial component swept caudally into and through the suprachiasmatic nucleus (SCN) and dorsally to the subparaventricular zone . Terminal label was seen in the medial preoptic region, peri-SCN area, retrochiasmatic area, periventricular nucleus, anterior and central parts of the anterior hypothalamic area, and the subparaventricular zone . In contrast to the more focused and symmetrical medial component, the lateral component was diffuse with light terminal label in the lateral preoptic region, olfactory tubercle, lateral hypothalamus, supraoptic nucleus, and medial and posteroventral medial amygdaloid nuclei . The striking exception to this diffuse pattern of the lateral component was an extremely dense columnar terminal field over the dorsal border of the supraoptic nucleus . Whereas the intensity of label in terminal fields of the medial component was often similar on the sides ipsilateral and contralateral to the injection, the lateral component was consistently asymmetrical with greater labeling on the side contralateral to the injection . In addition, a light projection arrived at several thalamic nuclei by returning toward the thalamus from the tectal or pretectal areas via stria medullaris, and thus was not a part of the RHT . Implications for circadian as well as noncircadian photobiologic effects are discussed. J Biol Chem, 1991 Apr 5, 266(10), 6447 - 55 Agonist-dependent, cholera toxin-catalyzed ADP-ribosylation of pertussis toxin-sensitive G-proteins following transfection of the human alpha 2-C10 adrenergic receptor into rat 1 fibroblasts . Evidence for the direct interaction of a single receptor with two pertussis toxin-sensitive G-proteins, Gi2 and Gi3; Milligan G et al.; A DNA encoding the human alpha 2-C10 adrenergic receptor was transfected into Rat 1 fibroblasts and clones selected on the basis of resistance to G418 sulfate . Two clones, one of which (1C) expressed some 3.5 pmol/mg membrane protein of the receptor as assessed by the specific binding of {3H}yohimbine and one (4D) which did not express detectable amounts of the receptor were selected for further study . When cholera toxin-catalyzed ADP-ribosylation was performed with {32P}NAD on membranes of these cells in the absence of added guanine nucleotides, radioactivity was incorporated into a polypeptide(s) of 40 kDa in addition to the 45- and 42-kDa forms of Gs alpha . Addition of the selective alpha 2 receptor agonist U.K.14304 enhanced markedly, in a dose-dependent manner, the cholera toxin-catalyzed {32P}ADP-ribosylation of the 40-kDa polypeptide(s), but not the 45- or 42-kDa polypeptides, in membranes of the 1C cells . Dose response curves for U.K.14304 enhancement of cholera toxin-labeling of the 40-kDa polypeptide(s) and stimulation of high affinity GTPase activity were identical . By contrast, U.K.14304 was ineffective in either assay in membranes from the 4D cells, demonstrating this effect to be dependent upon receptor activation . Furthermore, the alpha 2 receptor antagonist yohimbine blocked all effects of U.K.14304 . The agonist promotion of cholera toxin-catalyzed ADP-ribosylation of Gi was completely blocked by guanine nucleotides . Whether GDP or GDP + fluoroaluminate (as a mimic of GTP) was used, blockade of the agonist effect was complete and indeed both conditions prevented agonist-independent labeling by cholera toxin of the 40-kDa polypeptide(s) . Mg2+ produced an agonist-independent cholera toxin-catalyzed {32P}ADP-ribosylation of the 40-kDa polypeptide(s) but even in the presence of {Mg2+}, agonist-stimulation of cholera toxin-labeling of the 40-kDa polypeptide(s) was observed and was additive with the effect of {Mg2+} . Agonist stimulation of cholera toxin-catalyzed ADP-ribosylation of Gi was completely attenuated by pretreatment of the cells with pertussis toxin, which prevents contact between receptors and G-proteins which are substrates for this toxin . By contrast, pretreatment of the cells with concentrations of cholera toxin able to "down-regulate" essentially all of the membrane-associated Gs alpha did not prevent agonist stimulation of cholera toxin-catalyzed ADP-ribosylation of Gi.(ABSTRACT TRUNCATED AT 400 WORDS) Gastroenterology, 1991 Apr, 100(4), 986 - 97 Mucin and nonmucin secretagogue activity of Entamoeba histolytica and cholera toxin in rat colon; Chadee K et al.; Depletion of colonic mucus occurs before invasion of the colonic mucosa by Entamoeba histolytica trophozoites . It is hypothesized that E . histolytica releases a mucus secretagogue; this was studied in a rat colonic loop model . In colonic loops exposed to live amebae, mucus secretion was quantitated by release of acid-precipitable {3H}glucosamine-labeled luminal glycoprotein and by specific immunoassay . Mucus secretion increased in dose-dependent fashion in response to greater than or equal to 1 X 10(5) trophozoites; cholera toxin (20 micrograms per loop), a known mucus secretagogue, elicited a similar response . Thin-section histological analysis of amebae and cholera toxin-exposed loops showed increased mucus release and streaming from mucosal goblet cells with cellular cavitation compared with control loops . Sepharose-4B chromatography of amebae and cholera toxin-stimulated glycoproteins demonstrated secretion of mucins and an 80%-90% increase in low-molecular-weight proteins . E . histolytica trophozoites and cholera toxin enhanced the secretion of preformed and newly synthesized mucin glycoproteins and stimulated colonic glycoprotein synthesis . The level of mucus secretion elicited by axenic E . histolytica strains correlated with their virulence in vivo and in vitro . The amebic secretagogue was released into the culture medium and was heat stable . Mucus secretagogue activity of E . histolytica may contribute to depletion or alteration of the protective mucus blanket, facilitating pathogenesis of invasive amebiasis. Zh Mikrobiol Epidemiol Immunobiol, 1991 Apr, (4), 31 - 3 {An oral chemical vaccine from the hypertoxigenic strains of the causative agent of cholera KM-76 Inaba and KM-68 Ogawa}; Dzhaparidze MN et al.; The material on the development of chemical vaccine, prepared from two newly formed strains (KM-76 Inaba and KM-68 Ogawa) and intended for oral administration, is presented . The conditions for the submerged cultivation of these strains have been established, which makes it possible to increase the production of choleragen 8- to 10-fold and O-antigen 3- to 4-fold in comparison with V . cholerae natural strain 569B . The maximum accumulation of neuraminidase, protease, phospholipase, along with choleragen, has been registered in the logarithmic phase and that of O-antigen, in the stationary phase of growth . The use of strains KM-76 and KM-68 has led to the fourfold increase of the specific activity of the main immunogens, thus permitting the respective increase of the yield of the oral vaccine without changes in its high capacity for the formation of specific antibodies and its low residual toxigenicity. Rev Med Chil, 1991 Apr, 119(4), 481 - 4 {The first and unique epidemics of cholera in Chile (1886-1888)}; Costa-Casaretto C; The first and only cholera epidemics in Chile took place between 1886 and 1888 . It had originated in India in 1883, extended to Mecca and Alexandria, the Mediterranean, and reached Chile from Argentina . In spite of sanitary measures adopted by the government, the epidemics swept the country, with an estimated 56,838 patients and 23,395 dead (41% lethality rate) . Two outburst were observed: the first lasted 203 days (1886-87), the second 121 days . Duration varied from town to town, from 16 to a maximum of 288 days. Biochem J, 1991 Apr 1, 275 ( Pt 1), 175 - 81 Cholera toxin impairment of opioid-mediated inhibition of adenylate cyclase in neuroblastoma x glioma hybrid cells is due to a toxin-induced decrease in opioid receptor levels; McKenzie FR et al.; Cholera toxin treatment (up to 1 microgram/ml, 16 h) of neuroblastoma x glioma hybrid NG108-15 cells produced a decrease of some 35% in both delta opioid receptor-mediated stimulation of high-affinity GTPase activity and inhibition of forskolin-amplified adenylate cyclase . Coincident with these decreases was a down-regulation of some 35% in the delta opioid receptor population . A similar pattern of a decrease in signalling capacity was noted for the alpha 2B-adrenergic receptor in these cells after cholera toxin treatment . Half-maximal effects of cholera toxin on all of the parameters assayed were noted at concentrations between 2 and 5 ng/ml . Neither levels of Gi2, as assessed by immunoblotting with specific antisera, nor the intrinsic activity of the alpha subunit of the guanine-nucleotide-binding protein which acts as the inhibitory G-protein of the adenylate cyclase in these cells, as assessed by guanosine 5'-{beta gamma-imido}triphosphate (Gpp{NH}p)-mediated inhibition of adenylate cyclase, was lowered by cholera toxin treatment . Furthermore, levels of another pertussis toxin-sensitive G-protein (Go) expressed by these cells was also not lowered by cholera toxin treatment . However, as previously noted in other cells {Milligan, Unson & Wakelam (1989) Biochem . J . 262, 643-649}, marked down-regulation of the alpha subunit of the stimulatory G-protein (Gs) of the adenylate cyclase cascade was observed in response to cholera toxin treatment . Previous studies {Klee, Milligan, Simonds & Tocque (1985) Mol . Aspects Cell Regul . 4, 117-129} have shown that cholera toxin treatment can result in a decrease in the maximal effectiveness of agonists which function to inhibit adenylate cyclase . These data have been used as evidence to suggest a functional interaction between Gs and 'Gi' . The results provided herein demonstrate that such effects of the toxin can be explained adequately by a decrease in the number of receptors that function to produce inhibition of adenylate cyclase. Biochim Biophys Acta, 1991 Mar 19, 1092(1), 79 - 84 Protein synthesis is required for cholera toxin-induced stimulation of arachidonic acid metabolism; Peterson JW et al.; The molecular events in the mechanism of action of cholera toxin were analyzed using Chinese hamster ovary (CHO) cells . Cholera toxin stimulated both 3',5'-cyclic adenosine monophosphate (cAMP) synthesis and arachidonic acid metabolism in these cells . The turnover of phospholipid by cholera toxin-induced stimulation of phospholipase activity evoked the synthesis of PGE2 and other prostaglandins . Cholera toxin-induced release of both {3H}arachidonic acid and PGE2 was blocked by addition of either cycloheximide or actinomycin D . In contrast, accumulation of cAMP in cholera toxin-treated CHO cells was unaffected by adding these drugs . Further, dibutyryl cAMP or forskolin caused {3H}arachidonic acid release, which also was blocked by cycloheximide and actinomycin D . We concluded that the sequence of molecular events in cholera toxin-treated CHO cells first involved activation of adenylate cyclase, which caused an increase in cAMP . In turn, cAMP promoted transcription of mRNA that encoded either a specific phospholipase or a phospholipase-activating protein . The emerging arachidonic acid metabolites (e.g., PGE2 and PGF2 alpha) might be important mediators of cholera toxin's stimulatory effects on vascular permeability and smooth muscle contraction in the intestine during cholera. Eur J Biochem, 1991 Mar 14, 196(2), 313 - 20 Treatment of intact striatal neurones with cholera toxin or 8-bromoadenosine 3',5'-(cyclic)phosphate decreases the ability of pertussis toxin to ADP-ribosylate the alpha-subunits of inhibitory and other guanine-nucleotide-binding regulatory proteins, Gi and Go . Evidence for two distinct mechanisms; Maus M et al.; Using primary cultures of striatal neurones from the mouse embryo, we showed that treatment of intact cells with cholera toxin (5 micrograms/ml, 22 h) decreases the subsequent ADP-ribosylation of the alpha subunit of the guanine-nucleotide-binding regulatory protein Go (Go alpha) and the alpha subunit of the inhibitory guanine-nucleotide-binding regulatory protein (Gi alpha) of adenylate cyclase, which is catalyzed in vitro on neuronal membranes by pertussis toxin . The inhibitory effect of cholera toxin could not only be attributed to an increased production of cAMP in neurones . Treatment of cells with 0.1 microM 8-bromoadenosine 3',5'-(cyclic)phosphate (BrcAMP) for 16 h, or with 0.1 mM BrcAMP for 5 min, mimicked the effect of cholera toxin on the ADP-ribosylation of Go alpha and Gi alpha in vitro . However, the two agents seem to act through distinct mechanisms . The protein kinase inhibitor 1-(5-isoquinolinesulfonyl)-2-methylpiperazine prevented the action of Br8cAMP but not that of cholera toxin . In addition, measurements of the pI of the Go alpha deduced from immunoblots of two-dimensional gels performed using a specific antibody directed against Go alpha suggest that treatment of neurones with cholera toxin induces ADP-ribosylation of Go alpha in intact cells, while BrcAMP does not. Biochemistry, 1991 Mar 12, 30(10), 2563 - 70 Neoglycolipid analogues of ganglioside GM1 as functional receptors of cholera toxin; Pacuszka T et al.; We synthesized several lipid analogues of ganglioside GM1 by attaching its oligosaccharide moiety (GM1OS) to aminophospholipids, aliphatic amines, and cholesteryl hemisuccinate . We incubated GM1-deficient rat glioma C6 cells with each of the derivatives as well as native GM1 and assayed the cells for their ability to bind and respond to cholera toxin . On the basis of the observed increase in binding of 125I-labeled cholera toxin, it was apparent that the cells took up and initially incorporated most of the derivatives into the plasma membrane . In the case of the aliphatic amine derivatives, the ability to generate new toxin binding sites was dependent on chain length; whereas the C10 derivative was ineffective, C12 and higher analogues were effective . Increased binding was dependent on both the concentration of the neoglycolipid in the medium and the time of exposure . Cells pretreated with the various derivatives accumulated cyclic AMP in response to cholera toxin, but there were differences in their effectiveness . The cholesterol and long-chain aliphatic amine derivatives were more effective than native GM1, whereas the phospholipid derivatives were less effective . The distance between GM1OS and the phospholipid also appeared to influence its functional activity . The neoglycolipid formed by cross-linking the amine of GM1OS to phosphatidylethanolamine (PE) with disuccinimidyl suberate was less effective than the neoglycolipid formed by directly attaching GM1OS to PE by reductive amination . Furthermore, insertion of a C8 spacer in the former neoglycolipid rendered it even less effective.(ABSTRACT TRUNCATED AT 250 WORDS) Nature, 1991 Mar 7, 350(6313), 74 - 7 Pituitary hyperplasia and gigantism in mice caused by a cholera toxin transgene; Burton FH et al.; Cyclic AMP is thought to act as an intracellular second messenger, mediating the physiological response of many cell types to extracellular signals . In the pituitary, growth hormone (GH)-producing cells (somatotrophs) proliferate and produce GH in response to hypothalamic GH-releasing factor, which binds a receptor that stimulates Gs protein activation of adenylyl cyclase . We have now determined whether somatotroph proliferation and GH production are stimulated by cAMP alone, or require concurrent, non-Gs-mediated induction of other regulatory molecules by designing a transgene to induce chronic supraphysiological concentrations of cAMP in somatotrophs . The rat GH promoter was used to express an intracellular form of cholera toxin, a non-cytotoxic and irreversible activator of Gs . Introduction of this transgene into mice caused gigantism, elevated serum GH levels, somatotroph proliferation and pituitary hyperplasia . These results support the direct triggering of these events by cAMP, and illustrate the utility of cholera toxin transgenes as a tool for physiological engineering. Immunology, 1991 Mar, 72(3), 329 - 35 H-2-unrestricted adjuvant effect of cholera toxin B subunit on murine antibody responses to influenza virus haemagglutinin; Hirabayashi Y et al.; Cholera toxin B subunit (CTB) has been shown to augment the antibody responses to influenza virus haemagglutinin (HA) in BALB/c mice immunized with HA vaccine together with CTB . In this study, mouse strain differences in the adjuvant effect of CTB on anti-HA antibody responses were investigated along with those in the antibody responses to CTB or HA, using various inbred and H-2 congenic strains . The antibody responsiveness to CTB depended on the H-2 haplotype of the strain: strains with the H-2b haplotype were high responders, those with H-2a, H-2k and H-2s were low responders, and those with H-2d were intermediate . The responsiveness to HA was also related to the H-2 haplotype: H-2a and H-2k strains were high responders, H-2b and H-2s strains were low responders, and H-2d strains were intermediate . However, the degree of the adjuvant effect of CTB on anti-HA antibody responses was almost constant, regardless of the H-2 haplotype or other genetic backgrounds of the strain . The lack of genetic restriction of the adjuvant effect would be favourable for application of CTB-combined HA vaccine to humans, who are genetically diverse . Moreover, these results suggest that the immunogenicity and adjuvanticity of CTB differ essentially in their mechanisms. Infect Immun, 1991 Mar, 59(3), 996 - 1001 Antibody-producing cells in peripheral blood and salivary glands after oral cholera vaccination of humans; Czerkinsky C et al.; We examined whether immunization with a newly developed oral cholera vaccine would elicit gut-derived antibody-producing cells in the blood and in distant mucosal tissues, such as the minor salivary glands, in 30 adult Swedish volunteers . The results of this study demonstrated that this vaccine indeed induced production of specific antibody-producing cells against the cholera toxin B subunit in both peripheral blood and salivary glands . The response in blood, which after primary and booster immunizations comprised both immunoglobulin A (IgA) and IgG antibody-forming cells, was highly transient and preceded the response in salivary glands; the latter response was restricted to the IgA isotype . The results provide further evidence of the existence of a common mucosal immune system in humans . Furthermore, these findings support previous observations that in animals, the cholera toxin B subunit may be a useful carrier protein for preparing enteric vaccines against pathogens encountered at intestinal and extraintestinal mucosal sites. Immunology, 1991 Mar, 72(3), 323 - 8 Mucosal priming of T-lymphocyte responses to fed protein antigens using cholera toxin as an adjuvant; Clarke CJ et al.; Cholera toxin is widely recognized as a potent stimulator of mucosal IgA responses after oral feeding . However, comparatively little is known of its ability to stimulate cellular responses . This is due in part to the direct inhibitory effects of cholera toxin on T lymphocytes, which can confound attempts to study primed T-cell responses in vitro . We have avoided this problem by using cholera toxin as an adjuvant to enhance the response to an unrelated protein fed simultaneously . Thus the simultaneous feeding of 10 micrograms of cholera toxin and 5 mg of keyhole limpet haemocyanin (KLH) results in the priming of T cells in the spleen, mesenteric lymph nodes, Peyer's patch and lamina propria that proliferate when restimulated with KLH in vitro . The feeding of KLH alone does not result in such responses . Both CD4- and CD8-positive antigen-specific T cells were involved in the response and the cells produced both interleukins 2 and 4 on antigen restimulation in vitro. J Virol, 1991 Feb, 65(2), 589 - 97 Structural proteins of hog cholera virus expressed by vaccinia virus: further characterization and induction of protective immunity; Rumenapf T et al.; A cDNA fragment covering the genomic region that encodes the structural proteins of hog cholera virus (HCV) was inserted into the tk gene of vaccinia virus . Expression studies with vaccinia virus/HCV recombinants led to identification of HCV-specific proteins . The putative HCV core protein p23 was demonstrated for the first time by using an antiserum against a bacterial fusion protein . The glycoproteins expressed by vaccinia virus/HCV recombinant migrated on sodium dodecyl sulfate-gels identically to glycoproteins precipitated from HCV-infected cells . A disulfide-linked heterodimer between gp55 and gp33 previously detected in HCV-infected cells was also demonstrated after infection with the recombinant virus . The vaccinia virus system allowed us to identify, in addition to the heterodimer, a disulfide-linked homodimer of HCV gp55 . The vaccinia virus/HCV recombinant that expressed all four structural proteins induced virus-neutralizing antibodies in mice and swine . After immunization of pigs with this recombinant virus, full protection against a lethal challenge with HCV was achieved . A construct that lacked most of the HCV gp55 gene failed to induce neutralizing antibodies but induced protective immunity. Mol Biol Med, 1991 Feb, 8(1), 129 - 33 Effect of cholera toxin on L-{14C}glycine uptake and intestinal cell enzymes in rabbit; Yi-Yi-Myint et al.; The uptake of L-{14C}glycine and the activities of intracellular marker enzymes of enterocytes were studied in ligated small intestinal segments of rabbits during experimental cholera induced by intra-intestinal injection of pure cholera toxin (CT) . No significant difference was observed in the active uptake of L-{14C}glycine between the CT-injected small intestinal segments and the saline-injected control segments, indicating that there is an intact active transport system for intestinal absorption of L-{14C}glycine during experimental cholera in rabbits . Apart from a significant increase in the activity of a brush border marker enzyme (alkaline phosphatase), there was no significant difference between the activities of marker enzymes for lysosomes (acid phosphate), microsomes (glucose-6-phosphatase), mitochondria (succinate dehydrogenase), and a cytosol enzyme (proteinase) in mucosal homogenates of CT-injected small intestinal segments compared to controls . The finding of an intact mitochondrial marker enzyme together with intact L-{14C}glycine absorption provides a scientific basis for considering the use of glycine and other monoamino monocarboxylic amino acids in "improved" oral rehydration solutions for the treatment of acute diarrhea, including cholera. Tierarztl Prax, 1991 Feb, 19(1), 54 - 7 {A sensitive enzyme-linked immunosorbent assay (ELISA) for the serological detection of antibodies against the European hog cholera virus}; Schagemann G et al.; An ELISA for the detection of antibodies against hog cholera virus (HCV) was developed . The HCV-specific glycoprotein gp53 served as diagnostic antigen after immobilization using a monoclonal capture antibody . Due to the higher affinity of HCV-specific antibodies to the viral gp53, sera cross reacting with bovine viral diarrhea (BVD) virus were discriminated by the slope of the titration curves. Biochem Biophys Res Commun, 1991 Jan 15, 174(1), 372 - 8 The B subunit of cholera toxin enhances DNA synthesis in rat hepatocytes induced by insulin and epidermal growth factor; Mitsui H et al.; The B subunit of cholera toxin, which binds to ganglioside GM1, enhanced DNA synthesis in rat hepatocytes in primary culture induced by insulin and/or epidermal growth factor . The effect was dose-dependent, and whole cholera toxin, activating adenylate cyclase, showed a higher effect than the B subunit alone . The B subunit acted additively with other agents that also increase cyclic AMP levels . A competitive antagonist of cyclic AMP could not suppress the effect of the B subunit completely . These data suggest that the effect is independent of the cyclic AMP signal pathway, and that GM1 plays a role in hepatocyte proliferation. Arch Virol Suppl, 1991, 3, 209 - 15 Differentiation of pestiviruses by a hog cholera virus-specific genetic probe; Schelp C et al.; A hog cholera virus (HCV)-specific genetic probe has been generated after cloning of the genomic viral RNA . This probe distinguished between HCV and the closely related bovine viral diarrhoea virus (BVDV) . Furthermore, it detected a broad spectrum of HCV strains and isolates which differ in their phenotype such as virulence. Arch Virol Suppl, 1991, 3, 7 - 18 Molecular characterization of hog cholera virus; Rumenapf T et al.; An efficient tissue culture system was established which allowed to obtain substantial quantities of hog cholera virus (HCV) from the cell free tissue culture supernatant . After preparation of viral RNA and cDNA synthesis, the complete HCV genome was cloned and sequenced . Comparison with published BVDV sequences revealed a surprisingly high homology between HCV and BVDV at both the nucleotide and the amino acid level . In addition host cellular sequences were identified in BVDV genomes . The genomic localization of HCV glycoproteins was determined by the use of sequence specific antisera directed against bacterial fusion proteins . The order on the HCV genome was determined as follows: N-gp44/48-gp33-gp55-C . HCV gp33 and HCV gp55 were shown to be intracellularly linked by disulfide bridges . A cDNA fragment covering the genomic region that encodes the structural proteins of HCV was inserted into a vaccinia recombination vector . Expression studies with vaccinia/HCV recombinants led to identification of HCV specific glycoproteins which migrated on sodium dodecyl sulfate-gels identically to glycoproteins precipitated from HCV-infected cells . The vaccinia virus/HCV recombinant that expressed all four structural proteins induced virus-neutralizing antibodies in mice and swine . After immunization of pigs with this recombinant virus, full protection against a lethal challenge with HCV was achieved . A construct that lacked most of the HCV gp55 gene failed to induce neutralizing antibodies but induced protective immunity. Int Immunol, 1991 Jan, 3(1), 95 - 103 Ig isotype switching in B lymphocytes . The effect of T cell-derived interleukins, cytokines, cholera toxin, and antigen on isotype switch frequency of a cloned B cell lymphoma; Whitmore AC et al.; The murine B cell lymphoma CH12.LX, which bears cell surface IgM specific for the phosphatidyl choline epitope of sheep red blood cells, is capable of spontaneous isotype switching in vitro . Switching to IgG3, IgG1, IgG2b, and IgA has been observed and variants expressing those isotypes have been isolated and cloned . We have developed a procedure for precise numerical evaluation of the frequency of switching to the several isotypes to which CH12.LX can switch . We have used a modified Poisson method which can distinguish between treatments which change isotype switch frequency and those which affect, in an isotype-specific fashion, growth or secretion rates of cells which have already switched . In this report we examine the effect of several cytokines, cholera toxin, hydroxyurea, and antigen on the isotype switch frequency of CH12.LX . The strongest effect observed was that of transforming growth factor-beta, which increases switch frequency 40-fold to an absolute switch frequency of 0.04 switch events (from IgM to IgA expression) per cell division . Interleukin-4 (IL-4) and cholera toxin also increase the switch frequency of CH12.LX while IL-5, IL-6 (with or without antigen), antigen (SRBC) alone, interferon-gamma, or hydroxyurea have no effect . We have shown that none of the cytokines studied change the relative frequency of switching to the available isotypes, only the absolute frequency of switching . We infer from this that the factors tested do not 'instruct' CH12.LX to switch to a particular isotype, but rather they deliver a 'go' signal to cells committed to switching to IgA at high frequency, rarely to IgG3, IgG1, or IgG2b, and never to IgG2a or IgE. J Cell Physiol, 1991 Jan, 146(1), 81 - 5 Prostaglandins do not mediate the actions of cholera toxin on pancreatic acini or gastric chief cells from the guinea pig; Raufman JP et al.; Recent reports suggest that prostaglandins, rather than cAMP, play a major role in mediating cholera toxin-induced water and electrolyte secretion from rabbit intestinal loops . We examined the role of prostaglandins in mediating toxin-induced pancreatic and gastric exocrine secretion . In these tissues, indomethacin, a potent inhibitor of prostaglandin synthesis, did not alter the stimulatory effects of cholera toxin on increases in cellular cAMP or enzyme secretion . Moreover, the addition of cholera toxin did not alter prostaglandin E2 release from either tissue . In contrast to their effects in rabbit intestinal loops, prostaglandins do not regulate cholera toxin-induced enzyme secretion from the guinea pig pancreas or stomach. Life Sci, 1991, 48(18), 1721 - 7 Cholera toxin and pertussis toxin on opioid- and alpha 2-mediated supraspinal analgesia in mice; Sanchez-Blazquez P et al.; Cholera toxin, an agent that impairs the function of Gs transducer proteins, was injected (0.5 microgram/mouse, icv) and the antinociceptive activity of opioids and clonidine was studied 24h later in the tail-flick test . In these animals, an enhancement of the analgesic potency of morphine, beta-endorphin and clonidine could be observed . Cholera toxin did not modify the antinociception evoked by the enkephalin derivatives DAGO and DADLE . Pertussis toxin that catalyses the ADP ribosylation of alpha subunits of Gi/Go regulatory proteins was given icv (0.5 microgram/mouse) . This treatment reduced the analgesic effect of opioids and clonidine . However, while the analgesia elicited by DAGO, DADLE and clonidine was greatly decreased, the effect of morphine and beta-endorphin was reduced to a moderate extent . It is concluded that Gi/Go regulatory proteins functionally coupled to opioid and alpha 2 receptors are implicated in the efficacy displayed by opioids and clonidine to produce supraspinal analgesia . Moreover, these two receptors are susceptible to regulation by a process that might involve a Gs protein. J Physiol (Paris), 1991, 85(4), 181 - 7 In vitro effects of tetrodotoxin and hexamethonium on electrolyte transport in rabbit ileum treated with cholera toxin; Ben Mansour A et al.; To determine if there was a role for the submucosal nerves in cholera toxin (CT)-induced secretion, we studied the effects of serosal addition of two neurotoxins, the nerve conduction blocking agent, tetrodotoxin (TTX), and the nicotinic ganglionic blocking agent, hexamethonium (HXM), on electrolyte secretion in control isolated rabbit ileum and in that stimulated by CT . 1) . In the absence of CT, the short circuit current (Isc) decreased after TTX (10(-7) M) (P less than 0.01) and was unaltered by HXM (10(-5) M) . In the presence of CT, Isc increased but was not modified by 10(-7) M TTX or 10(-5) M HXM . 2) In control tissues the mean isotopic Na+ and Cl- fluxes were not significantly altered by TTX addition . Cl- absorption alone was significantly reduced by HXM (delta JCl- = 1.95 +/- 0.81 microEq.hr-1.cm-2; P less than 0.02) . After stimulation with CT, TTX significantly inhibited Na+ and Cl- secretion (delta JNa+ = 2.15 +/- 0.61 and delta JCl- = 2.15 +/- 0.76 microEq.hr-1.cm-2; P less than 0.01) . Similarly, HXM significantly inhibited CT-stimulated Na+ and Cl- secretion (delta JNa+ = 1.73 +/- 0.70 and delta JCl- = 1.46 +/- 0.62 microEq.hr-1.cm-2; P less than 0.02) . 3) In TTX and HXM treated tissues there was no difference in the increase in Isc caused by cAMP (2 x 10(-3) M), calcium ionophore A 23187 (4 x 10(-6) M) and glucose (10(-3) M) compared to the untreated tissues in the presence or absence of CT.(ABSTRACT TRUNCATED AT 250 WORDS) Dev Biol Stand, 1991, 73, 133 - 41 Monoclonal antibodies against the enzymatic subunit of both pertussis and cholera toxins; Kenimer JG et al.; A synthetic peptide corresponding to amino acids 6-17 of the A subunit of pertussis toxin was synthesised and used for the immunization of Balb/c mice and the subsequent production of monoclonal antibodies (MAbs) . This peptide contains a region of eight amino acids which is homologous to a region in the cholera toxin A subunit . The properties of two of the resultant MAbs are described . Both of the antibodies (CP7-3003F7, an IgG3 and CP7-3004G6X1, an IgG1) react in an ELISA with the peptide and with intact pertussis toxin, pertussis toxin A subunit and cholera toxin A subunit, but do not react significantly with pertussis toxin B subunit, intact cholera toxin, or cholera toxin B subunit . Competition ELISA assays in which the peptide, the intact toxins and the toxin subunits were compared with respect to their ability to inhibit the binding of the MAbs to peptide-coated ELISA plates demonstrated that only pertussis toxin A subunit was as active, on a molar basis, as the peptide . Western blot analyses of the holotoxins confirmed that both MAbs were reactive only with the toxin A subunits . The MAbs were unable to neutralize the activity of cholera toxin or pertussis toxin in a Chinese hamster ovary (CHO) cell assay . Both were also unable to neutralize either the ADP-ribosylation activity or the NAD-glycohydrolase activity of the pertussis toxin A subunit . The significance of these results with respect to the role of this conserved site in the activity of these two toxins is discussed. Trop Geogr Med, 1991 Jan-Apr, 43(1-2), 12 - 6 Serum ferritin and cholera . A prospective study; Alam AN et al.; An association has been shown between iron deficiency and a low gastric acidity while the latter is known to increase susceptibility to cholera . This study was undertaken to ascertain whether iron deficiency is a risk factor for contracting cholera . The subjects were 60 adult males-30 with cholera admitted to ICDDR,B and 30 controls matched for age, sex and socio-economic status from the same household or immediate neighbourhood of the index case . Fingerstick blood was taken from all subjects to estimate the haematocrit, and serum ferritin concentration by an ELISA . The mean ferritin level of the study group was 38.7 ng/100 ml, in the controls . There was a significant difference in the serum ferritin level between the groups (P less than 0.005), Wilcoxon Sign Rank test for matched pairs suggesting that cholera patients tend to have lower serum ferritin concentration . Further prospective studies are required to define the possible association between iron deficiency and cholera more accurately. J Hirnforsch, 1991, 32(2), 249 - 55 A comparative study of the transganglionic transport of cholera toxin-horseradish peroxidase (CT-HRP) and wheat germ agglutinin-horseradish peroxidase (WGA-HRP) in the trigeminal system of the guinea pig; Segade LA et al.; A comparative study of cholera toxin (CT) and wheat germ agglutinin (WGA) conjugated with horseradish peroxidase (HRP) was made on trigeminal central projections of the lower incisor gingiva afferent neurons in the guinea pig . Considerably more CT-HRP-labeled endings were observed in the trigeminal sensory nuclear complex (TSNC) and in the cervical spinal cord (C1-C8) . The substantia gelatinosa (lamina II) of both the caudal nucleus of the TSNC and C1-C2 was the only area where WGA-HRP labeled more terminals . CT-HRP-labeled fibers and endings were traced up to C7-C8, whereas with WGA-HRP were rare caudal to C5 . A comparison of the two methods currently in use, i.e . the 2-step glutaraldehyde and sodium periodate, showed that the latter yields conjugates which are more sensitive as neuroanatomical tracers. Immunology, 1991 Jan, 72(1), 89 - 93 Manipulation of intestinal immune responses against ovalbumin by cholera toxin and its B subunit in mice; Van der Heijden PJ et al.; We studied the effect of mucosal presentation of ovalbumin (OVA) conjugated to cholera toxin (CT) or cholera toxin B subunit (CTB) on the intestinal immune responses against OVA . Mice were primed intraperitoneally (i.p.) with OVA in a water-in-oil emulsion and boosted intraduodenally (i.d.) with OVA conjugated to CT or CTB in various molar ratios . Responses were evaluated by testing intestinal secretions for OVA-specific antibodies and by quantifying the OVA-specific antibody secreting cells (ASC) in the lamina propria of the small intestine . OVA-CT conjugates were tested in a molar ratio ranging from 1.8:1 to 4500:1 . OVA-CTB conjugates were tested in a molar ratio ranging from 0.25:1 to 500:1 . The optimum intestinal immune response was reached at a molar ratio of 1.8:1 for OVA-CT and 5:1 for OVA-CTB . The binding capacity of OVA-CTB, but not of OVA-CT, to GM1 ganglioside corresponded with the capacity to enhance the intestinal immune response . The effect of conjugating CTB or CT to OVA on the immune response against OVA was more striking when mice were not only boosted i.d., but also primed i.d . Both OVA-CT and OVA-CTB induced detectable immune responses, whereas free OVA did not . Therefore, the carrier effect of CT or CTB is essential to trigger a mucosal immune response against OVA when presented mucosally only . We conclude that enhancing antigen uptake greatly facilitates mucosal immune responses. Mol Cell Biol, 1991 Jan, 11(1), 102 - 7 Cholera toxin induces expression of the immediate-early response gene JE via a cyclic AMP-independent signaling pathway; Qureshi SA et al.; Cholera toxin (CT) activates expression of two immediate-early response genes (JE and TIS10) in quiescent BALB/c 3T3 cells . Increases in cyclic AMP (cAMP) levels in response to CT are likely responsible for the induction of TIS10 gene expression, since treatment with 8-Br-cAMP and increasing the intracellular levels of cAMP by treatment with forskolin induce TIS10 gene expression . In contrast, neither forskolin nor 8-Br-cAMP induces JE gene expression . 3-Isobutyl-1-methylxanthine, which stabilizes intracellular cAMP, potentiates CT-induced TIS10 gene expression but has no effect on CT-induced JE gene expression . Thus, induction of JE by CT is independent of the cAMP produced in response to CT . Induction of JE by CT does not require protein kinase C (PKC), since depleting cells of PKC activity has no effect on the induction of JE by CT . CT-induced expression of JE can be distinguished from CT-induced TIS10 gene expression by using protein kinase inhibitors and inhibitors of arachidonic acid metabolism, further suggesting distinct signaling pathways for CT-induced JE and TIS10 gene expression . Thus, induction of JE gene expression by CT results from the activation of an intracellular signaling pathway that is independent of cAMP production . This pathway is independent of PKC activity and uniquely sensitive to inhibitors of protein kinases and arachidonic acid metabolism. Cell Signal, 1991, 3(6), 599 - 606 Effect of fluoride, pertussis and cholera toxin on the release of arachidonic acid and the formation of prostaglandin E2, D2, superoxide and inositol phosphates in rat liver macrophages; Schulze-Specking A et al.; Fluoride elicited in liver macrophages a release of arachidonic acid and prostaglandins but not formation of inositol phosphates or superoxide . The effects of fluoride required extracellular calcium and were inhibited by staurosporine and by phorbol ester treatment of the cells . Furthermore, fluoride led to a translocation of protein kinase C from the cytosol to membranes . This indicates that the calcium-dependent protein kinase C is involved in the action of fluoride . Cholera toxin decreased the zymosan-induced release of arachidonic acid and prostaglandins but not of inositol phosphates or superoxide . Pertussis toxin ADP-ribosylated a 41,000 molecular weight membrane protein; enhanced specifically the zymosan-induced formation of prostaglandin(PG)E2 but did not affect the zymosan-induced release of arachidonic acid, PGD2, inositol phosphates or superoxide . These data suggest that activation of phospholipase (PL)A2, phosphoinositide (PI)-specific PLC and NADPH oxidase in liver macrophages is most probably not mediated by activation of guanine nucleotide binding (G)-proteins coupled directly to these enzymes. Tidsskr Nor Laegeforen, 1990 Dec 10, 110(30), 3854 - 9 {Cholera in Drammen 1832--the first time in Norway}; Oeding P; Cholera came to Norway for the first time in autumn 1832 . The outbreak was limited to the city of Drammen and some densely populated areas on the Drammen fjord . Mortality was low compared with later Norwegian epidemics . 80 persons died of cholera, 59 of them in Drammen . The morbidity is difficult to estimate, because cholera could not be distinguished from the frequent summer diarrhoeas . This article tries to present a picture of how the doctors interpreted the disease and what was done to prevent it from spreading . In the light of the available information the author discusses how cholera was imported to Drammen and how it spread in the city. Biochim Biophys Acta, 1990 Dec 6, 1036(3), 188 - 92 ADP-ribosylation of myelin basic protein by cholera toxin; Enomoto K et al.; Cholera toxin ADP-ribosylates four types of myelin basic proteins (MBPs) of Mr 14,000, 17,500, 19,000 and 22,000 in rat brain myelin . On an analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, MBP underwent mono- and multi-(ADP-ribosyl)ation by cholera toxin and thus modified MBP migrated on the gel as several discrete protein bands, the molecular masses of which were apparently larger by 500-2000 daltons than that of the corresponding untreated MBP . On average, 1.1 mol of ADP-ribosyl residue was incorporated into 1 mol of MBP . Four types of purified MBPs were also ADP-ribosylated by cholera toxin dependent on GTP and the protein factor for the ADP-ribosylation . The results show evidence that MBP is one of major and specific substrates of cholera toxin in brain membranes. Biochem J, 1990 Dec 1, 272(2), 327 - 31 Effects of beta-adrenergic receptor activation, cholera toxin and forskolin on human natural killer cell function; Whalen MM et al.; Membranes from highly purified natural killer (NK) cells were ADP-ribosylated by treatment with cholera toxin (CTX) . CTX resulted in a single band of specific 32P incorporation at Mr 43,600 . CTX treatment of intact NK cells caused a 9-fold increase in cyclic AMP (cAMP) concentrations . Pretreatment of NK cells with CTX diminished their ability to lyse K562 tumour cells by up to 79% . Forskolin treatment elevated NK cell cAMP levels 8-fold and decreased lysis of K562 cells by up to 45% . Adrenaline and isoprenaline (isoproterenol) both inhibited lysis of K562 cells by approx . 35% and elevated cAMP by at least 2.5-fold, and their inhibition of lysis was reversed by propranolol . These data suggest that the stimulatory guanine-nucleotide-binding protein GS coupled to beta-adrenergic receptors is involved in transducing signals which inhibit NK cell lysis of tumour cells . CTX and forskolin also diminish the ability of NK cells to bind K562 cells (binding is necessary for lysis) . This suggests that the NK-cell receptor(s) for the tumour cell may be altered as a consequence of cAMP-mediated events or by activation of GS. J Clin Invest, 1990 Dec, 86(6), 1904 - 12 Cholera toxin inhibits signal transduction by several mitogens and the in vitro growth of human small-cell lung cancer; Viallet J et al.; Cholera toxin (CT) inhibited the in vitro growth of three of four human small-cell lung carcinoma (SCLC) cell lines with a 50% inhibitory concentration of 27-242 ng/ml . Loss of surface membrane ruffling and the capacity of {Tyr4}-bombesin, vasopressin, and fetal calf serum to stimulate increases in intracellular free calcium clearly preceded effects on cellular metabolic activity and cell growth . 125I-{Tyr4}-bombesin binding was unaffected by CT treatment but {Tyr4}-bombesin stimulated phospholipase C activity was decreased in membranes from CT-treated SCLC cells . CT stimulated a rapid but transient increase in intracellular cyclic AMP ({cAMP}i) in SCLC . The effects of CT on susceptible SCLC were not reproduced by elevations of {cAMP}i induced by forskolin or cyclic AMP analogues . GM1 ganglioside, the cellular binding site for CT, was highly expressed in the CT-sensitive but not the CT-resistant SCLC cell lines . In contrast, expression of guanine nucleotide binding protein substrates for ADP-ribosylation by CT was similar . These data demonstrate the existence of a CT-sensitive growth inhibitory pathway in SCLC-bearing GM1 ganglioside . Addition of CT results in decreased responsiveness to several mitogenic stimuli . These results suggest novel therapeutic approaches to human SCLC. Vaccine, 1990 Dec, 8(6), 595 - 9 Cross-protection against influenza B type virus infection by intranasal inoculation of the HA vaccines combined with cholera toxin B subunit; Kikuta K et al.; The relationship between the antibody responses to various influenza B type virus HA vaccines and protection against live B virus infection was investigated in Balb/c mice which had been inoculated intranasally with a combination of the HA vaccines and B subunit of cholera toxin (CTB) 4 weeks previously . The inoculation of HA vaccine, prepared from B/Ibaraki/2/85 (B/Ibaraki), B/Nagasaki/1/87 (B/Nagasaki) or B/Aichi/5/88 (B/Aichi) viruses, combined with CTB induced high levels of both nasal IgA and serum HI antibodies to any of B/Ibaraki, B/Nagasaki and B/Aichi viral antigens . Simultaneous inoculation of each CTB-combined HA vaccine provided complete protection against B/Ibaraki virus infection which is demonstrated by both rapid clearance of pulmonary virus and complete survival . On the other hand, the inoculation of HA vaccine prepared from B/Yamagata/16/88 (B/Yamagata) virus together with CTB induced only a low level of nasal IgA antibodies, cross-reactive to B/Ibaraki, B/Nagasaki and B/Aichi viral antigens and protected only partially against B/Ibaraki virus challenge . The involvement of the B type virus-specific immunity in this protection was suggested by the absence of protection against B/Ibaraki virus infection in mice previously inoculated with both A/PR/8/34 (H1N1) virus HA vaccine and CTB . These results suggest that antibodies to various influenza B viruses are cross-reactive to each B type virus antigens and that cross-protection against B virus infection could be conferred depending on the degree of B type virus cross-reactive immunity including secretory IgA antibodies. Infect Immun, 1990 Dec, 58(12), 3966 - 72 Inhibition of cholera toxin binding to membrane receptors by pig gastric mucin-derived glycopeptides: differential effect depending on the ABO blood group antigenic determinants; Monferran CG et al.; The capacity of pig gastric mucin-derived glycopeptides to interfere with the binding of cholera toxin (CT) to membrane receptors was studied . Two types of glycopeptide preparations with or without human blood group A antigenic activity were assayed for comparison in a system in which the target for the toxin was rat erythrocyte ghosts . Blood group A-active glycopeptides (A+ glycopeptides) were more potent inhibitors for the toxin binding than those lacking group A activity (A- glycopeptides) . The mean values of the 50% inhibitory dose revealed that the A+ glycopeptide preparations were 6.6-fold-more potent inhibitors than the A- ones (P less than 0.001) . The inhibitory capacity of the different A+ glycopeptide preparations was not directly proportional to the group A antigenic titer . The A+ glycopeptides showed a higher capacity than the A- glycopeptides to interact with the toxin as revealed by CT-glycopeptide complex formation, which could be detected by Sephacryl S-400 chromatography . This result suggests that glycopeptide inhibition of CT binding to the erythrocyte ghosts is mediated by a competition between the GM1 receptors and the glycopeptides for the toxin . The differential effect between both types of glycoconjugates was independent of the way of measuring the amount of glycopeptides used (dry weight, carbohydrate or protein content) . The existence in the gastrointestinal tract of mucins not carrying or carrying different ABO blood group determinants, which could behave as more or less potent inhibitors of CT binding to membrane receptors, may help to explain the relationship between ABO blood groups and severity of cholera. FEBS Lett, 1990 Nov 26, 275(1-2), 143 - 5 Chloroquine inhibition of cholera toxin; Liang YF et al.; Cholera toxin (CT) stimulated adenylate cyclase and a phospholipase which elevated cellular levels of 3',5'-cyclic adenosine monophosphate (cAMP) and arachidonic acid (AA) . The AA was quickly converted to prostaglandins (PGs) via the cyclo-oxygenase pathway . Chloroquine exerted minimal inhibition of cAMP levels in CT-treated cells, although CT-induced release of {3H}AA and PGs was blocked completely when the drug was added in concentrations as low as 0.1 mM (50 micrograms/ml) . Inhibition of {3H}AA release was complete when chloroquine was added before or within 30 min after CT . The capacity of chloroquine to inhibit either phospholipase C (PLC) or phospholipase A2 (PLA2) could explain the antisecretory activity of this drug. Brain Res, 1990 Nov 26, 534(1-2), 209 - 24 Iontophoretic application of unconjugated cholera toxin B subunit (CTb) combined with immunohistochemistry of neurochemical substances: a method for transmitter identification of retrogradely labeled neurons; Luppi PH et al.; In this report, we demonstrate that cholera-toxin B subunit (CTb) is a very sensitive retrograde tracer in the central nervous system when recognized by streptavidin-peroxidase immunohistochemistry . We further show that: (1) injection of a small volume of CTb gives rise to small sharply defined injection sites limited to the cell group of interest associated with the labeling of all the known afferent projections, (2) CTb is taken up, and anterogradely as well as retrogradely transported in damaged but not intact fibers of passage, (3) CTb can be applied iontophoretically, allowing us to study the afferents to small cell groups without any evidence of tissue necrosis in the sites and therefore without artefactual labeling due to uptake by damaged fibers of passage, (4) the use of 4% paraformaldehyde fixative ideally suited for the preservation of most neural antigens, the addition of a 48 h colchicine treatment and the development of a double immunohistochemical method allow the biochemical characterization of the cell of origin of particular pathways in the CNS, (5) CTb is also anterogradely transported with an extensive filling of axons and axon terminals and thereby opens up the possibility of identifying simultaneously the afferents as well as the efferents of the group of cells studied and finally (6) the very long conservation of the preparation, the possibility of counterstaining it and of making camera lucida drawings allow easy and precise localization of the retrogradely labeled cells. J Immunol, 1990 Nov 15, 145(10), 3162 - 9 cAMP-independent effects of cholera toxin on B cell activation . I . A possible role for cell surface ganglioside GM1 in B cell activation; Francis ML et al.; 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. J Immunol, 1990 Nov 15, 145(10), 3316 - 24 Cholera toxin acts synergistically with IL-4 to promote IgG1 switch differentiation; Lycke N et al.; Previously, we reported that cholera toxin (CT) causes LPS-stimulated membrane (m)IgM+ B cells to undergo increased switch differentiation to IgG- and IgA-producing B cells . In this study we determined whether this effect is specific for one or several of the IgG subclasses and whether B cells exposed to CT respond differently to IL-4, a lymphokine with switching capabilities . In initial studies we found that in LPS-stimulated, mIgM+ B cell cultures, CT eightfold enhanced the formation of IgG1-producing B cells, whereas it only weakly enhanced, one- to twofold, the formation of IgG3-producing B cells . In addition, CT synergistically enhanced the induction of IgG1-producing B cells by IL-4, even at plateau concentrations of IL-4 . In contrast, IgM and IgG3 responses were suppressed in the CT plus IL-4-containing cultures as compared to those containing only LPS or LPS and CT . Furthermore, CT plus IL-4 had no enhancing effect on the formation of cells producing IgA; on the contrary, the presence of IL-4 led to a reversal of the stimulatory effect of CT on the IgA response . In further studies, we found that CT affected B cell differentiation at the gene level, before final gene recombination has occurred . Thus, CT together with LPS induced faint but detectable germline gamma 1 RNA transcripts not seen with cells cultured in LPS alone . However, more strikingly, CT enhanced by several-fold expression of germline gamma 1 RNA transcripts in LPS-stimulated B cell cultures containing optimal IgG1-inducing concentrations of IL-4 . In addition, despite its weakly positive effect on IgG3 production . CT inhibited expression of germline gamma 3 RNA transcripts in cultures containing LPS and caused a further decrease in such transcripts in cultures containing LPS and IL-4 . Finally, we found that CT enhanced the in vivo IgG1 but not the IgG3 or IgM anti-DNP serum antibody response of mice immunized with DNP-LPS . Taken together, these studies suggest that CT more strongly promotes B cell differentiation to IgG1 than to any other IgG subclass in LPS-stimulated cultures . CT acts alone or in synergy with IL-4, early in B cell differentiation to promote IgG1 expression in LPS-stimulated B cell cultures, probably by inducing early steps in the switch to this isotype such as the production of germline gamma 1 RNA transcripts. J Comp Neurol, 1990 Nov 8, 301(2), 262 - 75 Nuclei of origin of monoaminergic, peptidergic, and cholinergic afferents to the cat trigeminal motor nucleus: a double-labeling study with cholera-toxin as a retrograde tracer; Fort P et al.; The aim of the present study was to determine the brainstem afferents and the location of neurons giving rise to monoaminergic, cholinergic, and peptidergic inputs to the cat trigeminal motor nucleus (TMN) . This was done in colchicine treated animals by using a very sensitive double immunostaining technique with unconjugated cholera-toxin B subunit (CT) as a retrograde tracer . After CT injections in the TMN, retrogradely labeled neurons were most frequently seen bilaterally in the nuclei reticularis parvicellularis and dorsalis of the medulla oblongata, the alaminar spinal trigeminal nucleus (magnocellular division), and the adjacent pontine juxtatrigeminal region and in the ipsilateral mesencephalic trigeminal nucleus . We further observed that inputs to the TMN arise from the medial medullary reticular formation (the nuclei retricularis magnocellularis and gigantocellularis), the principal bilateral sensory trigeminal nucleus, and the dorsolateral pontine tegmentum . In addition, the present study demonstrated that the TMN received 1) serotonergic afferents, mainly from the nuclei raphe obscurus, pallidus, and dorsalis; 2) catecholaminergic afferent projections originating exclusively in the dorsolateral pontine tegmentum, including the Kolliker-Fuse, parabrachialis lateralis, and locus subcoeruleus nuclei; further, that 3) methionin-enkephalin-like inputs were located principally in the medial medullary reticular formation (nuclei reticularis magnocellularis and gigantocellularis and nucleus paragigantocellularis lateralis), in the caudal raphe nuclei (Rpa and Rob) and the dorsolateral pontine tegmentum; 4) substance P-like immunoreactive neurons projecting to the TMN were present in the caudal raphe and Edinger-Westphal nuclei; and 5) cholinergic afferents originated in the whole extent of the nuclei reticularis parvicellularis and dorsalis including an area located ventral to the nucleus of the solitary tract at the level of the obex . In the light of these anatomical data, the present report discusses the possible physiological involvement of TMN inputs in the generation of the trigeminal jaw-closer muscular atonia occurring during the periods of paradoxical sleep in the cat. Gut, 1990 Nov, 31(11), 1256 - 61 Modulation of fluid absorption and the secretory response of rat jejunum to cholera toxin by dietary fat; Sagher FA et al.; To study the effects of dietary fat on jejunal water and ion absorption and on cholera toxin-induced secretion, 3 week old Sprague Dawley rats were fed isocaloric diets . Forty per cent of the total calories were given as fat, as butter (high saturated fat), olive oil (high monounsaturated fat), or corn oil (high polyunsaturated fat), with one group on low fat (10% of calories) standard laboratory diet as controls . During in vivo jejunal perfusion studies we found that (i) a polyunsaturated fat (corn oil) supplemented diet improves jejunal absorption of water and electrolytes and these changes are independent of the observed concentrations of luminal prostaglandins; (ii) high dietary fat appreciably reduced the secretory response to cholera toxin, probably without fundamentally changing the mechanism by which cholera toxin induces secretion . We conclude that dietary fat composition altered the permeability and transport characteristics of the small intestine . This observation might have relevance to some human diarrhoeal disorders. Infect Immun, 1990 Nov, 58(11), 3711 - 6 Activation of cholera toxin-specific T cells in vitro; Elson CO et al.; Cholera toxin (CT) and its B subunit (CT-B) are potent oral immunogens in vivo, although both strongly inhibit polyclonal lymphocyte activation in vitro . In order to help understand this paradox, we have studied the activation and proliferation of CT-specific T cells in vitro, by using CT-B-primed lymph node T cells as responders, concanavalin A-stimulated peritoneal macrophages as antigen-presenting cells (APCs), and various forms of CT-B as antigen . The results indicate that in many ways CT-specific T cells respond in a manner similar to that of T cells specific for other protein antigens: the degree of proliferation was proportional to the dose of antigen and APCs in the cultures, was antigen specific, and was H-2 restricted . APCs from genetic high-responder strains to CT stimulated significantly more proliferation in F1 (high x low) responder T cells than did APCs from low responder strains . However, there was a marked difference in the activation of CT-specific T cells when different forms of CT-B were used . Native CT-B stimulated little or no T-cell proliferation, whereas denatured CT-B or CT-B blocked by its ligand, GM1 ganglioside, stimulated T cells well . Addition of native CT-B to cocultures of primed T cells, APCs, and these latter stimulatory forms of CT-B inhibited the specific proliferative response to CT-B to varying degrees, depending on the ratio of the two forms in culture . We conclude that the ability of CT-B to inhibit T cells extends even to T cells specific for CT itself . Because of these inhibitory properties, processing of CT to nonbinding molecular forms or fragments must be an important prerequisite for the immune response to CT to occur in vivo, and such processing is likely to be important in the immune response to a variety of other enterotoxins as well. Microb Pathog, 1990 Nov, 9(5), 345 - 53 Synthesis of prostaglandins in cholera toxin-treated Chinese hamster ovary cells; Peterson JW et al.; The prostaglandin (PG) and adenosine 3',5'-cyclic monophosphate (cAMP) responses of Chinese hamster ovary (CHO) cells were measured after cholera toxin (CT) exposure to evaluate dose and kinetic relationships . Release of prostaglandin E2 (PGE2) and the accumulation of cAMP were dependent on the dose of CT, with an effective dose of approximately 10-100 ng/ml within 4 h; the PGE2 response was about four- to six-fold more than that of PGE1 . CHO cells exposed to CT also released increased amounts of thromboxane B2 (TxB2), PGF2 gamma, and 6-keto PGF1 gamma (a non-enzymatic degradation product of prostacyclin) . Kinetic analysis of CT-treated cells revealed that small peaks of cAMP accumulation and of PGE1 and PGE2 release were detected at approximately 30 min, but larger, progressive PG and cAMP responses were measured 2-4 h later . Exposure of the cells to relatively high doses of membrane-permeable derivatives of cAMP (1 mM) and forskolin (10 microM) caused PGE2 release . Concomitantly, exogenous PGE2 (100 microM) increased intracellular levels of cAMP . We have considered the interrelationship of the cyclo-oxygenase and the cyclic nucleotide pathways relative to the molecular mechanism of CT. Brain Res, 1990 Oct 29, 531(1-2), 1 - 7 Cholera toxin-B subunit blocks excitatory effects of opioids on sensory neuron action potentials indicating that GM1 ganglioside may regulate Gs-linked opioid receptor functions; Shen KF et al.; In a previous study, we demonstrated that cholera toxin-A subunit, as well as the whole toxin, selectively blocks opioid-induced prolongation of the Ca2+ component of the action potential duration (APD) in dorsal root ganglion (DRG) neurons, indicating mediation of this excitatory effect by Gs-linked opioid receptors . The present study shows that pretreatment of DRG neurons with the B subunit of cholera toxin (1-10 ng/ml; greater than 15 min) can also block mu/delta and kappa opioid-induced APD prolongation, but not shortening . Since the B subunit binds selectively to GM1 ganglioside located on the cell surface, these results suggest that this ganglioside may regulate Gs-linked excitatory opioid receptor functions in DRG neurons . Possible contamination of purified B subunit preparations of cholera toxin with traces of the more potent A subunit was eliminated by heating the stock solution to 56 degrees C for 20 min . Exposure of DRG neurons to an affinity-purified anti-GM1 antiserum also blocked opioid-induced APD prolongation, providing further evidence that GM1 ganglioside may play an essential role in excitatory opioid modulation of the action potential of these cells . The blockade by cholera toxin-B subunit and anti-GM1 antibodies of opioid-induced APD prolongation is best accounted for by the following hypothesis: CTX-B interferes with an endogenous GM1 ganglioside component of the excitatory, but not inhibitory, opioid receptor complex on DRG neurons that may allosterically regulate coupling of the receptors via Gs to adenylate cyclase/cyclic adenosine monophosphate-dependent ionic conductances. J Immunol, 1990 Oct 15, 145(8), 2375 - 80 Effects of cholera toxin on human B cells . Cholera toxin induces B cell surface DR expression while it inhibits anti-mu antibody-induced cell proliferation; Anastassiou ED et al.; Experiments were performed to investigate the effect of cholera toxin (CT) on human B cell function . Highly purified (greater than 98% CD20+) human peripheral blood B cells were exposed to CT in the presence or absence of anti-mu antibody . Treatment of highly purified B cells with CT stimulated enhanced expression of surface DR molecules, whereas it did not enhance expression of other B cell surface activation markers including transferrin or IL-2R . Neither the A nor the B subunits of CT by themselves enhanced the expression of surface DR Ag . In addition, 8-bromo-cAMP alone or in combination with the B subunit did not increase the expression of human B cell surface DR Ag . These findings suggest that neither elevation of cAMP nor binding to GM1 ganglioside are sufficient to stimulate this activation parameter in B cells . Associated with CT-mediated enhanced expr