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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 expression of MHC class II molecules we found that CT-treated B cells also served as stronger stimulators, compared with control cells, of both autologous and allogeneic MLR responses in peripheral blood T cells . Although CT stimulated early events in B cell activation, it inhibited anti-mu antibody-induced B cell thymidine incorporation by 55 to 75% . Inhibitory effects of CT were observed even when CT was added to cultures as late as 36 h after the addition of the anti-mu antibody . These results suggest that CT has both a stimulatory and inhibitory effect on human B cells and that the stimulatory effect may be mediated via a cAMP-independent mechanism.

FEBS Lett, 1990 Oct 15, 272(1-2), 41 - 4
Effect of antipsychotic drugs on the molecular action of cholera toxin in rabbit intestinal epithelial cells; Longbottom D et al.; Antipsychotic drugs of known antidiarrhoeal and anticalmodulin activity inhibited the cholera-toxin-catalysed ADP-ribosylation of proteins of Mr 37,000, 40,000 and 45,000 (thought to be regulatory components of the adenylate cyclase complex) that was previously shown to occur in plasma membranes from rabbit intestinal epithelial cells {(1989) Biochim . Biophys . Acta 1014, 289-297} . There was no obvious correlation between the different activities of the drugs . The drugs also inhibited adenylate cyclase activity, but in this case the inhibition correlated well with the known IC50 values of the drugs for anticalmodulin activity and with their antidiarrhoeal activities.

Brain Res, 1990 Oct 8, 529(1-2), 324 - 8
Blockade of morphine analgesia by both pertussis and cholera toxins in the periaqueductal gray and locus coeruleus; Bodnar RJ et al.; Rats demonstrating analgesia following microinjection of morphine into the periaqueductal gray (PAG) or locus coeruleus (LC) were injected with either pertussis toxin, cholera toxin or saline into the same brain region . Both pertussis and cholera toxin blocked the analgesic effect of morphine at both injection sites for up to 7 days after toxin treatment . These results indicate that morphine analgesia is a complex response involving systems dependent upon Gs as well as Gi or Go proteins.

Nippon Juigaku Zasshi, 1990 Oct, 52(5), 1037 - 42
Studies on the binding and hemagglutinating properties of cholera toxin to human erythrocytes; Sugii S; The binding and hemagglutinating properties of cholera toxin (CT) were studied by competitive binding assays and hemagglutination inhibition . The binding of 125I-labeled CT to neuraminidase-treated human type B erythrocytes was most effectively inhibited by ganglioside GM1 among different inhibitors used . Other mono-, di-, and polysaccharides and glycoproteins were at least 10(5) times less potent inhibitors . On the other hand, hemagglutination of neuraminidase-treated human type B erythrocytes by CT was inhibited by lactose, galactose, hog A + H, bovine salivary mucin, porcine thyroglobulin, and fetuin, whereas that was not effectively inhibited by ganglioside GM1 at the highest concentration . These findings suggest that the predominant binding substance for CT on human type B erythrocytes is ganglioside GM1 and that hemagglutination requires some additional process since the interaction of CT with ganglioside GM1 is somehow different in hemagglutination.

Biochem J, 1990 Oct 1, 271(1), 107 - 11
Cyclic AMP accumulation in HeLa cells induced by cholera toxin . Involvement of the ceramide moiety of GM1 ganglioside; Masserini M et al.; The influence of ceramide composition on the rate of GM1 association to HeLa cells has been investigated by incubating the cells in the presence of either native ganglioside or molecular species carrying highly homogeneous long chain base moieties, fractionated from native GM1 . The GM1 ganglioside species carrying the unsaturated C18 long chain base moiety proved to have the fastest rate of association, whereas the saturated species carrying 20 carbon atoms had the slowest rate . After having increased the GM1 cell content (65-fold) by incubation with the various ganglioside species, the cells were incubated with cholera toxin and the time course of cyclic AMP accumulation was monitored . Remarkable differences among cells enriched with the various molecular species were found in the duration of the lag time preceding the accumulation of cyclic AMP, the shortest being displayed by the unsaturated C18 species . Moreover, the amount of cyclic AMP accumulated after a given time of incubation with cholera toxin was significantly higher when the C18:1-GM1 species was present than with native GM1 . Fluorescence anisotropy experiments, carried out using the probe 1,3-diphenylhexatriene, show that the GM1 ganglioside ceramide moiety was also modifying the cell membrane fluidity of the host.

FEMS Microbiol Lett, 1990 Oct, 60(1-2), 137 - 41
Inhibitory effect of aspirin on cholera toxin-induced phospholipase and cyclo-oxygenase activity; Liang YF et al.; Cholera toxin (CT) stimulated phospholipase activity and caused {3H}arachidonic acid (3H-AA) release in a murine macrophage/monocyte cell line . Pretreatment of cells with dexamethasone, a phospholipase A2 (PLA2) inhibitor, did not affect CT-induced 3H-AA release . In contrast, aspirin, which is an inhibitor of phospholipase C (PLC), blocked CT-induced 3H-AA release and subsequent prostaglandin (PC) synthesis . The inhibitory effect of aspirin was dose dependent, with 4 mM reducing the CT response by approximately 50% . Similarly, inhibition was time dependent, occurring when the drug was added to the culture medium as late as 30 min after CT . Brief exposure (30 min) of the cells to aspirin did not alter their subsequent response to CT, but 3H-AA release from cells exposed to aspirin for 2.5 h was irreversibly inhibited . The data suggested that CT stimulation of AA metabolism may involve increased PLC activity.

Immunol Invest, 1990 Oct-Dec, 19(5-6), 435 - 51
Anti-cholera toxin IgA-, IgG- and IgM-secreting cells in various rat lymphoid tissues after repeated intestinal or parenteral immunizations; Solbreux PM et al.; Single antibody-secreting spot-forming cells (SFC) of the 3 main isotypes were counted in lymphoid cells from the gut lamina propria (LP), Peyer's patches (PP), mesenteric nodes (MN) and spleen (SP) of rats immunized 2-6 times intraduodenally (ID) or intraperitoneally (IP) with cholera toxin (CT) . Responses for all isotypes peaked in all tissues after 4 ID- or IP-immunizations at much larger values than previously reported, and significantly decreased thereafter, except in LP and PP after IP-injections, where IgA- or IgG-SFC, but not IgM-SFC, only appeared or increased after 6 IP-doses . The highest IgA-SFC numbers (17% of tested cells) were in LP after 4 ID-doses . The isotype ratio was IgA greater than IgG greater than IgM in LP and PP after ID-injections, but IgG greater than IgA greater than IgM in MN and SP after both ID- and IP-routes . The isotype dispersion was much larger in LP and PP than in MN and SP . Our data show that at least 4 IP CT-doses were required to only elicit a few IgG- and almost no IgA-SFC in LP and PP, outlining the need for intestinal CT-immunizations to induce strong mucosal IgA-SFC responses . We also show good systemic responses elicited both by enteral and parenteral routes, and the small contribution of PP in total SFC, particularly after parenteral immunizations.

Biochem Biophys Res Commun, 1990 Sep 28, 171(3), 949 - 54
Endothelin/sarafotoxin receptor induced phosphoinositide turnover: effects of pertussis and cholera toxins and of phorbol ester; Galron R et al.; The induction of phosphoinositide hydrolysis (PI) by endothelin/sarafotoxin (ET/SRTX) receptors in rat heart myocytes was investigated by the use of bacterial toxins as well as a phorbol ester . Both pertussis- and choleratoxin enhanced the stimulation of PI hydrolysis . Phorbol ester treatment of the myocytes for short periods distinguished between two types of PI-hydrolysis, the one induced by endothelins and the other by sarafotoxins . The possible mediation of G-protein (s) in the induction by ET/SRTX receptors of PI-hydrolysis is discussed.

Biochem Biophys Res Commun, 1990 Sep 14, 171(2), 770 - 6
Coupling of adenosine A1 receptors to a G-protein in coated vesicles isolated from bovine brain: presence of pertussis and cholera toxin substrates; Ros M et al.; Adenosine A1 receptors have been described in coated vesicles isolated from bovine brain (Gonzalez-Calero et al., J . Neurochem . 1990, 55, 106-113) . Addition of non hydrolyzable GTP analogue (guanyl-5-yl-imidodiphosphate) caused a transition of the receptor from the high- to the low-affinity state, without any significant change in the total binding sites . The presence of G-proteins has been investigated by pertussis and cholera toxins catalyzed ADP-ribosylation . A band of Mr = 41,000 D, similar to the alpha Gi subunit, was specifically labeled in the presence of preactivated pertussis toxin . Bands of Mr = 42,000 D and Mr = 47,000 D were specifically labeled in the presence of preactivated cholera toxin . These results confirm the presence of GTP binding proteins (alpha Gi and alpha Gs) in coated vesicles isolated from bovine brain.

Biochemistry, 1990 Sep 4, 29(35), 8106 - 11
Structure, stability, and receptor interaction of cholera toxin as studied by Fourier-transform infrared spectroscopy; Surewicz WK et al.; The structure and thermal stability of isolated B and A subunits of cholera toxin, as well as the interaction of the B subunit with a ganglioside GM1 receptor, were studied by Fourier-transform infrared spectroscopy . The B subunit of the toxin is highly folded; its secondary structure consists predominantly of beta-sheets . The temperature dependence of the infrared spectrum indicates that the B subunit undergoes thermal unfolding in the temperature range between approximately 66 and 78 degrees C . Binding to the ganglioside GM1 receptor or to its oligosaccharide moiety results in only marginal, if any, change in the secondary structure of the B subunit; however, the receptor-associated subunit does show a markedly increased thermal stability . The secondary structure of the enzymatically active A subunit is less ordered and much less stable than that of the B subunit . The relatively loose folding of the A subunit is likely to be of importance for the effective membrane translocation of this subunit.

Biochem J, 1990 Sep 1, 270(2), 391 - 5
Treatment of cardiac myocytes with 8-(4-chlorophenylthio)-adenosine 3',5'-cyclic monophosphate, forskolin or cholera toxin does not stimulate cellular or heparin-releasable lipoprotein lipase activities; Carroll R et al.; Incubation of isolated cardiac myocytes with 500 microM-8-(4-chlorophenylthio)adenosine 3',5'-cyclic monophosphate (CPT-cAMP) or 100 microM-forskolin for 2 1/2 h did not increase the heparin-induced release of lipoprotein lipase (LPL) into the medium . When LPL activity in cardiac myocytes was depleted by treatment of rats with cycloheximide (2 mg/kg; 2.5 h) and inclusion of the protein-synthesis inhibitor in the isolation solutions, incubation with CPT-cAMP or forskolin did not influence the rate of repletion of LPL activity in cells or the recovery of heparin-releasable LPL activity . Although the administration of cholera toxin (0.5 mg/kg; 16-17 h) to rats increased LPL activity in a low-speed supernatant fraction from heparin-perfused hearts, LPL activity was not increased in cardiac myocytes from cholera-toxin-treated rat hearts, and the heparin-induced release of LPL was unchanged . Incubation of cultured ventricular myocytes with 1 microgram of cholera toxin/ml or 500 microM-CPT-cAMP for 24 h did not increase cellular LPL activity or LPL released into the culture medium after a 40 min incubation with heparin . Therefore interventions that stimulate adenylate cyclase activity (forskolin, cholera toxin) or incubation with CPT-cAMP do not increase cellular LPL activity or promote the translocation of LPL to a heparin-releasable fraction in cardiac myocytes.

Med J Malaysia, 1990 Sep, 45(3), 194 - 201
Cholera in Sarawak: a historical perspective (1873-1989); Yadav H et al.; Cholera has been in existence in Sarawak for many years and since 1873 many major epidemics have occurred . The epidemics usually occur during the dry months of May, June and July and the population affected are those in coastal areas . As in other outbreaks the areas affected were those which had poor environmental sanitation, poor water supply, poor refuse disposal and indiscriminate disposal of faeces . Malays are more affected as in Peninsular Malaysia outbreaks . The classical biotype was common prior to 1961 . In later years the El Tor (biotype) has been responsible for most outbreaks.

Med J Malaysia, 1990 Sep, 45(3), 187 - 93
Cholera outbreak in Tumpat, Kelantan-1990; Isa AR et al.; Two episodes of El Tor cholera outbreak occurred in Tumpat, Kelantan between the 13th of January and the 16th of May 1990 . Every case and carrier reported were investigated to determine the source and mode of transmission and to identify specific preventive measures to break the chain of transmission . There were 109 cases and 85 carriers involved in this study . The first episode of one case only was of Inaba serotype while the second episode was caused by the imported Ogawa serotype . Two foci of spread were identified from cluster occurrence but the majority of infection had no discernible link between them . The outbreak became both explosive and protracted indicating poor basic sanitation and personal hygiene . Person-to-person transmission via food and water was the main mode of spread . The Kelantan river water and river clams were confirmed sources of reservoir during the outbreak . Recommendations for prevention are intensified surveillance throughout the year,urgent upgrading of potable water supply and concerted effort in public health education especially against the use of river water and the consumption of raw food.

Eur J Immunol, 1990 Sep, 20(9), 1881 - 6
The G protein coupling T cell antigen receptor/CD3-complex and phospholipase C in the human T cell lymphoma Jurkat is not a target for cholera toxin; Sommermeyer H et al.; Intact Jurkat cells could be stimulated by monoclonal antibodies against the Tcell antigen receptor complex (OKT3 directed against the CD3 complex, BMA031 directed against constant framework epitopes in the alpha/beta heterodimer) . The accumulation of inositol phosphates was inhibited by prior incubation of the cells with cholera holotoxin . The inhibitory effect of cholera toxin (CT) was not cAMP mediated because forskolin (a direct activator of adenylate cyclase) did not mimic the inhibitory effect . When measuring phospholipase C (PLC) in a cell-free assay system by using {3H}inositol-labeled membranes, the enzyme could be stimulated by the poorly hydrolyzable GTP analogue guanosine 5'-O-(thiotriphosphate (GTP gamma S) . Both anti-receptor antibodies augmented the GTP gamma S stimulatory effect, while the antibodies alone had no stimulatory capacity . In membranes from CT-pretreated cells, whereas the antibodies lost their stimulatory effect on PLC as in untreated cells, whereas the antibodies lost their stimulatory capacity in the presence of GTP gamma S . These data imply that CT exerts its inhibitory effect on signaling by acting at the receptor level while the PLC regulating G protein is not a target for CT-mediated alterations . This assumption is supported by the finding that in intact Jurkat cells CT, which ADP ribosylated only the alpha-subunit of the stimulatory G protein of the adenylate cyclase, led to a loss of the T cell antigen receptor complex from the cell surface as demonstrated by a decrease of receptor density using flow cytometry analysis . Receptor loss could not be achieved by forskolin treatment or incubation of the cells with the binding subunit of the toxin alone.

Res Microbiol, 1990 Sep-Oct, 141(7-8), 971 - 9
Recombinant cholera toxin B subunit and gene fusion proteins for oral vaccination; Sanchez J et al.; The B subunit portion of cholera toxin (CTB) is a safe and effective oral immunizing agent in humans, affording protection against both cholera and diarrhoea caused by enterotoxigenic Escherichia coli producing heat-labile toxin (LT) (Clemens et al., 1986; 1988) . CTB may also be used as a carrier of various "foreign" antigens suitable for oral administration . To facilitate large-scale production of CTB for vaccine development purposes, we have constructed recombinant overexpression systems for CTB proteins in which the CTB gene is under the control of strong foreign (non-cholera) promoters and in which it is also possible to fuse oligonucleotides to the CTB gene and thereby achieve overexpression of hybrid proteins (Sanchez and Holmgren, 1989; Sanchez et al., 1988) . We here expand these findings by describing overexpression of CTB by a constitutive tacP promoter as well as by the T7 RNA-polymerase promoter, and also by describing gene fusions leading to overexpression of several hybrid proteins between heat-stable E . coli enterotoxin (STa)-related peptides to either the amino or carboxy ends of CTB . Each of the hybrid proteins, when tested as immunogens in rabbits, stimulated significant anti-STa as well as anti-CTB antibody formation, although the anti-STa antibody levels attained (c.a . 1-15 micrograms/ml specific anti-STa immunoglobulin) were too low to give more than partial neutralization of STa intestinal challenge in baby mice . The hybrid proteins also had a near-native conformation, as apparent from their oligomeric nature and their strong reactivity with both a neutralizing antibody against the B subunit and a neutralizing monoclonal antibody (mAb) against STa.(ABSTRACT TRUNCATED AT 250 WORDS)

Brain Res, 1990 Aug 20, 525(2), 225 - 31
Cholera toxin-A subunit blocks opioid excitatory effects on sensory neuron action potentials indicating mediation by Gs-linked opioid receptors; Shen KF et al.; Our previous studies indicated that opioid-induced prolongation of the Ca2+ component of the action potential duration (APD) in dorsal root ganglion (DRG) neurons is mediated by excitatory opioid receptors that are coupled to cyclic AMP-dependent voltage-sensitive ionic conductances . In the present study, DRG neurons were treated with cholera toxin (CTX), or with the A subunit of CTX, in order to determine if these excitatory opioid receptors are positively coupled via the GTP-binding protein Gs to the adenylate cyclase/cyclic AMP system . In contrast, inhibitory opioid receptors have been shown to be linked to pertussis toxin-sensitive Gi/Go regulatory proteins that mediate APD shortening responses . After pretreatment of DRG-spinal cord explants with remarkably low concentrations of CTX-A (1 pg/ml-1 ng/ml; greater than 15 min) or whole toxin (1 pg/ml-1 microgram/ml) the APD prolongation elicited in DRG neurons by 1-10 nM delta/mu (DADLE) or kappa (U-50,488H) opioids was blocked (29 out of 30 cells), whereas APD shortening by microM opioid concentrations was unaffected . Opioid-induced APD prolongation was blocked even when the initial treatment with CTX or CTX-A alone did not prolong the APD . The blocking effects of CTX and CTX-A were reversed in tests made 2 h after return to control medium . The mechanisms underlying the unusually potent blocking effects of CTX and CTX-A on opioid excitatory modulation of the APD of DRG neurons require correlative biochemical analyses.(ABSTRACT TRUNCATED AT 250 WORDS)

Endocrinology, 1990 Aug, 127(2), 949 - 56
Follicle-stimulating hormone receptor-mediated uptake of 45Ca2+ by cultured rat Sertoli cells does not require activation of cholera toxin- or pertussis toxin-sensitive guanine nucleotide binding proteins or adenylate cyclase; Grasso P et al.; We have previously reported that FSH stimulates flux of 45Ca2+ into cultured Sertoli cells from immature rats via voltage-sensitive and voltage-independent calcium channels . In the present study, we show that this effect of FSH does not require cholera toxin (CT)- or pertussis toxin (PT)-sensitive guanine nucleotide binding (G) protein or activation of adenylate cyclase (AC) . Significant stimulation of 45Ca2+ influx was observed within 1 min, and maximal response (3.2-fold over basal levels) was achieved within 2 min after exposure to FSH . FSH-stimulated elevations in cellular cAMP paralleled increases in 45Ca2+ uptake, suggesting a possible coupling of AC activation to 45Ca2+ influx . (Bu)2cAMP, however, was not able to enhance 45Ca2+ uptake over basal levels at a final concentration of 1000 microM, although a concentration-related increase in androstenedione conversion to estradiol was evident . Exposure of Sertoli cells to CT (10 ng/ml) consistently stimulated basal levels of androstenedione conversion to estradiol but had no effect on basal levels of 45Ca2+ uptake . Similarly, CT had no effect on FSH-induced 45Ca2+ uptake, but potentiated FSH-stimulated estradiol synthesis . PT (10 ng/ml) augmented basal and FSH-stimulated estradiol secretion without affecting 45Ca2+ influx . The adenosine analog N6-phenylisopropyladenosine, which binds to Gi-coupled adenosine receptors on Sertoli cells, inhibited FSH-stimulated androgen conversion to estradiol in a dose-related (1-1000 nM) manner, but FSH-stimulated 45Ca2+ influx remained unchanged . Our results show that in contrast to FSH-stimulated estradiol synthesis, the flux of 45Ca2+ into Sertoli cells in response to FSH is not mediated either directly or indirectly by CT- or PT-sensitive G protein, nor does it require activation of AC . Our data further suggest that the FSH receptor itself may function as a calcium channel.

J Biol Chem, 1990 Jul 25, 265(21), 12434 - 43
Hexose transport stimulation and membrane redistribution of glucose transporter isoforms in response to cholera toxin, dibutyryl cyclic AMP, and insulin in 3T3-L1 adipocytes; Clancy BM et al.; Exposure of 3T3-L1 adipocytes to 100 ng/ml of cholera toxin or 1 mM dibutyryl cyclic AMP caused a marked stimulation of deoxyglucose transport . A maximal increase of 10- to 15-fold was observed after 12-24 h of exposure, while 100 nM insulin elicited an increase of similar magnitude within 30 min . A short term exposure (4 h) of cells to cholera toxin or dibutyryl cyclic AMP resulted in a 3- to 4-fold increase in deoxyglucose transport which was associated with significant redistribution of both the HepG2/erythrocyte (GLUT1) and muscle/adipocyte (GLUT4) glucose transporters from low density microsomes to the plasma membrane fraction . Total cellular amounts of both transporter proteins remained constant . In contrast, cells exposed to cholera toxin or dibutyryl cyclic AMP for 12 h exhibited elevations in total cellular contents of GLUT1 (but not GLUT4) protein to about 1.5- and 2.5-fold above controls, respectively . Although such treatments of cells with cholera toxin (12 h) versus insulin (30 min) caused similar 10-fold enhancements of deoxyglucose transport, a striking discrepancy was observed with respect to the content of glucose transporter proteins in the plasma membrane fraction . While insulin elicited a 2.6-fold increase in the levels of GLUT4 protein in the plasma membrane fraction, cholera toxin increased the amount of this transporter by only 30% . Insulin or cholera toxin increased the levels of GLUT1 protein in the plasma membrane fraction equally (1.6-fold) . Thus, a greater number of glucose transporters in the plasma membrane fraction is associated with transport stimulation by insulin compared to cholera toxin . We conclude that: 1) at early times (4 h) after the addition of cholera toxin or dibutyryl cyclic AMP to 3T3-L1 adipocytes, redistribution of glucose transporters to the plasma membrane appears to contribute to elevated deoxyglucose uptake rates, and 2) the stimulation of hexose uptake after prolonged treatment (12-18 h) of cells with cholera toxin may involve an additional increase in the intrinsic activity of one or both glucose transporter isoforms.

FEBS Lett, 1990 Jul 16, 267(2), 221 - 5
Reduction of adenylyl cyclase activity by cholera toxin in myeloid cells . Long-term down-regulation of Gs alpha subunits by cholera toxin treatment; Hermouet S et al.; In IPC-81 cells, the adenylyl-cyclase activation by cholera toxin produces an elevation of cAMP that causes a rapid cytolysis . A resistant clone with deficient cholera toxin-induced cyclase activity (yet sensitive to cAMP) showed a rapid decrease in the amount of membrane-bound Gs alpha (42-47 kDa) detectable soon after ADP-ribosylation of these proteins; pertussis toxin-sensitive G proteins (41 kDa) were not affected . Resistant cells showed a rapid decrease of Gs alpha that is consistent with the finding that cAMP did not accumulate in these cells . Cholera toxin treatment of resistant cells had long-lasting effects (several weeks) on the level of Gs alpha in the cell membrane . The duration of Gs alpha decrease does not correspond to the probable life of catalytically active cholera toxin in the cells, and suggests a regulated process more complex than a proteolytic degradation targeted on ADP-ribosylated molecules.

Exp Cell Res, 1990 Jul, 189(1), 13 - 21
The bimodal growth response of Swiss 3T3 cells to the B subunit of cholera toxin is independent of the density of its receptor, ganglioside GM1; Buckley NE et al.; The B subunit of cholera toxin, a protein which binds specifically to cell surface ganglioside GM1, has been shown to have a bimodal effect on DNA synthesis in Swiss 3T3 fibroblasts . The B subunit induced cellular proliferation of confluent and quiescent cells while it inhibited the growth of the same cells when they were sparse and rapidly dividing . The amount of cell surface GM1 increased when the cells reached confluency . To examine the hypothesis that the variation in levels of GM1 was responsible for the bimodal effect, we increased GM1 levels in rapidly dividing cells by insertion of exogenous GM1 or by treatment of the cells with neuraminidase to convert polysialogangliosides to GM1 . Even after the level of GM1 was increased to levels similar to those found in confluent cells, the B subunit still inhibited, rather than stimulated, their growth . Therefore, this result indicates that the bimodal response to the B subunit is not solely a function of the concentration of cell surface GM1; rather it is the growth stage that determines the fate of the signal transduced by the interaction of the B subunit and ganglioside GM1.

Gastroenterology, 1990 Jul, 99(1), 83 - 9
5-HT2 and 5-HT3 receptor subtypes mediate cholera toxin-induced intestinal fluid secretion in the rat; Beubler E et al.; The mechanisms of diarrhea in Asiatic cholera have been studied extensively . Cyclic adenosine monophosphate, 5-hydroxytryptamine (5-HT), prostaglandins, and the function of neuronal structures have been implicated in the pathogenesis of cholera . To elucidate the action of 5-HT in mediating cholera secretion, in vivo experiments were performed in the rat jejunum . The inhibitory effects of the 5-HT2 receptor antagonist ketanserin and the 5-HT3 receptor antagonist ICS 205-930 were studied in cholera toxin- and 5-HT-induced fluid secretion . Both ketanserin and ICS 205-930 dose-dependently but only partially reduced the secretory effect of cholera toxin . The combination of the two blockers totally abolished cholera toxin-induced secretion without any influence on cholera toxin-induced increase in cyclic adenosine monophosphate . Prostaglandin E2- and bisacodyl-induced secretion was not affected by the combined administration of 5-HT2 and 5-HT3 antagonists . The present results provide evidence for an important role of 5-HT in cholera toxin-induced secretion . The data suggest a model in which cholera toxin may initiate the release of 5-HT from enterochromaffin cells . 5-Hydroxytryptamine may then cause prostaglandin E2 formation via 5-HT2 receptors and activation of neuronal structures via 5-HT3 receptors . These two effects may finally lead to the profuse fluid secretion which can be totally blocked by the combination of a 5-HT2 blocker and a 5-HT3 blocker.

Microbiologica, 1990 Jul, 13(3), 185 - 9
The lapinized Chinese strain of hog cholera virus (HCV) previously adapted in minipig kidney (MPK) cell line cultures, would also protect pigs to experimental infection with virulent HCV {corrected}; Rivero VB et al.; Two-month old piglets previously inoculated with different dilutions of the Lapinized Chinese (LC) strain of Hog Cholera Virus (HCV), adapted in a minipig kidney (MPK) cell line, resisted challenge infection with virulent HCV . All the animals remained healthy and the challenge virus was never recovered from any of them . In contrast, the pigs which served as controls for the challenging virus underwent the clinically lethal form of the disease and HCV was consistently recovered from their tissues.

J Reprod Fertil, 1990 Jul, 89(2), 553 - 63
Effects of follicle stimulating hormone, cholera toxin, pertussis toxin and forskolin on adenosine cyclic 3',5'-monophosphate output by granulosa cells from Booroola ewes with or without the F gene; McNatty KP et al.; The cAMP outputs by granulosa cells from 3-4.5 mm diameter (medium) follicles of Booroola FF ewes were similar to those by cells from greater than or equal to 5 mm diameter (large) follicles of ++ ewes with respect to time or dose of FSH, cholera toxin or forskolin . Likewise, the cAMP outputs by cells from 1-2.5 mm diameter (small) FF follicles were similar to those by cells from small and medium ++ follicles with respect to time or dose of FSH, cholera toxin or forskolin . At FSH, cholera toxin or forskolin doses of 1 microgram/ml, 0.5 microgram/ml and 10(-4) M respectively, the granulosa cell cAMP outputs of medium FF or large ++ follicles were approximately 2-fold (P less than 0.05) higher than in the respective small FF and medium ++ follicles . The effects of cholera toxin plus forskolin or FSH plus forskolin were additive irrespective of genotype or follicle size, with significant differences (P less than 0.05) observed between follicle sizes but not genotype . No differences were noted between cholera toxin plus forskolin or FSH plus forskolin on granulosa cell cAMP output . For the FSH and forskolin treatments, increased mean cAMP outputs were evident after 10 min, whereas after cholera toxin treatment they were not evident until after 20 min incubation . For all treatments the rate of cAMP production tended to slow down after 40-60 min . Pre-incubation of granulosa cells with pertussis toxin subsequently resulted in a significantly greater (P less than 0.05) FSH-induced output of cAMP relative to the untreated controls irrespective of follicle size . However, no gene-specific differences were noted when the cAMP outputs of cells from medium or small FF follicles were compared with cells from large or small-medium ++ follicles respectively . These results indicate that the activity (or composition) of the regulatory and catalytic components of adenylate cyclase in the FF granulosa cells change in a manner similar to those observed in ++ cells with the only difference being that the increases in cyclase in FF ewes occurs as follicles enlarge from 1-2.5 to 3-4.5 mm in diameter, whereas in ++ ewes they occur as follicles enlarge from 3-4.5 to greater than or equal to 5 mm in diameter . No evidence was found to link the F gene to the granulosa cell cAMP response independently of follicle size . It is suggested that the association between the F gene and the size-specific difference in follicle maturation may be unrelated to the FSH receptor/cAMP generating system.

J Exp Med, 1990 Jul 1, 172(1), 95 - 103
Cholera toxin discriminates between T helper 1 and 2 cells in T cell receptor-mediated activation: role of cAMP in T cell proliferation; Munoz E et al.; CD4+ T helper (Th) clones can be divided into interleukin 2 (IL-2)-secreting Th1 and IL-4-secreting Th2 cells . We show in the present report that these two Th subsets have different activation requirements for lymphokine production and proliferation: namely, cholera toxin (CT) as well as forskolin inhibit T cell receptor (TCR)-mediated IL-2 production and proliferation in Th1 cells, while the same reagents fail to block IL-4 production and proliferation in Th2 cells . In addition, CT and forskolin differentially influence the proto-oncogene mRNA expression in Th1 vs . Th2 cells after stimulation with Con A . Since both reagents lead to elevated levels of intracellular cAMP, it is likely that Th1 and Th2 cells differ in their sensitivity to an increase in cAMP . Our results indicate that the two Th subsets use different transmission signal pathways upon TCR-mediated activation.

Virology, 1990 Jul, 177(1), 184 - 98
Molecular cloning and nucleotide sequence of hog cholera virus strain Brescia and mapping of the genomic region encoding envelope protein E1; Moormann RJ et al.; Genomic RNA of hog cholera virus (HCV) strain Brescia was cloned and sequenced . The nucleotide sequence was deduced from overlapping cDNA clones and comprises 12,283 nucleotides . We cloned the complete 3' end of the HCV genome, but could not unequivocally prove that the cDNA sequence also completely covers HCV RNA at the 5' end . The HCV genome contained one large open reading frame, which spans the viral plus strand RNA and encodes an amino acid sequence of 3898 residues with a calculated molecular weight of 438,300 . To identify structural HCV glycoproteins, we prepared rabbit antisera against three synthetic peptides deduced from the sequence . Because one of these antisera reacted with a 51- to 54-kDa glycoprotein (envelope protein E1 of HCV) on Western blot, the genomic position of the sequence encoding gp51-54 could be clearly established . The amino acid sequence of Brescia was compared with that of HCV strain Alfort and that of BVDV strains NADL and Osloss . The degree of homology between the two HCV strains was 93%, and between Brescia and the BVDV strains about 70% . NADL contained an inserted sequence of 90 amino acids that was absent from the sequences of Brescia, Alfort, and Osloss, whereas Osloss contained an inserted sequence of 76 amino acids that was absent from the sequences of Brescia, Alfort, and NADL . Sequences in p80, the most homologous protein among pestiviruses, showed similarity to six sequence motifs found conserved in helicase-like proteins represented by eIF-4A . Furthermore, a trypsin-like serine protease domain detected in p80 of BVDV was also found conserved in HCV, suggesting that pestivirus p80 may be bifunctional.

Biochem Biophys Res Commun, 1990 Jun 29, 169(3), 1099 - 104
Interleukin (IL)-8-induced in vitro human lymphocyte migration is inhibited by cholera and pertussis toxins and inhibitors of protein kinase C; Bacon KB et al.; To further investigate the intracellular mechanisms involved in IL-8-induced human mixed peripheral blood lymphocyte (PBL) migration, the effects of pertussis toxin (PTX), cholera toxin (CTX), and protein kinase C (pkC) inhibitors were investigated . Potent inhibition of IL-8-induced PBL migration was observed following exposure of PBL to PTX and CTX (1 pM to 0.1 microM), 8-bromo cyclic adenosine monophosphate (cAMP; 1 nM to 1 microM), H7 (1 pM to 0.1 microM), sphingosine (0.1 microM to 100 microM) and the novel pkC inhibitors Ro 31-7549 and Ro 31-8220 (10 pM to 1 microM) for 10 min . Following incubation of the lymphocytes for 30 min in the presence of the direct activators of pkC, 1-oleoyl-2-acetyl-sn-glycerol (OAG) and 1,2-dioctanoyl-sn-glycerol (DOG; 10nM to 100 microM), there was a reversal of the effects of a suboptimal dose of the specific pkC inhibitors Ro 31-7549 and Ro 31-8220 . These results suggest that intracellular signals transduced during IL-8-induced in vitro PBL migration may involve pertussis and cholera toxin-sensitive G protein subunits and activation of pkC, processes which are characteristically linked to receptor binding.

Vet Microbiol, 1990 Jun, 23(1-4), 185 - 91
Nucleotide sequence of hog cholera virus RNA: properties of the polyprotein encoded by the open reading frame spanning the viral genomic RNA; Moormann RJ et al.; Hog cholera virus RNA was cloned and sequenced . A single major open reading frame (ORF), encoding an amino acid sequence of 3898 residues, was found in the second reading frame of the sequence of one of the cDNA strands . We demonstrated that the ORF spans the length of the viral sense RNA, which implies that it is translated into a precursor polyprotein . Several properties of this polyprotein, like hydrophobicity, position of putative protease cleavage sites, distribution of N-linked glycosylation sites, distribution of cysteines and distribution of acidic and basic residues are described and discussed.

Cell Calcium, 1990 Jun-Jul, 11(6), 419 - 23
Cholera toxin and its B subunit do not change cytosolic free calcium concentration; Astashkin EI et al.; Using the fluorescent Ca2+ probe Quin-2 it has been reported that cholera toxin (CT) and its B subunit (B-CT) increase cytosolic free Ca2+ concentration ({Ca2+}i) in entherocytes, thymocytes and fibroblasts . In this work we show, however, that the fluorescence increases of Quin-2-loaded cells (rat thymocytes, mouse splenocytes, P-388 macrophages and 3T3 fibroblasts) observed upon addition of CT or B-CT are not caused by an increase in {Ca2+}i . The observed effect appears to be accounted for by EDTA-2Na admixtures (present as conservation agent in all CT and B-CT preparations) which 'unquenches' the fluorescence of Quin-2 acid leaked out from the cells into the extracellular medium and produces influorescent complexes with contaminating heavy metal ions . Thus the mitogenic effect of B-CT is not obviously connected with the cytosolic free Ca2+ increase but is probably due to ganglioside-mediated protein phosphorylation.

Vaccine, 1990 Jun, 8(3), 243 - 8
Comparison of intranasal inoculation of influenza HA vaccine combined with cholera toxin B subunit with oral or parenteral vaccination; Hirabayashi Y et al.; The antibody responses to influenza virus A/PR/8/34 HA vaccine and protection against virus challenge in mice given the vaccine together with the B subunit of cholera toxin (CTB) intranasally were compared with those in mice given the vaccine with CTB perorally, intraperitoneally or subcutaneously . Intranasal vaccination induced remarkably higher levels of antiviral IgA antibodies in both respiratory washings and serum than did other routes of vaccination . The titres of antiviral IgG antibodies in respiratory washings and serum, and haemagglutination-inhibiting (HI) antibodies in serum, were similar after intranasal and parenteral vaccination . Oral vaccination, however, induced low levels of antiviral IgG antibodies but no detectable HI antibodies . Moreover, intranasal immunization elicited significantly higher titres of antiviral IgA antibodies in intestinal secretions in comparison with oral immunization . In contrast, parenteral immunization failed to induce these IgA antibodies . In virus challenge studies, a greater protective effect was seen after intranasal and intraperitoneal vaccination than after other routes of vaccination . These results suggest that intranasal inoculation of combined HA vaccine and CTB is superior to oral or parenteral inoculation in protecting mice . Furthermore, the intestinal antiviral IgA responses suggest that intranasal administration of CTB-combined vaccines could be effective not only against respiratory pathogens but also against enteropathogens.

Microb Pathog, 1990 Jun, 8(6), 421 - 31
Stimulation of phosphorylation of rat brush-border membrane proteins by Escherichia coli heat-stable enterotoxin, cholera enterotoxin and cyclic nucleotides, and its inhibition by protein kinase inhibitors, isoquinolinesulfonamides; Hirayama T et al.; Stimulation of phosphorylation of rat brush-border membrane proteins by the heat-stable enterotoxin of Escherichia coli (STh), cholera enterotoxin, cGMP and cAMP was demonstrated . Among at least 14 proteins examined, a protein with a molecular mass of 81,000 Da (81 kDa protein) was phosphorylated most in the presence of both STh and cholera enterotoxin, as well as in the presence of cGMP and cAMP . This phosphorylation was inhibited by N-{2-(methylamino)ethyl}-5-isoquinolinesulfonamide (H-8) or N-(2-aminoethyl)-5-isoquinoline-sulfonamide (H-9), which suggests that the phosphorylation occurs through cGMP- and cAMP-dependent protein kinases.

Can J Microbiol, 1990 Jun, 36(6), 452 - 4
Binding specificity of the combining site of cholera toxin for human erythrocytes; Sugii S; The reactivity of cholera toxin (CT) with blood-group determinant(s) on human erythrocytes was studied by competitive binding assays . 125I-labeled CT was found to bind more efficiently to pronase- and neuraminidase-treated human type A, B, and O erythrocytes than their untreated ones . The binding of 125I-labeled CT to neuraminidase-treated human type B erythrocytes was effectively inhibited by ganglioside GM1, but not by porcine gastric mucin with both A and H determinants (hog A + H), blood group specific lectins, and other substances at the highest concentrations used . Ganglioside GM1 was at least 10(5) times more potent than other inhibitors . These findings strongly suggest that the predominant binding substance for CT on human erythrocytes is not the blood-group determinant(s) but ganglioside GM1.

Biochim Biophys Acta, 1990 May 16, 1034(2), 195 - 9
Mechanism of cholera toxin activation by a guanine nucleotide-dependent 19 kDa protein; Noda M et al.; Cholera toxin causes the devastating diarrheal syndrome characteristic of cholera by catalyzing the ADP-ribosylation of Gs alpha, a GTP-binding regulatory protein, resulting in activation of adenylyl cyclase . ADP-ribosylation of Gs alpha is enhanced by 19 kDa guanine nucleotide-binding proteins known as ADP-ribosylation factors or ARFs . We investigated the effects of agents known to alter toxin-catalyzed activation of adenylyl cyclase on the stimulation of toxin- and toxin subunit-catalyzed ADP-ribosylation of Gs alpha and other substrates by an ADP-ribosylation factor purified from a soluble fraction of bovine brain (sARF II) . In the presence of GTP, sARF II enhanced activity of both the toxin catalytic unit and a reduced and alkylated fragment ('A1'), as a result of an increase in substrate affinity with no significant effects on Vmax . Activation of toxin was independent of Gs alpha and was stimulated 4-fold by sodium dodecyl sulfate, but abolished by Triton X-100 . sARF II therefore serves as a direct allosteric activator of the A1 protein and may thus amplify the pathological effects of cholera toxin.

J Biol Chem, 1990 May 5, 265(13), 7673 - 8
Generation of cell surface neoganglioproteins . GM1-neoganglioproteins are non-functional receptors for cholera toxin; Pacuszka T et al.; GM1 (II3Neu5Ac-GgOse4Cer)-oligosaccharide was prepared from the ganglioside by ozonolysis and alkaline fragmentation, reductively aminated and coupled to the heterobifunctional cross-linker succinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate . The resulting derivative reacted with free sulfhydryl groups and readily cross-linked to cell surface components on rat glioma C6 cells which are GM1-deficient . Attachment of the GM1-oligosaccharide derivative, which was monitored by increased binding of 125I-cholera toxin to the cells, was both time- and concentration-dependent . Prior treatment of the cells with dithiothreitol enhanced the attachment by generating additional free sulfhydryl groups . The affinity of cholera toxin for cells treated with the GM1-oligosaccharide derivative or with GM1 was similar . The nature of the newly generated toxin receptors was determined by Western blotting . Membranes from derivatized cells were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the resolved components were electrophoretically transferred to a nitrocellulose sheet which was overlain with 125I-cholera toxin . The toxin bound to a wide variety of membrane proteins, most of which were trypsin-sensitive . No such binding was observed using membranes from control cells . Although the GM1-neoganglioproteins newly generated on the surface of rat glioma C6 cells readily bound cholera toxin, the cells did not become more responsive to the toxin as measured by increased production of cyclic AMP or activation of adenylate cyclase . In contrast, cells exposed to GM1 became highly responsive to the toxin . Thus, neoganglioproteins on the cell surface appear to behave as nonfunctional receptors for cholera toxin.

J Reprod Fertil, 1990 May, 89(1), 325 - 33
The effects of cholera toxin, pertussis toxin, sodium fluoride and alpha-interferon on prostaglandin production by the guinea-pig endometrium; Leckie CM et al.; The outputs of prostaglandin (PG) F-2 alpha, 6-keto-PGF-1 alpha and PGE-2 from Day-7 and Day-15 guinea-pig endometrium were neither stimulated nor inhibited by cholera toxin and pertussis toxin . This indicates that PG synthesis by guinea-pig endometrium is not controlled by toxin-sensitive G-proteins . Short-term treatment of guinea-pig endometrium in culture with sodium fluoride stimulated PG output, suggesting that endometrial PG synthesis may be regulated by a fluoride-sensitive G-protein . Long-term treatment of guinea-pig endometrium in culture with sodium fluoride inhibited endometrial PG synthesis, and this was due to an inhibition of endometrial protein synthesis . Human alpha-interferon had no inhibitory effect on the outputs of PGF-2 alpha, 6-keto-PGF-1 alpha and PGE-2 from Day-15 guinea-pig endometrium in culture . It appears that the anti-luteolytic factor secreted by guinea-pig conceptus is not an alpha-interferon and is therefore probably different from ovine trophoblast protein-1.

Jpn J Cancer Res, 1990 May, 81(5), 520 - 6
Combination effects of interferon-gamma and cholera toxin on induction of differentiation of an insensitive U-937 clone; Iwanami M et al.; We examined the combination effect of interferon-gamma (IFN-gamma) and cholera toxin and the role of cAMP in the induction of differentiation of a differentiation-insensitive U-937 clone, in which the reactivity to differentiation-inducers was decreased . IFN-gamma (100 units/ml) or cholera toxin (10(-9) M) alone only marginally induced various differentiation-associated characteristics such as NBT-reducing activity, phagocytic activity, a-naphthyl acetate esterase activity and surface markers . However, when combined with each other, they significantly induced these markers . Other cAMP-inducing agents such as prostaglandin E2, forskolin, epinephrine and isoproterenol did not induce NBT-reducing activity, either alone or in combination with IFN-gamma . However, all these cAMP-inducing agents significantly increased intracellular cAMP levels . Tumor necrosis factor, interleukin 6 or granulocyte/macrophage colony-stimulating factor alone did not induce NBT-reducing activity, but they could induce activity when combined with cholera toxin . These results suggest that enhancement of induction of differentiation by cholera toxin in combination with IFN-gamma or other cytokines may not be merely due to increased cAMP levels . There seems to be a transduction signal other than cAMP coupling with cholera toxin to stimulate induction of differentiation of an insensitive U-937 clone.

Gene, 1990 Apr 30, 89(1), 47 - 52
Expression in Escherichia coli of two mutated genes encoding the cholera toxin B subunit; L'hoir C et al.; To allow subsequent genetically mediated fusion of foreign antigens to cholera toxin B subunit (CTB), two mutated CTB encoding genes (ctxB) were constructed and overexpressed in Escherichia coli . The signal peptide coding sequence was deleted and restriction sites were created at both ends of the modified sequence . Both synthesized CTBs contain additional amino acid(s) at the N terminus (one and three) . They were purified as insoluble products and refolded into the natural pentameric CTB structure by a denaturation-renaturation cycle . After renaturation, both recombinant proteins recovered CTB antigenicity and the ability to bind to GM1 gangliosides, as shown by in vitro analysis . Preliminary data indicated that both properties were unaltered by fusion of a foreign peptide to the mutated CTBs.

Eur J Pharmacol, 1990 Apr 25, 188(4-5), 203 - 9
Chronic exposure of rat glioma C6 cells to cholera toxin induces loss of the alpha-subunit of the stimulatory guanine nucleotide-binding protein (Gs); Carr C et al.; Rat glioma C6 BU1 cells were treated in tissue culture with cholera toxin . Incubation of membranes derived from these cells with fresh cholera toxin and {32P}NAD+ failed to promote incorporation of radioactivity into polypeptides corresponding to forms of Gs alpha . This is generally assumed to reflect prior ADP ribosylation of these polypeptides in vivo using endogenous NAD+ as substrate . However, immunological studies with anti-peptide antisera which identify all forms of Gs alpha demonstrated that concentrations of this polypeptide were now substantially reduced in the membranes . This effect was specific for Gs alpha as neither the alpha-subunits of the pertussis toxin-sensitive G-proteins Gi2 and Gi3, nor the beta subunit common to the various G-proteins were lost in parallel . Pertussis toxin-catalysed ADP ribosylation did not cause the downregulation of Gs alpha nor of the alpha-subunits of Gi2 or Gi3 although it did cause ADP ribosylation of the entire complement of both Gi2 and Gi3 in the membranes . Despite the reduction in levels of immunoreactive Gs alpha from the membranes of cholera toxin-treated cells, no alterations in levels of mRNA corresponding to this G-protein were noted.

J Comp Neurol, 1990 Apr 8, 294(2), 179 - 91
Cholera toxin B-gold, a retrograde tracer that can be used in light and electron microscopic immunocytochemical studies; Llewellyn-Smith IJ et al.; The purpose of this study was to test whether a new retrograde tracer, the B subunit of cholera toxin conjugated to colloidal gold particles (CTB-gold), was taken up and transported by neurons in the central nervous system of the rat . Retrograde transport of CTB-gold was assessed from axon terminals, from damaged nerve fibers, and from axons of passage . For light microscopy, CTB-gold was visualized by silver intensification; for electron microscopy, sections were silver-intensified with or without subsequent gold toning . Retrogradely transported CTB-gold was detected in neurons after survival times of 12 hours to 42 days and appeared as black punctate deposits in perikarya and proximal dendrites at the light microscope level . Ultrastructurally, the deposits were usually associated with lysosomes . Injections of CTB-gold into the caudal ventrolateral medulla or into the lateral horn of the spinal cord gave small well-defined injection sites and resulted in retrograde labelling in medullary neurons in the same locations as similarly placed injections of wheat germ agglutinin-horseradish peroxidase . When injected into the superior cervical ganglion, CTB-gold was transported to nerve cell bodies in the spinal cord, but application of CTB-gold to the cut cervical sympathetic trunk did not label neurons in the spinal cord . Injection of CTB-gold into the nodose ganglion retrogradely labelled neurons in the dorsal motor nucleus of the vagus and the nucleus ambiguus . CTB-gold was not transported anterogradely from injections sites within the medulla . Nerve fibers and cell bodies containing neuropeptides, monoamines, or neurotransmitter-synthesizing enzymes were readily immunostained after silver intensification of retrogradely transported CTB-gold . Immunoreactivity for neuropeptides and enzymes was also demonstrated ultrastructurally after silver intensification and gold toning . These results show that CTB-gold is retrogradely transported from nerve terminals and fibers of passage but not from damaged axons . CTB-gold gives well-localized injection sites and persists in neurons for weeks . Transported CTB-gold is easily visualized and its detection is compatible with light and electron microscopic immunocytochemistry . These properties make CTB-gold a valuable tool for studying the connectivity and neurochemistry of pathways in the central nervous system.

Scand J Immunol, 1990 Apr, 31(4), 443 - 51
Whole cholera toxin and B subunit act synergistically as an adjuvant for the mucosal immune response of mice to keyhole limpet haemocyanin; Wilson AD et al.; Cholera toxin (CT) is a potent stimulator of IgA responses when administered orally and has been shown to promote IgA responses to a second protein such as keyhole limpet haemocyanin (KLH) if this is fed simultaneously . In this paper we show that whilst feeding 5 mg KLH with either 0.5 micrograms CT or 10 micrograms B subunit fails to stimulate a mucosal IgA response to KLH, feeding 0.5 microgram CT and 10 micrograms B subunit together with 5 mg KLH produces a local IgA anti-KLH response as great as that produced by 10 micrograms of whole CT . In addition to stimulating IgA responses in the lamina propria, preliminary results indicate that cellular responses are also stimulated, as we have demonstrated KLH antigen-driven proliferation of cells isolated from groups of mice fed either 10 micrograms CT + 5 mg KLH or 0.5 micrograms CT + 10 micrograms CTB + 5 mg KLH but not mice fed KLH alone or with either 10 micrograms CTB or 0.5 micrograms CT . These results indicate that the mucosal adjuvant action of CT is due to a synergistic effect involving both the GM1 binding of the B subunit and adenylate cyclase activation by the A subunit.

Immunol Invest, 1990 Apr, 19(2), 119 - 32
Use of anti-cholera toxin IgA-secreting hybridoma cells for the elaboration of an antigen-specific IgA-ELISPOT-assay; Solbreux PM et al.; Hybridoma anti-cholera toxin (CT) IgA-secreting cells, because of their monoclonal character, were used to establish and optimize an ELISPOT-assay for CT-specific IgA-secreting cells . The use of hybridoma cells easily and quickly allowed the elaboration of a sensitive, specific and reproducible ELISPOT-assay, which was successfully applied to enumerate intestinal lamina propria lymphoid cells secreting anti-CT IgA in mice immunized twice intraintestinally with CT . Monoclonal antibody-secreting hybridoma cells, if available against a given antigen, are suggested as ideal cell suspensions to elaborate new ELISPOT-assays to study antibody responses at the cellular level.

Eur J Biochem, 1990 Mar 30, 188(3), 567 - 76
Cholera toxin differentially decreases membrane levels of alpha and beta subunits of G proteins in NG108-15 cells; Klinz FJ et al.; Treatment of NG108-15 neuroblastoma x glioma cells (24 h) with cholera toxin (0.1-10 micrograms/ml) resulted in a concentration-dependent reduction of the membrane levels of subunits of GTP-binding regulatory proteins (G proteins), as determined by quantitative immunoblot procedures . The extent of reduction differed for different types of subunits: the levels of Go alpha and G beta 1 were reduced by 40-50%, whereas those of G alpha common immunoreactivity and Gi2 alpha were only reduced by 10-20% following treatment with 10 micrograms/ml cholera toxin . This effect of the toxin could not be mimicked by incubation with the resolved B oligomer of cholera toxin, nor by exposure of cells to agents able to raise the intracellular levels of cAMP . Basal adenylate cyclase was stimulated in a biphasic manner by cholera toxin, being stimulated at low concentrations (0.01-10 ng/ml) and then decreased at high (0.1-10 micrograms/ml) concentrations . Thus, the down regulation of G-protein subunits produced by cholera toxin requires its (ADP-ribosyl)transferase activity but does not result from a cAMP-mediated mechanism . The toxin-mediated decrease of Go alpha in the membrane was correlated with a diminution of opioid-receptor-mediated stimulation of high-affinity GTPase activity, suggesting that opioid receptors interact with Go in native membranes of NG108-15 cells . Northern-blot analysis of cytoplasmic RNA prepared from cells treated with cholera toxin showed that the levels of mRNA coding for G beta 1 did not change . Thus, the cholera-toxin-induced decrease of G-protein subunits may not result from an alteration in mRNA levels, but may involve a direct effect of the toxin on the process of insertion and/or clearance of G proteins into and/or from the membrane . These data indicate that cholera toxin, besides catalyzing the ADP-ribosylation of Gs and Gi/Go types of G proteins, can also reduce the steady state levels of Go alpha and G beta 1 subunits in the membrane and thus alter by an additional mechanism the function of inhibitory receptor systems.

Z Gesamte Hyg, 1990 Mar, 36(3), 150 - 1
{Hospital hygiene measures in the care of cholera patients}; Schiller WG et al.; The hygienic precautions taken by a hospital are presented by the example of a cholera infected patient from Vietnam . Also in the future the import of single cholera cases will be inevitable as a consequence of the fast modern communication . But the hygienic standards valid for the GDR health service are well suited to prevent cholera from distribution.

Dig Dis Sci, 1990 Mar, 35(3), 353 - 9
Effect of cholera toxin on small intestinal motor activity in the fed state; Cowles VE et al.; We sought to determine the effect of cholera toxin on small intestinal transit and motor activity in the fed state . Mean transit time increased after cholera toxin, but there was no change in mean amplitude, duration, and area of contractions . In contrast, there was a reduction in the total amplitude, duration, and area of contractions, and this was due to a decrease in frequency of contractions . The reduction in the total parameters of all contractions could be accounted for by a reduction in the same parameters for propagating contractions . The parameters of nonpropagating contraction were not different after cholera toxin . Also, there was a decrease in the distance of propagation of contractions . Our findings demonstrate that during the secretory state due to cholera toxin the small intestinal motor activity works in a compensatory mode to decrease transit and allow more time for absorption.

Am J Epidemiol, 1990 Mar, 131(3), 400 - 11
Breast feeding and the risk of severe cholera in rural Bangladeshi children; Clemens JD et al.; The association between breast feeding and the risk of severe cholera was examined in a case-control study of rural Bangladeshi children under 36 months of age who were studied in 1985-1986 during a field trial of killed oral cholera vaccines . A total of 116 cases who were treated for severe cholera were compared with 464 age-matched community controls without severe cholera . Overall, the odds ratio relating breast feeding to severe cholera (0.30, p less than 0.0001) reflected a 70% reduction in the risk of severe cholera among breast-fed children . The estimated reduction of risk declined with age, but was clearly evident in children up to 30 months of age . Although the association between breast feeding and a reduced risk of severe cholera was not significantly greater in children of mothers who had received cholera vaccine than in children whose mothers had received placebo during the trial, maternal vaccination per se was suggestively associated with a reduced risk of severe cholera in their nonvaccinated children (odds ratio = 0.53, p = 0.05) . These results indicate that breast feeding was associated with a substantial reduction of the risk of severe cholera and raise the possibility that vaccination of mothers may provide protection to their young children in endemic settings.

Vopr Med Khim, 1990 Mar-Apr, 36(2), 18 - 9
{The effect of indomethacin on the activity of Na+,K+- and HCO3--ATPases and prostaglandin levels in the rat ileum mucosa after exposure to cholera exotoxin}; Dolmatova LS; Preadministration of indomethacin (10 mg/kg of rat body mass), 1 hr before choleraic toxin injection, removed the toxin inhibitory effect on activity of Na+, K(+)-ATPase and decreased distinctly its inhibitory action on HCO3(-)-ATPase . At the same time, the indomethacin premedication caused a decrease in content of prostaglandins PGE, 6-keto-PGF1 alpha and of thromboxane B2 as compared with the choleraic toxin effect and controls . The data obtained corroborate the hypothesis on antisecretory effect of indomethacin occurring via PG-dependent mechanism.

J Cell Biochem, 1990 Mar, 42(3), 143 - 52
Cautionary note on the use of the B subunit of cholera toxin as a ganglioside GM1 probe: detection of cholera toxin A subunit in B subunit preparations by a sensitive adenylate cyclase assay; Spiegel S; The use of the B subunit of cholera toxin, a protein that binds specifically to ganglioside GM1, has provided a new paradigm for studying physiological functions of ganglioside GM1 . The B subunit inhibited the growth of rat glioma C6 cells that had been pretreated with ganglioside GM1 . In some preparations of the B subunit, the inhibition was independent of adenylate cyclase activation and was due to the binding of the B subunit to ganglioside GM1 inserted onto the cell surface . However, in other preparations of the B subunit, there was an additional inhibitory effect due to small contaminations with the A subunit, which caused increases in intracellular cyclic adenosine monophosphate (cAMP) levels and concomitant growth inhibition . This vanishingly small contamination with the A subunit could not be detected by conventional protein sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis but could be measured utilizing a sensitive adenylate cyclase activation assay . Thus caution must be used to ensure that any biological effects of the B subunit are not due to contaminating A subunit and are due solely to the binding of the B subunit to ganglioside GM1 exposed on the cell surface . This is especially important in cyclic nucleotide-sensitive systems.

Proc Soc Exp Biol Med, 1990 Mar, 193(3), 181 - 4
Stimulatory effects of cholera toxin on arachidonic acid metabolism in Chinese hamster ovary cells; Reitmeyer JC et al.; Cholera toxin (CT) stimulated the release of arachidonic acid (AA) from Chinese hamster ovary cells with no apparent lag period . CT-induced release of {3H}AA or its metabolites was dose dependent during a 4-hr period of toxin exposure with a minimum effective dose of 0.1 ng/ml . CT-induced release of {3H}AA metabolites began within 15 min of toxin addition and became maximal after approximately 5 hr . Neither CT-A subunit nor CT-B subunit alone caused {3H}AA release . Furthermore, {3H}AA release was not caused by addition of dibutyryl cAMP to the culture medium, indicating that the observed effect of CT on arachidonate metabolism appeared to be independent of cAMP . The effect of CT on AA metabolism is proposed as a possible mechanism leading to the synthesis of prostaglandin E and fluid secretion during cholera.

Proc Natl Acad Sci U S A, 1990 Mar, 87(6), 2206 - 10
Selective amplification of an mRNA and related pseudogene for a human ADP-ribosylation factor, a guanine nucleotide-dependent protein activator of cholera toxin; Monaco L et al.; ADP-ribosylation factors (ARFs) are approximately 20-kDa proteins that act as GTP-dependent allosteric activators of cholera toxin . With deoxyinosine-containing degenerate oligonucleotide primers corresponding to conserved GTP-binding domains in ARFs, the polymerase chain reaction (PCR) was used to amplify simultaneously from human DNA portions of three ARF genes that include codons for 102 amino acids, with intervening sequences . Amplification products that differed in size because of differences in intron sizes were separated by agarose gel electrophoresis . One amplified DNA contained no introns and had a sequence different from those of known ARFs . Based on this sequence, selective oligonucleotide probes were prepared and used to isolate clone psi ARF 4, a putative ARF pseudogene, from a human genomic library in lambda phage EMBL3 . Reverse transcription-PCR was then used to clone from human poly(A)+ RNA the cDNA corresponding to the expressed homolog of psi ARF 4, referred to as human ARF 4 . It appears that psi ARF 4 arose during human evolution by integration of processed ARF 4 mRNA into the genome . Human ARF 4 differs from previously identified mammalian ARFs 1, 2, and 3 . Hybridization of ARF 4-specific oligonucleotide probes with human, bovine, and rat RNA revealed a single 1.8-kilobase mRNA, which was clearly distinguished from the 1.9-kilobase mRNA for ARF 1 in these tissues . The PCR provides a powerful tool for investigating diversity in this and other multigene families, especially with primers targeted at domains believed to have functional significance.

Eur J Biochem, 1990 Feb 14, 187(3), 683 - 9
Regulation by butyrate of the cAMP response to cholera toxin and forskolin in pituitary GH1 cells; Aranda A et al.; In pituitary GH1 cells, a rat growth hormone-producing cell line, butyrate elicited a dose-dependent increase in cholera toxin receptors as measured by an increased binding of 125I-labeled cholera toxin to the intact cells . Butyrate did not alter the affinity of cholera toxin binding, the dissociation constant being 0.4 nM for both control and butyrate-treated cells . Despite the increased binding, the cAMP response to cholera toxin was strongly reduced after exposure to butyrate . This reduction was dose-dependent and with butyrate 1--5 mM, intracellular and extracellular (medium) cAMP levels were decreased by more than 70% in cells incubated for 24 h with 1 nM cholera toxin . Forskolin (30 microM) elicited a cAMP response similar to that found with the toxin, and a similar inhibition of cAMP was also found after incubation of GH1 cells with butyrate . Butyrate also affected basal cAMP levels which were reduced by 40--60% in cells cultured for 24--48 h with the fatty acid . In order to study whether butyrate influenced cAMP synthesis and/or cAMP degradation, adenylyl cyclase and phosphodiesterase activities were determined in control cells and in cells incubated for 24 h with cholera toxin or forskolin . Butyrate had a dual effect since, besides activating phosphodiesterase by more than twofold, it also inhibited the cyclase by 40--50% in all groups . The in vitro response of adenylyl cyclase to stimulatory (NaF) and inhibitory (carbachol and adenosine) effectors was also examined . The absolute activity of the cyclase was always 40--50% lower in the cells incubated with butyrate, but the percentage change of activity obtained in butyrate-treated and untreated cells was unaltered . In addition, ADP-ribosylation of the guanine nucleotide stimulatory component of the cyclase (Gs) was not affected in the cells incubated with butyrate . These results suggest that the catalytic (C) subunit of adenylyl cyclase and/or its interaction with the regulatory components might be altered in butyrate-treated GH1 cells . The inhibition of the cAMP response in GH1 cells was accompanied by an inhibition of a biological action of the nucleotide, namely growth hormone (somatotropin) production which is primarily controlled by thyroid hormones in these cells . Forskolin alone did not affect the somatotropin levels but potentiated the growth hormone response to triiodothyronine . Butyrate produced a dose-dependent inhibition of this response, which was totally abolished at concentrations of butyrate higher than 1 mM.

Lancet, 1990 Feb 3, 335(8684), 270 - 3
Field trial of oral cholera vaccines in Bangladesh: results from three-year follow-up; Clemens JD et al.; The protective efficacy (PE) of B subunit killed whole-cell (BS-WC) and killed whole-cell-only (WC) oral cholera vaccines was assessed in a randomised double-blind field trial among children aged 2-15 years and women over 15 years in rural Bangladesh . Among the 62 285 subjects who received three doses of BS-WC, WC, or Escherichia coli K12 strain placebo, cumulative PE at 3 years of follow-up was 50% for BS-WC and 52% for WC . PE was similar against severe and non-severe cholera, but was significantly lower in children who were vaccinated at 2-5 years (26% for BS-WC; 23% for WC) than in older persons (63% for BS-WC; 68% for WC) . Among persons vaccinated at 2-5 years, protection at 4-6 months of follow-up was similar to that for older persons, but rapidly waned thereafter and was not evident during the third year of follow-up . In contrast, persons vaccinated at older ages were protected even in the third year of follow-up (PE 40% for BS-WC; 62% for WC) . PE was substantially higher against classical cholera (58% for BS-WC; 60% for WC) than against El Tor cholera (39% and 40%).

Eur J Immunol, 1990 Feb, 20(2), 433 - 6
Cholera holotoxin and its B subunit enhance Peyer's patch B cell responses induced by orally administered influenza virus: disproportionate cholera toxin enhancement of the IgA B cell response; Chen KS et al.; In these studies we analyzed the adjuvant effect of cholera holotoxin or cholera toxin (CT) B subunit on the B cell response to mucosal antigens . Purified Peyer's patch B cells obtained from mice at varying periods of time after oral administration of inactivated influenza virus, with or without a CT preparation, were stimulated in vitro in the absence or presence of various lymphokines . Responses were measured by an antigen- and isotype-specific ELISPOT assay . In this system cultures containing a combination of lymphokines {interleukin 5 (IL 5), interferon-gamma (IFN-gamma), IL 4} gave comparable responses to those containing T cells from immunized mice or supernatant of concanavalin A-stimulated T cells and therefore were assumed to express optimum or near optimum B cell responses . Administration of a CT preparation along with influenza virus increased the number of B cells producing anti-influenza antibodies of both the IgM and IgA isotypes, with the effect on the IgA response at least threefold greater than the effect on the IgM response . These results thus indicate that CT preparations enhance the memory B cells response in Peyer's patches and, in addition, suggest that CT enhances isotype switching . In this antigen-specific B cell system IL 4 augmented responses in cultures containing IL 5 but not IFN-gamma; in addition, IL 5 and IFN-gamma acted in an additive fashion . Thus, these findings suggest that the effects of IL 5 and IFN-gamma are at least in part, mediated via different cellular differentiation pathways.

Dtsch Tierarztl Wochenschr, 1990 Feb, 97(2), 77 - 9
Development and properties of a cell culture produced vaccine for hog cholera based on the Chinese strain; Terpstra C et al.; The Chinese strain of hog cholera virus (HCV) was adapted to suspension cultures of the established swine kidney cell line SK6 . The strain designated "Cedipest", is produced on the basis of a seedlot system . The masterseed virus was identified in vitro and in vivo, and was found free from extraneous pig pathogenic viruses by repeated animal inoculation followed by appropriate serological tests . A distinct and reproducible relationship was ascertained between infectivity in vitro and protection . Pigs inoculated with 400-600 TCID50 of the Cedipest strain proved fully protected against challenge with greater than 100 pig LD50 of a virulent strain of HCV at 7 days and at 6 month post vaccination.

Transplantation, 1990 Feb, 49(2), 277 - 80
Immune function in transplanted small intestine . Total secretory IgA production and response against cholera toxin; Xia WY et al.; Secretory IgA is the dominant immunoglobulin produced in the small intestine and one important component of the local defense against dietary and infectious agents present in the gut lumen . The effect of small intestine transplantation on total production of sIgA and on the response to a newly presented antigen, cholera toxin, was determined in a rat segmental heterotopic intestinal transplant model . Lewis x Brown Norway F1 (LBNF1) allografts in Lewis hosts made normal amounts of sIgA, when compared with LBNF1 Thiry-Vella loops or LBNF1 isografts . In contrast, the allografts failed to make a significant specific sIgA response when immunized with cholera toxin at days 0 and 7 following transplantation . This failure was not the result of surgical manipulation, as isografts made normal amounts of specific sIgA directed against cholera toxin . Cyclosporine immunosuppression delayed, but did not prevent, the secretion of specific antibody in isografts . This failure to respond to a new antigen may have important implications for the safety of small bowel transplantation.

Dtsch Tierarztl Wochenschr, 1990 Feb, 97(2), 91 - 3
A new approach for the diagnosis of hog cholera; Moennig V et al.; Pestiviruses were isolated from seven cases of suspect hog cholera . Using peroxidase conjugates of monoclonal antibodies (Mabs) six isolates were identified as hog cholera viruses (HCV), while one isolate was of ruminant origin, possibly bovine viral diarrhea virus . In parallel attempts were made to develop an ELISA for the detection of HCV-specific antibodies in pig sera . The Mab HCTC26 coated to polystyrol plates efficiently captured the major viral glycoprotein gp53 from crude antigen suspensions prepared from infected cells . The immobilized gp53 served as diagnostic antigen . Five pigs experimentally infected with the HCV strain Glentorf were sequentially bled and the development of antibodies was monitored by neutralization tests and the ELISA . Results showed that both tests detected antibodies simultaneously after infection . Titres measured by ELISA were slightly higher than those registered by neutralization.

Dtsch Tierarztl Wochenschr, 1990 Feb, 97(2), 79 - 81
Antigenic comparisons of hog cholera virus isolates from Europe, America and Asia using monoclonal antibodies; Edwards S et al.; Nineteen monoclonal antibodies (MAbs) with specificity for hog cholera virus (HCV) were prepared . They were used in an immune binding (peroxidase linked) assay to determine the reaction patterns of HCV isolates from Europe, Brazil, USA, Japan and Malaysia, as well as laboratory reference strains of the virus . A further panel of 17 MAbs raised against bovine virus diarrhoea virus (BVDV) was included in the study, together with 5 MAbs raised against a non-HCV pestivirus of porcine origin . All the MAbs were also tested against representative strains of BVDV and border disease virus . Six MAbs were HCV-specific, reacting with all isolates of HCV and none of the ruminant viruses . Among the other HCV MAbs geographical variation in reaction patterns was observed . There was evidence of antigenic distinction between recent European isolates, and archive material originally isolated more than 10 years ago.

Biochem J, 1990 Feb 1, 265(3), 799 - 807
Cholera toxin modulation of angiotensin II-stimulated inositol phosphate production in cultured vascular smooth muscle cells; Socorro L et al.; Activation of phospholipase C by angiotensin II in vascular smooth muscle has been postulated to be mediated by an unidentified GTP-binding protein (G-protein) . Using a permeabilized preparation of myo-{3H}inositol-labelled cultured vascular smooth muscle cells, we examined the ability of a non-hydrolysable analogue of GTP, guanosine 5'-{gamma-thio}triphosphate (GTP{S}), to stimulate inositol phosphate formation . GTP{S} (5 min exposure) stimulated inositol polyphosphate release by up to 3.8-fold in a dose-dependent manner, with an EC50 (concn . producing half-maximal stimulation) of approx . 50 microM . Inositol bisphosphate (IP2) and inositol trisphosphate (IP3) accumulations were also stimulated by NaF (5-20 mM) . Furthermore, angiotensin II-induced inositol phosphate formation could be potentiated by a submaximal concentration of GTP{S} (10 microM), and this treatment appeared to interfere with the normal termination mechanism of the initial hormonal signal . The G-protein mediating angiotensin II-stimulated phospholipase C activation was insensitive to pertussis toxin at an exposure time and concentration which were sufficient to completely ADP-ribosylate all available substrate (100 ng/ml, 16 h) . In contrast, a similar incubation with cholera toxin markedly inhibited angiotensin II-stimulated IP2 and IP3 release by 67 +/- 6% and 62 +/- 6% respectively . Cholera toxin appeared to inhibit angiotensin II stimulation of phospholipase C by a dual mechanism: it caused a 45% decrease in angiotensin II receptor number, and also inhibited G-protein transduction as assessed by GTP{S}-stimulated IP2 formation . This latter inhibition may be secondary to an increase in cyclic AMP, since it could be simulated by addition of dibutyryl cyclic AMP . Thus angiotensin II-stimulated inositol phosphate formation is cholera-toxin-sensitive, and is mediated by a pertussis-toxin-insensitive G-protein, which may be involved directly in termination of early signal generation.

J Physiol, 1990 Feb, 421, 399 - 409
Reversal and inhibition of cholera toxin-induced secretion in isolated rabbit ileum; Falk G et al.; 1 . Cholera toxin (1 microgram/ml) abolished net fluid absorption by everted sacs of rabbit ileum, leading to net fluid secretion . This action occurred via the toxin-catalysed ADP ribosylation of the stimulatory GTP-binding protein Gs which is linked to adenylate cyclase . Nicotinamide (10 mM), a reaction product of ADP ribosylation, reversed cholera toxin-induced secretion, restoring absorption . Lower concentrations of nicotinamide induced partial reversal . 2 . Nicotinamide (1 mM) blocked the secretory action of cholera toxin applied to ileal sacs . This inhibitory action was more effective in the presence of methionine (1 mM) . 3 . Other inhibitors of ADP ribosylation, benzamide and adenine, blocked the secretory action of cholera toxin . Hypoxanthine, an analogue and metabolite of adenine, was similarly effective . 4 . Nicotinamide was not, however, effective in blocking or reversing the secretory action of theophylline (10 mM) or of heat-stable E . coli enterotoxin STa . This indicates that nicotinamide has a highly specific action against ADP ribosylating toxins . 5 . It is proposed that nicotinamide reverses the secretory action of cholera toxin by reversing ADP ribosylation, simply by the law of mass action . This counters the established idea that the effects of cholera and other ADP-ribosylating toxins are irreversible under physiological conditions.

Biochemistry, 1990 Jan 30, 29(4), 855 - 61
Mechanism of activation of cholera toxin by ADP-ribosylation factor (ARF): both low- and high-affinity interactions of ARF with guanine nucleotides promote toxin activation; Bobak DA et al.; Activation of adenylyl cyclase by cholera toxin A subunit (CT-A) results from the ADP-ribosylation of the stimulatory guanine nucleotide binding protein (GS alpha) . This process requires GTP and an endogenous guanine nucleotide binding protein known as ADP-ribosylation factor (ARF) . One membrane (mARF) and two soluble forms (sARF I and sARF II) of ARF have been purified from bovine brain . Because the conditions reported to enhance the binding of guanine nucleotides by ARF differ from those observed to promote optimal activity, we sought to characterize the determinants influencing the functional interaction of guanine nucleotides with ARF . High-affinity GTP binding by sARF II (apparent KD of approximately 70 nM) required Mg2+, DMPC, and sodium cholate . sARF II, in DMPC/cholate, also enhanced CT-A ADP-ribosyltransferase activity (apparent EC50 for GTP of approximately 50 nM), although there was a delay before achievement of a maximal rate of sARF II stimulated toxin activity . The delay was abolished by incubation of sARF II with GTP at 30 degrees C before initiation of the assay . In contrast, a maximal rate of activation of toxin by sARF II, in 0.003% SDS, occurred without delay (apparent EC50 for GTP of approximately 5 microM) . High-affinity GTP binding by sARF II was not detectable in SDS . Enhancement of CT-A ADP-ribosyltransferase activity by sARF II, therefore, can occur under conditions in which sARF II exhibits either a relatively low affinity or a relatively high affinity for GTP . The interaction of GTP with ARF under these conditions may reflect ways in which intracellular membrane and cytosolic environments modulate GTP-mediated activation of ARF.

Biochemistry, 1990 Jan 30, 29(4), 921 - 8
Antibodies against a peptide of cholera toxin differing in cross-reactivity with the toxin differ in their specific interactions with the peptide as observed by 1H NMR spectroscopy; Anglister J et al.; The interactions between the aromatic residues of the monoclonal antibody TE34, and its peptide antigen CTP3, have been studied by 2D TRNOE difference spectroscopy . The sequence of CTP3 corresponds to residues 50-64 of the B subunit of cholera toxin (VEVPGSQHIDSQKKA) . Unlike two previously studied anti-CTP3 antibodies (TE32 and TE33), the TE34 antibody does not bind the toxin . The off-rate of CTP3 from TE34 was found to be too slow to measure strong TRNOE cross-peaks between the antibody and the peptide . Much faster off-rates, resulting in a strong TRNOE, were obtained for two peptide analogues: (a) CTP3 with an amide in the C-terminus (VEVPGSQHIDSQKKA-NH2) and (b) a truncated version of the peptide (N-acetyl-IDSQKKA) . These modifications do not interfere significantly either with the interactions of the unmodified part of the peptide with the antibody or with intramolecular interactions occurring in the epitope recognized by the antibody . The combined use of these peptides allows us to study the interactions between the antibody and the whole peptide . Two tyrosine residues and one or more tryptophan and phenylalanine residues have been found to interact with histidine-8, isoleucine-9, aspartate-10, lysine-13 and/or lysine-14, and alanine-15 of the peptide . In the bound peptide, we observe interactions of a lysine residue with aspartate-10 beta protons . While the peptide epitope recognized by TE34 is between histidine-8 and the negatively charged C-terminus, that recognized by TE32 and TE33 is between residues 3 and 10 of the peptide.(ABSTRACT TRUNCATED AT 250 WORDS)

J Comp Neurol, 1990 Jan 22, 291(4), 621 - 36
Musculotopic organization of the facial motor nucleus in Macaca fascicularis: a morphometric and retrograde tracing study with cholera toxin B-HRP; Welt C et al.; Morphometric and retrograde tracing methods were used to determine the location and number of motoneurons innervating individual facial muscles in Macaca fascicularis . Intramuscular injections of the cholera toxin B subunit-horseradish peroxidase conjugate produced discrete labeling patterns in the ipsilateral facial motor nucleus with good definition of somata and their processes . The facial nucleus extended rostrocaudally in the pons for about 2 mm, varying in shape and cross-sectional area along this axis . Motoneurons were clustered in subnuclei, but their boundaries were not sharp and they were not segregated by fiber bundles . The length, number, and area of subnuclei varied with rostrocaudal location . Retrograde labeling patterns revealed that individual muscles were innervated by longitudinal columns of motoneurons with each muscle region represented at all rostrocaudal levels of its column . The columns began at different rostrocaudal levels and varied in length . Columns for closely related muscles, such as the orbicularis oris and mentalis of the lower lip, tended to overlap, whereas columns for disparate muscles, such as the perioral and orbital, did not overlap . The dendritic processes of most motoneurons branched extensively among several different columns or subnuclei . Some dendrites extended outside of the nucleus into the surrounding tegmentum . Mean soma diameter (10.4-42.2 microns) was distributed unimodally, reflecting the absence of gamma motoneurons and lack of muscle spindles in the facial muscles . Large and small motoneurons were found in all regions of the nucleus, but the largest ones were located caudally and innervated muscles of the upper and lower lip . The perioral muscles also had more neurons, longer columns, and a lower cell density than the other muscle groups examined . These features may reflect the functions of the perioral muscles in facial expression and vocalization.

Biochim Biophys Acta, 1990 Jan 19, 1037(1), 92 - 9
Meta-iodobenzylguanidine (MIBG), a novel high-affinity substrate for cholera toxin that interferes with cellular mono(ADP-ribosylation); Loesberg C et al.; Meta-iodobenzylguanidine (MIBG) is a guanidine analogue of the neurotransmitter norepinephrine . Radioiodinated {131I}MIBG is clinically used as a tumor-targeted radiopharmaceutical in the diagnosis and treatment of adrenergic tumors . Moreover, non-radiolabelled MIBG exerts several cell-biological effects, tentatively ascribed to interference with cellular mono(ADP-ribosyl) transferases (Smets, L.A., Bout, B . and Wisse, J . (1988) Cancer Chemother . Pharmacol . 21, 9-13; Smets, L.A., Metwally, E.A.G., Knol, E . and Martens, M . (1988) Leukemia Res . 12, 737-743) . In the present study it was investigated whether MIBG could serve as an acceptor for the ribosyl transferase activity of cholera toxin and of erythrocyte membranes . MIBG appeared a substrate for the cholera toxin-catalyzed transfer of the ADP-ribose moiety of NAD to arginine-like residues with the highest affinity for this enzyme reported as yet (Km = 6.5 microM) . MIBG was also ADP-ribosylated by the mono(ADP-ribosyl)transferase(s) of turkey erythrocyte membranes . Moreover, the drug appeared a potent affector of the ADP-ribose linkage to membrane proteins by these enzymes . Interference by MIBG was stronger than by related guanyltyramine, the monoamine precursors of MIBG, meta-iodobenzylamine had no effect at all . In contrast, the drug failed to affect endogenous, O-linked poly(ADP-ribose) polymerase, induced in nuclei of S49-leukemia cells by deoxyribonuclease . Since MIBG is the first described drug that specifically interferes with the cellular N-linked mono(ADP-ribosyl) transferase reactions, it may be an important tool to elucidate the physiological role of this posttranscriptional protein modification.

BMJ, 1990 Jan 6, 300(6716), 25 - 6
Overprescription of cholera vaccine to travellers by general practitioners; Mott A et al.; A questionnaire describing five hypothetical patients intending to travel to different countries was sent to 113 general practitioners, who were asked to state which patients they would recommend cholera vaccination to . The response rate was 80% . The general practitioners' recommendations were compared with those of the Liverpool School of Tropical Medicine . Sixty three of 86 respondents recommended cholera vaccination when it would probably have been unnecessary . A review of common sources of information on cholera vaccination showed that general practitioners are given confusing or inappropriate advice . General practitioners should be educated about when cholera vaccination is necessary; alternatively, the vaccine should be available only through special centres.

Microbiol Immunol, 1990, 34(3), 337 - 46
Enhancement of DTH response by cholera toxin B subunit inoculated intranasally together with influenza HA vaccine; Kikuta K et al.; The effects of B subunit of cholera toxin (CTB) on delayed-type hypersensitivity (DTH) response to influenza vaccine derived from influenza virus A/PR/8/34 (PR-8, HlNl) virus were investigated in B10 mice that were immunized intranasally with both influenza vaccine and CTB . The result showed that intranasal inoculation of this combination augmented DTH response to influenza vaccine, which reached its peak 6 days after inoculation, and also induced accelerated DTH response upon a second inoculation of influenza vaccine alone 4 weeks later, that the cross-reactive DTH response to PR-8 vaccine was elicited by the injection of the different influenza A-type virus vaccine into the footpad of the vaccinated mice, but was not by influenza B-type virus vaccine, that the DTH-mediating T cells were detected selectively in the lungs of mice that received the nasal inoculation of the vaccine and CTB together, but that subcutaneous inoculation of this combination failed to induce DTH-mediating T cells in the lungs . These results, together with the previous papers (Tamura et al, Vaccine 7: 257-262; 314-320, 1989), suggest that CTB could augment both humoral and DTH responses against influenza vaccine in the respiratory mucosal tract.

Acta Physiol Scand, 1990 Jan, 138(1), 75 - 84
Studies of cholera toxin-induced changes of alkaline secretion and transepithelial potential difference in the rat intestine in vivo; Tantisira MH et al.; A pH-stat technique was used to investigate the effects of cholera toxin (CT) on alkaline secretion from denervated intestines (jejunum, ileum, colon) in anaesthetized rats . Transepithelial potential difference (PD) was also followed in some experiments . CT, given intraluminally, caused a marked increase in jejunal alkaline secretion, whereas only a small effect was observed in the ileum and no apparent effect was noted in the proximal colon . The pronounced increase in jejunal alkaline secretion was found to be inhibited by 10-25% by hexamethonium (10 mg kg-1 body wt i.v.) and similarly by serosal application of lidocaine, whereas atropine (0.25 mg kg-1 body wt i.v.) had no effect . Thus the cholera toxin-induced alkaline secretion in the jejunum is attributed mainly to a non-nervous mechanism . The small effect of CT on ileal alkaline secretion observed in this study contrasts with the high ileal bicarbonate concentration reported in cholera by authors who estimated the concentration from the total carbon dioxide/bicarbonate contents . This discrepancy may be explained by a CT-evoked increased transport of the coupled Na+/H+ and Cl-/HCO3- exchangers, which cannot be measured with the pH-stat technique used in this study.

Brain Res, 1990 Jan 1, 506(1), 159 - 65
Neurons in the sacral parasympathetic nucleus that project to the hypothalamus do not also project through the pelvic nerve--a double labeling study combining Fluoro-gold and cholera toxin B in the rat; Burstein R et al.; We recently reported that neurons in the sacral parasympathetic nucleus (SPN) project directly to the hypothalamus . In the present study, we examined the possibility that individual neurons in SPN send both an axon into the pelvic nerve and an ascending projection to the hypothalamus . We used a new double-labeling technique in which two sensitive retrograde tracers (Fluoro-gold and cholera toxin subunit B immunocytochemically stained with rhodamine-labeled antibodies) were combined . The effectiveness of this combination for singly and doubly labeling neurons was established in experiments in which both tracers were injected into overlapping areas of the tongue or ventrobasal thalamus . These injections doubly labeled large numbers of neurons in the hypoglossal or dorsal column nuclei, respectively . In studies of the projections of neurons in the SPN, injection of one tracer into the hypothalamus and the other into the pelvic nerve and/or pelvic ganglion singly labeled many neurons (more than 3300 in the 7 examined cases) . However, no SPN neurons were doubly labeled . These findings indicate that the SPN in the rat consists of at least two distinct groups of cells, parasympathetic preganglionic neurons and neurons that project to the hypothalamus.

Virology, 1990 Jan, 174(1), 286 - 9
Genomic localization of hog cholera virus glycoproteins; Stark R et al.; A polyspecific antiserum has been used to identify four different glycoproteins in hog cholera virus (HCV)-infected cells termed gp55, gp48, gp44, and gp33 (Rumenapf et al, 1989, Virology 171, 18-27) . Fusion proteins containing parts of the putative HCV-encoded glycoproteins were expressed in bacteria and served for the preparation of specific antibodies . These were used in radioimmunoprecipitation assays which revealed that gp48 and gp44 most likely share a common protein backbone . The order of the glycoproteins on the HCV genome was determined as follows: NH2-gp44/gp48-gp33-gp55-COOH.

J Clin Epidemiol, 1990, 43(12), 1361 - 7
Low gastric acid as a risk factor for cholera transmission: application of a new non-invasive gastric acid field test; Van Loon FP et al.; Although gastric acid is thought to be an important host defense against certain enteric infections, field studies of the role of gastric acid in preventing enteric infections have been hampered by the lack of a suitable non-invasive test . Because low gastric acid output (GAO) is an established risk factor for cholera, we assessed after validation, whether a new non-invasive test which estimates GAO by measuring breath hydrogen excess after ingestion of magnesium and a stimulant of gastric acid secretion, could discriminate between persons at high and at low risk of developing cholera . Fifteen age-matched pairs, participants in the field trial of two oral cholera vaccines in rural Bangladesh, were tested . In each pair the "case" was a person who had recovered from severe cholera at least 6 months before testing and the "control" was a person who resided in the home of a cholera patient but remained uninfected . The stimulated breath hydrogen was higher in controls (median hydrogen excess = 369 mumol/80 min) than in cases (median hydrogen excess = 150 mumol/80 min) (p less than 0.05) and was higher in controls in 12 out of 15 pairs . The results, which are consistent with past invasive assessments of the association between hypochlorhydria and cholera, suggest that this non-invasive test may be useful in evaluating GAO in epidemiological field studies.

Neuroscience, 1990, 37(1), 77 - 100
Afferents to the median raphe nucleus of the rat: retrograde cholera toxin and wheat germ conjugated horseradish peroxidase tracing, and selective D-{3H}aspartate labelling of possible excitatory amino acid inputs; Behzadi G et al.; Afferents to the median-paramedian raphe nuclear complex, which contains the B8 serotonergic cell group, were investigated in the rat with neuroanatomical and transmitter-selective retrograde labelling techniques . Injection of sensitive retrograde tracers, cholera toxin genoid or wheat germ agglutinin conjugated horseradish peroxidase into the median raphe resulted in labelling of neurons in a large number of brain regions . Projections from 26 of these regions are supported by available orthograde tracing data; the cingulate cortex, bed nucleus of stria terminalis, medial septum and diagonal band of Broca, ventral pallidum, medial and lateral preoptic areas, lateral hypothalamus, dorsomedial nucleus of hypothalamus, lateral habenula, interpeduncular nucleus, substantia nigra, central (periaqueductal) gray, and laterodorsal tegmental nucleus seem to represent major sources of afferents to the median-paramedian raphe complex . Retrogradely labelled cells were also observed in a number of regions for which anterograde tracing data are not available, including the perifornical hypothalamic nucleus, ventral premammillary nucleus, supramammillary and submammillothalamic nuclei and the B9 area . Possible excitatory amino acid afferents were identified with retrograde D-{3H}aspartate labelling . Microinjection of D-{3H}aspartate at a low concentration, 10(-4) M in 50 nl, resulted in retrograde labelling of a limited number of median raphe afferents . The most prominent labelling was observed in the lateral habenula and the interpeduncular nucleus, but retrogradely labelled cells were also noted in the medial and lateral preoptic areas, lateral and dorsal hypothalamus, ventral tegmental area, laterodorsal tegmental nucleus, medial parabrachial nucleus, and the pontine tegmentum . After injections of 10(-3) M D-{3H}aspartate selective labelling also appeared in more distant afferent regions, including cells in cingulate cortex, and in some regions located at shorter distances, such as the supramammillary nucleus . Injections of D-{3H}aspartate at high concentration, 10(-2) M, resulted in the appearance of weakly to moderately labelled cells in most afferent areas which were devoid of labelled cells after injections of lower concentrations, suggesting that this labelling may be non-specific . It was concluded that the median-paramedian raphe receives afferents from a large number of forebrain and hypothalamic regions, while relatively few brain stem regions project to this nuclear complex . The selectivity of retrograde labelling with D-{3H}aspartate was found to be concentration dependent, and it is suggested that the connections showing high affinity for D-{3H}aspartate may use excitatory amino acids as transmitters . Excitatory amino acid inputs from lateral habenula and interpeduncular nucleus may play predominant roles in the control of ascending serotonergic and non-serotonergic projections originating in the median and paramedian raphe nuclei.

Int Arch Allergy Appl Immunol, 1990, 91(4), 348 - 53
Expression and distribution of Ia antigen in the murine small intestine . Influence of environment and cholera toxin; Wilson AD et al.; Density and distribution of Ia antigen in the small intestine of C57bl/6 mice showed substantial differences, depending on the environment in which they were reared and maintained . Isolator-reared mice expressed low levels of epithelial Ia antigen in comparison to mice reared in a conventional environment . Oral administration of 10 micrograms of cholera toxin had no effect on the level of epithelial Ia expression . We conclude, therefore, that the mechanism whereby cholera toxin is able to potentiate mucosal responses to fed antigens is not related to changes in the level of epithelial Ia expression.

Bull World Health Organ, 1990, 68(3), 303 - 12
Development of vaccines against cholera and diarrhoea due to enterotoxigenic Escherichia coli: memorandum from a WHO meeting; The erratic evolution of cholera therapy: from folklore to science; Division of Biology and Medicine, Brown University, Providence, Rhode IslandCholera is an exceptionally frightening epidemic disease because it kills its victims so very rapidly . The development of cholera therapy is traced from the early 19th century purges and bloodletting to the current use of oral rehydration solutions.

Ann Rech Vet, 1990, 21(2), 119 - 29
A blocking ELISA to differentiate hog cholera virus antibodies in pig sera from those due to other pestiviruses; Leforban Y et al.; The blocking ELISA technique was extended to comparative serology by using 3 different pestivirus strains: Hog cholera virus (HCV) Alfort strain propagated in PK15 cell line, Border disease virus (BDV) Aveyron strain in PK15 and BVD NADL** strain in fetal calf kidney (FCK) primary cells . Rabbit antisera to the Alfort HCV strain and Aveyron BDV strain were raised for use in the test . A bovine hyperimmune serum to BVD virus was also used for detecting antibodies specific to BVD virus . The ELISA was compared with the neutralisation test on various groups of field and experimetnal porcine sera . The results obtained with the ELISA were well correlated with the neutralisation test . Therefore the ELISA may be recommended as a differential serological test between HCV and other pestivirus antibodies in pig sera.

Cell Signal, 1990, 2(2), 139 - 51
Biphasic regulation of adenylate cyclase by cholera toxin in neuroblastoma x glioma hybrid cells is due to the activation and subsequent loss of the alpha subunit of the stimulatory GTP binding protein (GS); Macleod KG et al.; Exposure of neuroblastoma x glioma hybrid (NG108-15) cells to low concentrations of cholera toxin produced a stimulation of both basal and forskolin-amplified adenylate cyclase activity in membranes prepared from these cells . Higher concentrations of cholera-toxin reversed this effect . Mn2+ activation of adenylate cyclase indicated that this effect was not due to a modification of the intrinsic activity of this enzyme . Cholera toxin was demonstrated to produce a concentration and time-dependent loss of GS alpha from membranes of these cells . Loss of GS alpha from membranes of these cells was preceded by its ADP-ribosylation . The effects of cholera toxin were specific for GS alpha, as no alterations in levels of the pertussis toxin-sensitive G-proteins Gi2, Gi3 and Go, were noted in parallel . Equally, no alteration in levels of G-protein beta-subunit were produced by the cholera toxin treatment . These experiments demonstrate that cholera toxin-catalysed ADP-ribosylation does not simply maintain an activated population of GS at the plasma membrane and that alterations in levels of GS at the plasma membrane can modify adenylate cyclase activity.

J Mol Cell Cardiol, 1990 Jan, 22(1), 73 - 82
Improvement of cholera toxin-catalyzed ADP-ribosylation by endogenous ADP-ribosylation factor from bovine brain provides evidence for an unchanged amount of Gs alpha in failing human myocardium; Schnabel P et al.; The aim of the present study was to investigate whether or not alterations of Gs alpha can be detected with cholera toxin-induced ADP-ribosylation in myocardial membranes from patients with heart failure . Therefore, Gs alpha was radiolabeled by cholera toxin-catalzyed (32P)ADP-ribosylation with (32P)NAD as substrate . In membranes from left ventricular myocardium of six patients with dilated cardiomyopathy classified as NYHA IV and three samples from two non-failing donor hearts, labeling was too weak to allow detection of possible changes in the amount of Gs alpha . Therefore, the cytosolic small molecular weight G protein ARF (ADP-ribosylation factor), a cofactor for cholera toxin-induced ADP-ribosylation of Gs alpha, was partially purified from bovine cerebral cortex . ARF activity was quantified by its ability to enhance auto-ADP-ribosylation of cholera toxin A1-subunit . Gs alpha was identified by comparing the ADP-ribosylation patterns of myocardial membranes, membranes prepared from human leukemia (HL 60) and S 49 mouse lymphoma wild type cells (45 kDa-band present) with membranes of the Gs alpha-deficient S 49 variant cyc- (45 kDa-band missing) . In the presence of ARF, specific radiolabeling of the Mr 45,000 subtype of Gs alpha was markedly enhanced . The amounts of Gs alpha as measured by cholera toxin-dependent (32P)-ADP-ribosylation in the presence of ARR were similar in failing and nonfailing human hearts . It is concluded that factors other than Gs alpha are responsible for the altered regulation of the adenylate cyclase complex in heart failure . Moreover, by enhancing cholera toxin-catalyzed ADP-ribosylation, endogenous ADP-ribosylation factor from bovine brain appears to be a useful tool to study Gs alpha even in tissues in which the labeling of Gs alpha is rather weak.

Reg Immunol, 1990-91, 3(3), 131 - 8
Induction of an enteric Ig-response against ovalbumin and stimulation of the response by cholera toxin and its B-subunit in mice; Bianchi AT et al.; We induced ovalbumin (OA)-specific antibody responses in the murine small intestine and quantitated the responses by counting ovalbumin-specific antibody-containing cells (OACC) in the intestinal lamina propria, and by measuring anti-OA Ig titers in intestinal secretions . OA is a "weak" mucosal antigen and we could only induce intestinal anti-OA responses by intraperitoneal, primary injection of OA in water-in-oil emulsion and a mucosal booster with OA . Double staining of OACC for OA- and isotype-specificity demonstrated that 95% of all mucosal (intestinal) OACC produced IgA, whereas 95% of all systemic (splenic) OACC produced IgG . The intestinal anti-OA response could be stimulated by addition of cholera toxin (CT) or cholera toxin B-subunit (CTB) during the mucosal booster immunization . The stimulatory effect of CT did not depend on covalent coupling of CT and OA, while the stimulatory effect of CTB required covalent coupling of CTB with OA . Mucosal (intraduodenal) application of OA as such does not prime for a mucosal and systemic OACC response but induces tolerance . We demonstrated that coupling of OA with CT or CTB could overcome the inability of mucosal application of OA to prime for a mucosal OACC response . Although CT proved to be much more effective than CTB in stimulation of mucosal immune responses and possibly in prevention of tolerance induction, our results indicate that CTB is a useful (and safer) alternative.

Stain Technol, 1990, 65(6), 293 - 7
A sensitive method to quantitate gangliosides of the gangliotetraose series directly on chromatograms using peroxidase conjugated cholera toxin; Cambron LD et al.; A method is described whereby ganglioside GM1 can be quantitated directly on thin-layer chromatograms using cholera toxin subunit B conjugated to horseradish peroxidase and visualized with chloronaphthol . Overlay and color development were performed after separating gangliosides on nano-TLC plates, and fixing with polyisobutylmethacrylate . Absolute quantitation was realized using a Shimadzu CS-9000 integrating spectrodensitometer, scanning at 580 nm . A correlation coefficient of 0.98 was obtained in a linear range of detection from 10(-11) to 10(-16) moles . Statistical analysis revealed good reproducibility and over 99% of the added gangliosides remained with the chromatogram during all overlay and washing procedures . By comparison, standard chemical visualization by resorcinol-HCl was linear in the nanomole range with a detection limit of only 10(-10) moles . Since the carbohydrate portion of gangliosides immobilized in this manner is susceptible to the action of enzymes including neuraminidase, this technique can be applied to all structures of the gangliotetraose series.

Reg Immunol, 1990-91, 3(5), 217 - 22
Cholera toxin as a mucosal adjuvant: III . Antibody responses to nontarget dietary antigens are not increased; Nedrud JG et al.; The effect of cholera toxin, a potent mucosal adjuvant, upon the murine immune response to both soybean meal, a regular dietary antigen, and ovalbumin, a simulated dietary antigen, was examined . Mice were orally immunized with a target antigen, Sendai virus, with and without cholera toxin as a mucosal adjuvant . Both serum and intestinal IgA and IgG anti-Sendai virus antibody responses were significantly increased when cholera toxin was used . For soybean meal, the major protein source in laboratory animal chows, however, no increases in serum antibody titers were found in animals which received oral cholera toxin plus virus . In addition, in mice orally immunized with ovalbumin plus cholera toxin (and virus), both serum and intestinal IgA and IgG antibody titers against ovalbumin were increased . Oral pre-immunization with ovalbumin prior to oral ovalbumin plus cholera toxin, however, resulted in no increase in either serum or intestinal anti-ovalbumin antibody responses . Thus, the use of cholera toxin as an oral adjuvant does not increase antibody responses against dietary antigens if there has been pre-exposure to the dietary antigen.

J Dermatol Sci, 1990 Jan, 1(1), 7 - 13
Cholera toxin- and forskolin-induced cyclic AMP accumulations of pig skin (epidermis) . Modulation by chemicals which reveal the beta-adrenergic augmentation effect; Matsuo S et al.; Effects of cholera toxin and forskolin on pig epidermal adenylate cyclase system were investigated . Both agents increased cyclic AMP levels of epidermis . Marked accumulations were observed in the presence of cyclic AMP phosphodiesterase inhibitor, isobutylmethylxanthine (IBMX) . It has been known that hormone-stimulated adenylate cyclase responses are modified by various chemical treatments . Following long term incubation with hydrocortisone, Ro10-1670, and colchicine, the epidermal beta-adrenergic adenylate cyclase response was increased without the alteration of cyclic AMP phosphodiesterase activity . Adenosine-, and histamine-adenylate cyclase responses were unchanged by hydrocortisone treatment, and were decreased by Ro10-1670 and colchicine treatments . Following the long term incubation with these chemicals, effects of cholera toxin and forskolin were investigated . Colchicine-treated skin revealed the increased cholera toxin-, and forskolin-induced cyclic AMP accumulations . Neither hydrocortisone- nor Ro10-1670-treated skin revealed alterations of cholera toxin-, and forskolin-effect . The stimulatory effect of colchicine on the cholera toxin-, and forskolin-effect was observed at doses of the beta-adrenergic augmentation effect . Our results indicate that among the chemicals which reveal the beta-adrenergic augmentation effect, colchicine is unique in that it also increases cholera toxin-, and forskolin-induced cyclic AMP accumulations of epidermis.

Arch Virol, 1990, 111(3-4), 213 - 25
Identification of conserved epitopes on a hog cholera virus protein; Greiser-Wilke I et al.; Eight monoclonal antibodies directed against the hog cholera virus (HCV) strain Alfort/187 and displaying broad cross-reactivity with other HCV strains were characterized . An enzyme immunoassay on fixed monolayers of porcine or bovine cells infected with 14 different strains and isolates of HCV and 12 bovine viral diarrhea viruses (BVDV), respectively, showed that all antibodies reacted with HCV only . Seven antibodies recognized all HCV tested, thus indicating that they were directed against conserved epitopes . All antibodies neutralized the homologous strain and different patterns of the other HCV tested . Radioimmunoprecipitation analysis showed that the monoclonal antibodies were directed against a doublet of 56-60 kDa, presumably representing the major envelope glycoprotein of HCV . The results of reciprocal antibody blocking assays allowed the mapping of two distinct conserved antigenic domains on this protein.

Biochem Biophys Res Commun, 1989 Dec 15, 165(2), 554 - 60
Cholera toxin ADP-ribosylates the receptor-coupled form of pertussis toxin-sensitive G-proteins; Klinz FJ et al.; Cholera toxin catalyzes the ADP-ribosylation of 40 kDa pertussis toxin substrates in membranes from NG108-15 cells, which is increased in the presence of the opioid agonist DADLE . The basal ADP-ribosylation can be abolished by the opioid antagonist ICI 174864, suggesting that unoccupied opioid receptors interact spontaneously with the pertussis toxin substrates Gi/Go in the membrane . Treatment of NG108-15 cells with the opioid agonist DADLE leads to a reduction of agonist-stimulated and basal ADP-ribosylation of 40 kDa substrates catalyzed by cholera toxin . This indicates that the spontaneous interaction between opioid receptors and G-proteins is decreased in membranes of cells in which the receptor was desensitized by prolonged exposure to the agonist.

J Biol Chem, 1989 Dec 15, 264(35), 21394 - 400
Chemotactic peptide receptor-supported ADP-ribosylation of a pertussis toxin substrate GTP-binding protein by cholera toxin in neutrophil-type HL-60 cells; Iiri T et al.; A 40-kDa protein, in addition to the alpha-subunits of Gs (a GTP-binding protein involved in adenylate cyclase stimulation), was {32P}ADP-ribosylated by cholera toxin (CT) in the membranes of neutrophil-like HL-60 cells, only if formyl Met-Leu-Phe (fMLP) was added to the ADP-ribosylation mixture . The 40-kDa protein proved to be the alpha-subunit of Gi serving as the substrate of pertussis toxin, islet-activating protein (IAP) . No radioactivity was incorporated into this protein in membranes isolated from HL-60 cells that had been exposed to IAP . Gi-alpha purified from bovine brain and reconstituted into IAP-treated cell membranes was ADP-ribosylated by CT plus fMLP . Gi-alpha was ADP-ribosylated by IAP, but not by CT plus fMLP, in membranes from cells that had been pretreated with CT plus fMLP . When membrane Gi-alpha {32P}ADP-ribosylated by CT plus fMLP or IAP was digested with trypsin, the radiolabeled fragments arising from the two proteins were different from each other . These results suggest that CT ADP-ribosylates Gi-alpha in intact cells when coupled fMLP receptors are stimulated and that the sites modified by two toxins are not identical . CT-induced and fMLP-supported ADP-ribosylation of Gi-alpha was favored by Mg2+ and allow concentrations of GTP or its analogues but suppressed by GDP . The ADP-ribosylation did not occur at all, even in the presence of ADP-ribosylation factor that supported CT-induced modification of Gs, in phospholipid vesicles containing crude membrane extract in which Gi was functionally coupled to stimulated fMLP receptors . Thus, Gi activated via coupled receptors is the real substrate of CT-catalyzed ADP-ribosylation . This reaction may depend on additional factor(s) that are too labile to survive the process of membrane extraction.

Biochim Biophys Acta, 1989 Dec 14, 1014(3), 289 - 97
The activation of rabbit intestinal adenylate cyclase by cholera toxin; Longbottom D et al.; Brush-border and basal-lateral membranes were prepared from rabbit intestinal epithelial cells by differential centrifugation and MgCl2 precipitation . The ADP-ribosylation of proteins in these fractions when incubated with {adenylate-32P}NAD+ and cholera toxin was investigated . Three proteins of molecular mass 45, 40 and 37 kDa were labelled in a toxin-dependent manner in each membrane fraction . The incorporation of 32P-labelled ADP-ribose was 18-fold greater in brush-border membranes than in basal-lateral membranes, comparable to the enrichment of sucrase (marker enzyme for the brush border) in these membranes . There was a 20% release of the 40 and 45 kDa proteins from the brush-border membrane following this ADP-ribosylation . Activation of adenylate cyclase by both cholera toxin and sodium fluoride was 2.7- and 2.3-fold greater, respectively, in basal-lateral membranes than in brush-border membranes, comparable to the enrichment of Na+/K+-ATPase (marker enzyme for the basal-lateral membrane) in these membranes . The effect of sodium fluoride on membranes pretreated with cholera toxin revealed no increase in adenylate cyclase activity above that due to the toxin . This presumably means that both toxin and fluoride activate adenylate cyclase by the same regulatory protein . The results show that cholera toxin catalyzes the ADP-ribosylation of regulatory proteins in the brush-border membrane, and these proteins then migrate to the basal-lateral membrane where they activate the catalytic component of adenylate cyclase.

Infect Immun, 1989 Dec, 57(12), 3823 - 7
Comparative immunogenicities of Vi polysaccharide-protein conjugates composed of cholera toxin or its B subunit as a carrier bound to high- or lower-molecular-weight Vi; Szu SC et al.; The effect of molecular weight or size of the components on the immunogenicity of polysaccharide-protein conjugates prepared with the native Vi capsular polysaccharide (Vi) (approximately 3 x 10(3) kilodaltons) or lower-molecular-weight Vi (Vis; approximately 46 kilodaltons) abound to cholera toxin (CT) or to its B subunit (CTB) was studied . In mice, Vi-CT, Vi-CTB, and Vis-CTB elicited higher Vi antibody levels than the Vi alone (P less than 0.0001) . Vi-CT and Vi-CTB were more immunogenic than Vis-CTB (P less than 0.01) . CT or Vi-CT elicited higher levels of CT antibodies, as measured by enzyme-linked immunosorbent assay, than did CTB or Vi-CTB . In rhesus monkeys, the Vi conjugates elicited higher Vi antibody levels than the Vi alone (P less than 0.01) . Vi-CTB elicited higher levels of Vi antibody after each injection than did Vis-CTB . Similar levels of CT antibodies, as measured by enzyme-linked immunosorbent assay, were elicited by all three conjugates . In contrast, Vi-CT elicited higher levels of neutralizing antibodies than Vi-CTB or Vis-CTB when either CT or the related heat-labile toxin of Escherichia coli was used as the antigen . These results indicate that the holotoxin and the native Vi provide the most immunogenic components for conjugates designed to induce both Vi and CT antibodies.

Regul Pept, 1989 Dec, 26(3), 241 - 52
Effects of cholera toxin, Escherichia coli heat stable toxin and sodium deoxycholate on neurotensin release from the ileum in vivo; Eklund S et al.; Neurotensin (NT) is a biologically active peptide found in specialized epithelial cells (N-cells) in the distal small intestine . In this study we tested the hypothesis that NT may be released by luminal secretagogues, i.e., cholera toxin, Escherichia coli heat-stable toxin and sodium deoxycholate . Cholera toxin elicited net fluid secretion in anesthetized cats . This secretion was accompanied by an increased release of NT-like immunoreactivity (NTLI) into the mesenteric vein when NTLI was measured with either a C-terminally or a N-terminally directed antibody . An increasing plasma NTLI concentration (N-terminally directed antibody) was recorded in the mesenteric vein and femoral artery in cholera experiments . These results indicate that cholera toxin releases NT from the small intestine . Since neurotensin causes intestinal fluid secretion at least in part via an activation of enteric nerves we propose that the N-cell functions as a 'receptor cell' which activates an intramural secretory reflex upon luminal stimulation by cholera toxin . This study does not support a similar role for NT in the secretion elicited by the heat stable toxin of Escherichia coli or by sodium deoxycholate since we were unable to demonstrate any intestinal release of NTLI after exposing the intestine to these secretory agents.

Vaccine, 1989 Dec, 7(6), 503 - 5
Effectiveness of cholera toxin B subunit as an adjuvant for nasal influenza vaccination despite pre-existing immunity to CTB; Tamura S et al.; In a previous paper, it was shown that cholera toxin B subunit (CTB) augments the production of protective antibodies to influenza virus when CTB is inoculated intranasally into Balb/c mice together with influenza HA vaccine . The present study was designed to determine whether the effectiveness of CTB as a potent adjuvant for nasal vaccination could be limited by pre-existing immunity to CTB . Mice were sensitized by intranasal inoculation of either 1 or 0.05 microgram of CTB 2, 4 and/or 6 weeks before nasal vaccination . They were then vaccinated by either a single inoculation of vaccine together with 1 microgram of CTB or a two-dose regimen, composed of a primary inoculation of vaccine together with 0.05 microgram of CTB and a subsequent inoculation of vaccine alone . Levels of nasal IgA antibodies to CTB increased with the increase of the dose of CTB and the frequency of CTB-inoculation . Pre-existing immunity to CTB, however, did not significantly reduce the levels of both nasal IgA and serum haemagglutination-inhibiting (HI) antibodies to influenza virus and did not change the ability of the vaccinated mice to resist viral challenge . These results suggest that a relatively low dose of CTB could be inoculated repeatedly into animals as an adjuvant for nasal vaccination.

Eur J Immunol, 1989 Dec, 19(12), 2387 - 90
Cholera toxin modulates the T cell antigen receptor/CD3 complex but not the CD2 molecule and inhibits signaling via both receptor structures in the human T cell lymphoma Jurkat; Sommermeyer H et al.; The human T cell lymphoma Jurkat can be activated by stimuli directed either against the T cell antigen receptor-CD3 antigen complex (TcR/CD3) or the CD2 molecule . Stimulation of cells via the TcR/CD3-complex or via the CD2 molecule increases inositol phosphates and cytoplasmic free calcium . Pretreatment of Jurkat cells with cholera toxin leads to a decrease of TcR/CD3 expression on the surface of the cells, while the expression of CD2 is unaffected . In contrast to this distinct effect on the receptor expression, signaling via both pathways is inhibited by cholera toxin . The most convincing explanation for the cholera toxin-mediated inhibition of signaling is that cholera toxin interrupts the signaling pathways at a point where both, stimulation via TcR/CD3 and via CD2, use the same route . The earliest common point of the two signaling pathways, at least in the Jurkat cell line, seems to be the CD3 complex because after its down-regulation (and functional inactivation) both pathways of activation are interrupted.

J Immunol, 1989 Dec 1, 143(11), 3647 - 52
Cholera toxin inhibits resting human T cell activation via a cAMP-independent pathway; Anderson DL et al.; The catalytic subunit of cholera toxin (CT) can chemically modify the alpha polypeptides of certain G-binding proteins and thus alter their function . In order to study the involvement of CT-sensitive G proteins in T cell activation, we have utilized CT in an in vitro system in which purified, resting human peripheral T cells are activated by anti-CD3 antibodies and rIL-2 . Perturbation of the TCR/CD3 molecular complex by anti-CD3 antibodies causes changes in membrane phospholipids and induces a rise in cytoplasmic Ca2+ . These events, however, are insufficient to allow progression into cellular proliferation and addition of IL-2 is required . Under these conditions, treatment of cells with a low concentration of CT (2 ng/ml) causes a significant inhibition of the anti-CD3-induced calcium event as well as the anti-CD3 plus IL-2-stimulated proliferation . Under our experimental conditions, inhibition of both proliferation and intracellular Ca2+ elevation by CT requires the involvement of the TCR/CD3 complex . This is supported by the observation that the toxin does not inhibit either the proliferation triggered by ionomycin and PMA or the Ca2+ influx induced by the ionophore . These data suggest that in TCR/CD3-mediated T cell activation CT acts at a point between TCR/CD3 perturbation and the generation of intracellular Ca2+ . In view of the ability of CT to activate the alpha subunit of the G protein that stimulates adenyl cyclase (G alpha s), it is possible that the effect of CT on T cells is secondary to intracellular elevation of cAMP . However, measurement of cAMP levels both early after CT addition and at later time points, when proliferation is maximal, reveals lack of cyclic nucleotide accumulation . The presented data are consistent with the interpretation that the CT-mediated inhibition is caused by the modification of a G-binding protein that is either directly or indirectly associated with triggering of T cells via the TCR/CD3 molecular complex . The data also suggest that this protein is not G alpha s and it probably represents an as yet unidentified moiety or one of the several G proteins that have been recently described as regulators of phospholipase C activation.

Am J Physiol, 1989 Nov, 257(5 Pt 1), C920 - 5
Effects of cholera toxin on gene expression in brown preadipocytes differentiating in culture; Herron D et al.; To investigate the cellular control of the recruitment process in brown adipose tissue, the ability of cholera toxin to influence the differentiation of brown preadipocytes developing in culture was investigated . Stromalvascular cells obtained from the brown adipose tissue of 3-wk-old rats were grown in culture for 6-7 days in the presence or absence of cholera toxin . It was found that cholera toxin treatment decreased the expression of the actin gene (indicating an increased degree of differentiation), while at the same time promoting the expression of the genes coding for the mitochondriogenesis marker cytochrome-c oxidase and for the adipocyte conversion marker lipoprotein lipase (all followed at the mRNA level) . Chronic cholera toxin treatment also increased the total amount of protein per cell in culture, and a specific cholera toxin-induced 35-kDa protein was identified . It was concluded that (in contrast to the case suggested for white preadipocytes) cholera toxin treatment of brown preadipocytes may not only affect the activity of catabolic enzymes but may also directly promote the differentiation process, indicating that this process is under beta-adrenergic control in the adapting animal.

Immunology, 1989 Nov, 68(3), 319 - 24
Cholera toxin-induced fluid secretion in rat gut ligated loops: influence of bile from normal or cholera toxin-immunized rats; Pierre PG et al.; Fresh normal rat bile premixed with cholera toxin (CT) did not significantly affect the CT-induced fluid accumulation in rat jejunal ligated loops . Bile from rats intrajejunally (i.j.) immunized three times with CT definitely inhibited CT-induced fluid secretion . Bile duct ligature (BDL) for 1-4 days in unimmunized rats, in contrast with mice, did not significantly affect subsequent CT-elicited fluid secretion in their ligated loops . BDL for 4 days in rats i.j . immunized with CT, only slightly decreased the CT-neutralizing ability of their gut loops . Passive transfer during 24 hr of bile from i.j.-immunized rats, but not from normal rats, into gut of normal recipient rats with BDL, significantly protected loops made in such recipients . The affinity-purified antibodies of immune bile, mixed with CT, neutralized its effect . Our data show that, unlike mice, rat bile acids are not required for expression of the CT effect in gut loops . In addition, bile from i.j.-immunized rats contains enough anti-CT antibodies to be protective on its own, but is not necessary for substantial gut protection against CT in i.j.-immunized BDL rats . Our results confirm a major and complementary role of both biliary and intestinal secretory IgA antibodies in protection of the rat gut mucosa against CT-induced fluid secretion.

Berl Munch Tierarztl Wochenschr, 1989 Nov 1, 102(11), 378 - 81
{Molecular characterization of the hog cholera virus}; Thiel HJ et al.; The molecular biology of hog cholera virus (HCV) was studied . After reverse transcription of the viral RNA the cDNA was cloned in the expression vector lambda gt11 . HCV clones were identified using antibodies as a probe . Partial sequencing of one HCV-derived cDNA clone revealed a high degree of homology to a portion of the bovine viral diarrhea virus (BVDV) . One goal of these studies is the preparation of defined, effective and safe vaccines against pestiviruses.

Vet Microbiol, 1989 Nov, 21(1), 9 - 20
Antigenic differentiation of pestivirus strains with monoclonal antibodies against hog cholera virus; Wensvoort G et al.; Thirty-one bovine viral diarrhoea virus (BVDV) or border disease virus (BDV) strains and 94 hog cholera virus (HCV) strains were grown in cell culture, and characterized by immunostaining with 13 monoclonal antibodies (MAbs) and one polyclonal serum (PAb) against HCV . All 125 strains were recognized by the PAb . None of the BVDV or BDV strains were detected by the 13 MAbs . Seven MAbs detected all 94 HCV strains . Six other MAbs detected heterogeneity among and within HCV strains . The MAbs are useful tools in differentiating between HCV and BVDV infections in pigs, and can also be used to differentiate infections induced by HCV field strains from infections induced by the "Chinese" strain of vaccine virus.

Br J Pharmacol, 1989 Nov, 98(3), 717 - 20
Cholera and pertussis toxins amplify prostacyclin synthesis in aortic smooth muscle cells; Demolle D et al.; Pretreatment of bovine aortic smooth muscle cells in culture with pertussis toxin (PT) or cholera toxin (CT) potentiated the synthesis of prostacyclin (PGI2) induced by 5-hydroxytryptamine (5-HT) and phorbol-12-myristate, 13-acetate (PMA) . The production of PGI2 by explants from the bovine aortic media was also synergistically stimulated by 5-HT and CT, whereas PT was inactive . These data are consistent with the hypothesis that guanosine 5'-triphosphate binding proteins are directly involved in the control of phospholipases which release free arachidonic acid for prostaglandin synthesis.

J Gen Virol, 1989 Nov, 70 ( Pt 11), 2865 - 76
Topographical and functional mapping of epitopes on hog cholera virus with monoclonal antibodies; Wensvoort G; Competitive binding studies and antigen capture assays were done with monoclonal antibodies (MAbs) raised against hog cholera virus (HCV) to map the corresponding epitopes . A model was constructed in which the 13 epitopes were situated in four distinct antigenic domains: A, B, C and D . Domain A was subdivided into A1, A2 and A3 . The functional relevance of this model was assessed by the characterization of pestivirus strains, by neutralization studies with the MAbs, and by isolation of variants that escaped neutralization . The topographical arrangement of the epitopes, as constructed in the model, was corroborated by the functional assays . The MAbs that defined domains A1 and A2 recognized all 94 HCV strains tested . Domains A3, B, C and D varied among the HCV strains . Neutralization was observed with MAbs defining domains A1, B and C . Synergistic neutralization occurred using MAbs against domains A1 and B, and A1 and C, but not within the domains . With MAbs defining A1, B or C, variants could be isolated that escaped neutralization and immunostaining by these MAbs.

Biochim Biophys Acta, 1989 Oct 9, 1013(3), 206 - 11
Inhibition of murine leukemia (WEHI-3B and L1210) proliferation by cholera toxin B subunit; Pessina A et al.; Cholera holotoxin produces both stimulation and inhibition of the growth of different cell populations . These opposite effects were both attributed to the enzymatic activity of the subunit A that activates adenylate cyclase, increasing the intracellular level of cAMP . We observed that the B subunit of cholera toxin produced by itself an inhibition of the 'in vitro' growth of two murine leukemia cell lines (L1210 limphoid leukemia and WEHI-3B myelomonocytic leukemia) . The sensitivity of WEHI-3B cells towards cholera toxin was about 5000-times higher than that of the L1210 cells, whereas the two leukemias showed an identical sensitivity to the B subunit (IC50 = 5.10(-10) M for L1210 and 10(-10) M for WEHI-3B) . The inhibition produced by the B subunit was neutralized by GM1 and in a minor degree by type II gangliosides . The two leukemias showed a remarkable difference in their gangliosides contents (L1210 cells contained GM1 (80.6%) and GM2 (19.4%), while WEHI-3B cells contained GM1 (28.2%), Fuc-GM1 (44.9%) and a band (26.9%) with a chromatographic mobility between GD1a and GD1b) . The inhibition could be explained by a competitive mechanism between the B subunit and some autocrine factor binding GM1-containing receptors . Our data strengthen the suggestion to consider gangliosides as very important pleiotropic biomodulators.

J Biol Chem, 1989 Oct 5, 264(28), 16512 - 7
Inhibition of protein kinase C-dependent cellular proliferation by interaction of endogenous ganglioside GM1 with the B subunit of cholera toxin; Spiegel S; In quiescent Swiss 3T3 fibroblasts, the B subunit of cholera toxin, a protein which binds specifically to ganglioside GM1 on the cell surface, stimulates DNA synthesis and potentiates the effects of several other growth factors such as insulin, epidermal growth factor, bombesin, and even unfractionated serum . In contrast to its synergistic effect with other known growth factors, the B subunit markedly inhibited DNA synthesis induced by the phorbol ester, 12-O-tetradecanoyl-phorbol 13-acetate (TPA) . The inhibitory effect of the B subunit was observed even in the presence of insulin, which greatly potentiates the mitogenic response to TPA or the B subunit . In contrast to the effect of the B subunit, calcium ionophores and cholera toxin stimulated DNA synthesis induced by TPA . The antagonism between the B subunit and TPA is not simply due to their abilities to modify their mutual binding sites or known effector systems . TPA did not block the early rise in cytosolic free calcium in response to the B subunit, and conversely, the B subunit did not modify the ability of TPA to activate protein kinase C . However, in protein kinase C-deficient cells, the antagonistic effect between TPA and the B subunit was abolished . In addition, there was no indication for the involvement of a pertussis toxin-sensitive G protein in the antagonism . Maximum inhibition was found when the B subunit was added 2 h after the addition of TPA . Significant inhibition was still evident when the time of addition of the B subunit was delayed until 6 h after the addition of TPA . This suggests that the cross-talk between signal transduction induced through endogenous gangliosides and protein kinase C is a late step in mitogenesis.

J Pharmacol Exp Ther, 1989 Oct, 251(1), 71 - 6
The role of serotonin in the canine secretory response to cholera toxin in vivo; LaRosa CA et al.; This study was initiated to evaluate the role of serotonin in cholera toxin-induced jejunal secretion of water and electrolytes . Chronic Thiry-Vella loops, constructed in six dogs, were perfused with an isosmotic neutral perfusate containing {14C}polyethylene glycol as the recovery marker . Fluxes of water, sodium, chloride and potassium were calculated and immunoreactive serotonin levels were measured in blood and effluent perfusates . Intraluminal application of 20 micrograms of cholera toxin induced secretion; fluxes of water (basal, 32.3 +/- 11.1; 6 hr, -541 +/- 35 microliter/min), sodium (basal, 9.0 +/- 2.8; 6 hr, -78.3 +/- 5.6 microEq/min), chloride (basal, 3.8 +/- 1.5; 6 hr, -65.7 +/- 4.0 muEq/min) and potassium (basal, 0.10 +/- 0.08; 6 hr, -2.80 +/- 0.18 muEq/min) were all significantly different from basal . Serum electrolytes remained normal, except that potassium fell from 4.9 +/- 0.5 to 3.9 +/- 0.2 mEq/l . Although circulating serotonin levels did not change from base line (180.9 +/- 29.3 ng/ml), effluent concentrations increased significantly from 68.2 +/- 4.6 to 81.1 +/- 5.0 ng/ml (at 3 hr) and jejunal outputs increased from 136.6 +/- 10.2 to 205.1 +/- 10.1 ng/min (at 6 hr) . In a separate set of experiments, verapamil was infused i.v . (12.5 micrograms/kg/min) during the 4th hr in four dogs exposed to cholera toxin . The lower dose of toxin (5 micrograms) induced secretion which was unaffected by the calcium channel blocker . In another series of studies, ketanserin (a 5-HT2 receptor blocker) was infused i.v . at 33 micrograms/kg/min during the 4th hr in four additional dogs exposed to the lower dose of cholera toxin . This potent serotonin antagonist failed to inhibit cholera toxin-induced jejunal secretion.(ABSTRACT TRUNCATED AT 250 WORDS)

Trop Geogr Med, 1989 Oct, 41(4), 377 - 82
Cholera epidemic and natural disasters; where is the link; Siddique AK et al.; In May 1985, a cyclone and tidal surge devastated Sandwip, an island off the Southern Corner of Bangladesh . Within one week after this disaster a cholera epidemic broke out . It resulted in 12,194 registered cases and 51 deaths . The factors contributing to the occurrence of the epidemic are analyzed.

J Med Assoc Thai, 1989 Oct, 72(10), 583 - 8
Several sporadic outbreaks of El Tor cholera in Sunpathong, Chiang Mai, September-October, 1987; Swaddiwudhipong W et al.; From September through October 1987, a cholera outbreak involving 59 cases of biotype El Tor, serotype Inaba occurred in Sunpathong district, Chiang Mai . No cases died . Twenty-seven cases were males and 32 were females . The age ranged between 4 months and 85 years, with a median of 36 years . The outbreak affected 7 small communities, and showed different vehicles of infection . Six housewives and one girl were infected with cholera in the first localized outbreak . The transmission of infection appeared due to the consumption of packed food contaminated by an infected food handler . In the second localized outbreak, 6 young males acquired cholera after eating uncooked fish harvested from a canal contaminated with cholera organisms . Another outbreak of cholera with 24 culture-confirmed cases occurred among guests at a funeral held in one rural village . The source of infection was traced to uncooked pork contaminated from an infected butcher: Early detection of infected persons, rapid identification of possible vehicles of transmission, and prompt implementation of control measures effectively curtailed the extension of these outbreaks.

J Cell Sci, 1989 Sep, 94 ( Pt 1), 33 - 42
Induction of DNA synthesis by cholera toxin in the temperature-sensitive cell-cycle mutants of rat 3Y1 fibroblasts at a restrictive temperature; Kabemura M et al.; Four temperature-sensitive mutants of rat 3Y1 fibroblasts representing separate complementation groups (3Y1tsD123, 3Y1tsF121, 3Y1tsG125 and 3Y1tsH203) are arrested at a restrictive temperature of 39.8 degrees C mainly with a G1-phase DNA content (temperature arrest) . Cholera toxin (CT) (3 micrograms ml-1) induced DNA synthesis at 39.8 degrees C in the temperature-arrested cultures of two mutants (3Y1tsD123 and 3Y1tsG125) . This effect of CT was not mimicked by other agents known to elevate the cellular level of cyclic AMP, such as dibutyryl-cyclic AMP, prostaglandin E1 and forskolin, suggesting that the elevation of cellular cyclic AMP level per se is not responsible for the induction of DNA synthesis by CT . Addition of the B subunit of CT to the temperature-arrested cultures of 3Y1tsD123 and 3Y1tsG125 did not induce DNA synthesis at 39.8 degrees C, indicating that the binding of CT to the cell surface alone is insufficient for the induction . The CT-treated cell membrane fraction prepared from temperature-arrested 3Y1tsG125 cells had similar activity for {32P}ADP-ribosylation of the 45 X 10(3) Mr protein to that prepared from cells proliferating at a permissive temperature of 33.8 degrees C . All these results suggest that 3Y1tsG125 cells utilize a CT-responsive signal transduction pathway, different from adenylate cyclase cascade, for preparation for entry into S phase in the temperature-arrested 3Y1tsG125.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochem J, 1989 Sep 1, 262(2), 643 - 9
Cholera toxin treatment produces down-regulation of the alpha-subunit of the stimulatory guanine-nucleotide-binding protein (Gs); Milligan G et al.; The effect of activation of the alpha-subunit(s) of the stimulatory guanine-nucleotide-binding protein, Gs, on levels of this polypeptide(s) associated with the plasma membrane of L6 skeletal myoblasts was ascertained . Incubation of these cells with cholera toxin led to a time- and concentration-dependent 'down-regulation' of both 44 and 42 kDa forms of Gs alpha as assessed by immunoblotting with an anti-peptide antiserum (CS1) able to identify the extreme C-terminus of Gs . The effect of cholera toxin was specific for Gs; levels of Gi alpha in membranes of cholera toxin-treated cells were not different from untreated cells . Down-regulation of Gs was absolutely dependent upon prior ADP-ribosylation, and hence activation of Gs and was not mimicked by other agents which elevate intracellular levels of cyclic AMP . Pretreatment with pertussis toxin, which catalyses ADP-ribosylation of Gi but not of Gs, did not down-regulate either Gi or Gs, demonstrating that covalent modification by ADP-ribosylation is alone not a signal for removal of G-proteins from the plasma membrane.

Immunol Lett, 1989 Sep, 22(3), 227 - 34
Receptor binding-like specificity of two anti-cholera toxin monoclonal antibodies detected by latex agglutination immunoassay; Lucas GP et al.; Six mouse monoclonal IgG antibodies (mAbs) reacting with the cholera toxin B-subunit (CT-B) were obtained from the spleen cells of mice immunized with CT . They were divided into two groups according to their different reactivities with CT in latex (Lx) agglutination . One group of two mAbs was called "non-repetitive" (NR) because they apparently recognized a single epitope on CT, which was unable to agglutinate Lx coated with these NRmAbs, in contrast with the other group of four mAbs that we called "repetitive" (RmAbs) . The two NRmAbs, but not the four RmAbs, failed to react with CT when CT was bound first to its GM1-receptor . Thus, the NRmAbs seemed directed at an epitope close to the GM1-binding site of CT-B . These NRmAbs could represent valuable immunogenic candidates for anti-idiotypic immunization against CT and related enterotoxins.

Science, 1989 Aug 25, 245(4920), 857 - 9
Role of prostaglandins and cAMP in the secretory effects of cholera toxin; Peterson JW et al.; The role of adenosine 3',5'-monophosphate (cAMP) and prostaglandins in the pathogenesis of experimental cholera was evaluated . Fluid accumulated in the rabbit intestinal loop model after 16 hours of incubation with cholera toxin, prostaglandin E1, or prostaglandin E2, but not with membrane-permeable derivatives of cAMP or forskolin . Dibutyryl cAMP triggered a small, transient intestinal fluid accumulation response by 4.5 hours; however, the fluid was completely absorbed by 9 hours . After exposure of intestinal loops to cholera toxin, prostaglandin E was released into the intestinal lumen in a concentration-dependent manner independent of cAMP . Thus, not only cAMP, but also prostaglandins may regulate water and electrolyte secretion in cholera.

Nippon Naibunpi Gakkai Zasshi, 1989 Aug 20, 65(8), 743 - 9
{Role of a guanine nucleotide regulatory protein in exocrine pancreatic secretion--effects of cholera toxin and pertussis toxin on cholecystokinin action}; Matozaki T et al.; We have recently shown that F- can mimic the actions of cholecystokinin (CCK) on amylase release, Ca2+ mobilization and inositol phosphate generation in pancreatic acinar cells . We have concluded, therefore, that pancreatic CCK receptors may be coupled to the activation of polyphosphoinositide hydrolysis by a guanine nucleotide regulatory protein (N protein), which seems to be sensitive to F- . In the present study, in order to further characterize this N protein coupled to pancreatic CCK receptors, we have examined the effects of bacterial toxins, pertussis toxin (PT) and cholera toxin (CT) on both CCK- and NaF-induced cellular responses in isolated rat pancreatic acini . Neither PT or CT pretreatment of acini affected both CCK- and NaF-stimulated increases in intracellular Ca2+ concentration monitored by quin2 . Furthermore, pretreatments of acini with PT and CT didn't alter the effects of CCK on inositol phosphate generation in acini . Similarly, NaF-induced inositol phosphate generation was not changed by these toxin treatments . However, pretreatment procedures employed in this study were considered to catalyze complete ADP-ribosylation of alpha-subunit of the stimulatory (Ns) and inhibitory (Ni) N protein . These results, therefore, strongly suggest that a N protein coupling pancreatic CCK receptors to the breakdown of polyphosphoinositide may be distinct from Ns or Ni like protein.

Vaccine, 1989 Aug, 7(4), 314 - 20
Protection against influenza virus infection by a two-dose regimen of nasal vaccination using vaccines combined with cholera toxin B subunit; Tamura S et al.; The effectiveness of the two-dose regimen, composed of a primary intranasal inoculation of influenza A-type virus HA vaccine together with B subunit of cholera toxin (CTB) and the subsequent intranasal inoculation of vaccine alone 4 weeks later, was examined . In mice given a relatively high dose of virus A/PR/8/34 (PR-8, H1N1) HA vaccine (1.5 micrograms) both as a primary antigen with CTB (1 microgram) and as the second antigen, the secondary responses of both antiviral IgA antibodies in nasal wash and haemagglutination-inhibiting (HI) antibody in serum were much higher than those of primary responses and persisted for greater than 12 weeks after the second inoculation . Even in mice that received reduced amounts of a primary vaccine (0.03 microgram) {prepared from virus PR-8, A/Yamagata/120/86 (H1N1) or A/Fukuoka/C29/85 (H3N2)} together with reduced amounts of CTB (0.05 microgram), the subsequent inoculation of PR-8 vaccine produced both nasal IgA and serum HI antibodies and provided complete protection against homologous A-type virus (PR-8) infection . Moreover, the combination of the reduced amounts of heterologous A-type virus vaccine (A/Yamagata or A/Fukuoka) with CTB for primary inoculation and the secondary heterologous A-type virus vaccine {A/Yamagata, A/Kumamoto/37/79 (H1N1), or A/Fukuoka} resulted in high levels of cross-reactive IgA antibodies and partial cross-protection against PR-8 infection . On the other hand, a second inoculation of B/Ibaraki/2/85 vaccine failed to produce cross-reactive antibodies and to protect against PR-8 infection.(ABSTRACT TRUNCATED AT 250 WORDS)

Clin Sci (Lond), 1989 Aug, 77(2), 161 - 6
An investigation of the role of possible neural mechanisms in cholera toxin-induced secretion in rabbit ileal mucosa in vitro; Moriarty KJ et al.; 1 . Cholera toxin stimulates intestinal secretion in vitro by activation of mucosal adenylate cyclase . However, it has been proposed that cholera toxin promotes secretion in vivo mainly through an indirect mechanism involving enteric neural reflexes . 2 . We examined this hypothesis further by studying the influence of neuronal blockade on cholera toxin-induced changes in fluid transport across rabbit ileum in vitro . Mucosa, stripped of muscle layers, was mounted in flux chambers and luminal application of crude cholera toxin (2 micrograms/ml) caused a delayed but sustained rise in the short-circuit current, electrical potential difference and Cl- secretion . Pretreatment with the nerve-blocking drug, tetrodotoxin (5 x 10(-6) mol/l serosal side), failed to influence the secretory response to cholera toxin, and addition of tetrodotoxin at the peak response to cholera toxin also had no effect . 3 . That tetrodotoxin could block neurally mediated secretagogues was confirmed by the demonstration that the electrical responses to neurotensin (10(-7) mol/l and 10(-8) mol/l) were blocked by tetrodotoxin (5 x 10(-6) mol/l) . Furthermore, the response to cholera toxin of segments of ileum, which included the myenteric, submucosal and mucosal nerve plexuses, was not inhibited by tetrodotoxin . 4 . We conclude that cholera toxin-induced secretion in rabbit ileum in vitro is not mediated via a neurological mechanism.

Virology, 1989 Aug, 171(2), 555 - 67
Molecular cloning and nucleotide sequence of the genome of hog cholera virus; Meyers G et al.; A cDNA clone derived from genomic RNA of hog cholera virus (HCV) was identified using an oligonucleotide complementary to the RNA encoding a hexapeptide from the putative RNA-dependent RNA polymerase of the closely related bovine viral diarrhea virus (BVDV) . This clone served as a probe for screening different size-selected cDNA libraries . After molecular cloning and nucleotide sequencing the HCV genome was shown to consist of 12,284 nucleotides containing one long open reading frame . Sequence comparison revealed a high degree of homology between HCV and BVDV genomic RNAs . With respect to HCV the genome of BVDV contains an insertion coding for 90 amino acids.

J Trop Med Hyg, 1989 Aug, 92(4), 290 - 4
Gastric emptying of oral rehydration solutions in acute cholera; Collins BJ et al.; Gastric emptying of rice powder electrolyte solution and of glucose electrolyte solution was measured by a marker dilution double sampling technique in 14 and in 16 adult patients respectively after intravenous rehydration during an attack of acute cholera . Six patients who received rice powder electrolyte solution and seven who received glucose electrolyte solution re-attended for a repeat study with the same test meal 16 days later, when fully recovered from cholera . No differences in gastric emptying patterns of the two electrolyte solutions were observed, either in the acute or in the recovered patients . Similarly, gastric emptying of both solutions was rapid during acute cholera and comparable to that observed in recovered patients . This study indicates that gastric emptying is not impaired in acute cholera and that the rate of emptying of oral rehydration solutions is adequate to account for their observed clinical efficacy in fast purging patients with acute cholera.

Mol Cell Endocrinol, 1989 Aug, 65(1-2), 27 - 33
The actions of forskolin, cholera toxin and iloprost on casein secretion by lactating doe mammary glands; Ollivier-Bousquet M; The secretagogue effects of prolactin (PRL) and of various agents acting on cAMP levels, forskolin, cholera toxin and iloprost (a stable analogue of prostaglandin I2) have been assessed in lactating doe mammary gland fragments in vitro . Forskolin (10 microM), cholera toxin (1 microgram/ml) and iloprost (10 mM) stimulated milk casein secretion . The effects of forskolin (10 microM) and cholera toxin (1 microgram/ml) were potentiated by PRL (10 micrograms/ml) . Conversely, the action of iloprost (10 microM) was not amplified by PRL (10 micrograms/ml) . Forskolin (10 microM) and cholera toxin (1 microgram/ml) stimulated the intracellular accumulation of cAMP . Neither PRL nor iloprost, at concentrations which stimulated casein secretion, modified the accumulation of cAMP . These results demonstrate that PRL does not act directly by any increase in intracellular cAMP levels . However, stimulating effects of forskolin and cholera toxin on casein secretion and intracellular cAMP levels suggest that various transduction signals are effective in the mammary cells.

Proc Natl Acad Sci U S A, 1989 Aug, 86(16), 6101 - 5
Molecular cloning, characterization, and expression of human ADP-ribosylation factors: two guanine nucleotide-dependent activators of cholera toxin; Bobak DA et al.; ADP-ribosylation factors (ARFs) are small guanine nucleotide-binding proteins that enhance the enzymatic activities of cholera toxin . Two ARF cDNAs, ARF1 and ARF3, were cloned from a human cerebellum library . Based on deduced amino acid sequences and patterns of hybridization of cDNA and oligonucleotide probes with mammalian brain poly(A)+ RNA, human ARF1 is the homologue of bovine ARF1 . Human ARF3, which differs from bovine ARF1 and bovine ARF2, appears to represent a newly identified third type of ARF . Hybridization patterns of human ARF cDNA and clone-specific oligonucleotides with poly(A)+ RNA are consistent with the presence of at least two, and perhaps four, separate ARF messages in human brain . In vitro translation of ARF1, ARF2, and ARF3 produced proteins that behaved, by SDS/PAGE, similar to a purified soluble brain ARF . Deduced amino acid sequences of human ARF1 and ARF3 contain regions, similar to those in other G proteins, that are believed to be involved in GTP binding and hydrolysis . ARFs also exhibit a modest degree of homology with a bovine phospholipase C . The observations reported here support the conclusion that the ARFs are members of a multigene family of small guanine nucleotide-binding proteins . Definition of the regulation of ARF mRNAs and of function(s) of recombinant ARF proteins will aid in the elucidation of the physiologic role(s) of ARFs.

FEBS Lett, 1989 Jul 31, 252(1-2), 88 - 90
Homologous sequences in cholera toxin A and B subunits to peptide domains in myelin basic protein; Caamano CA et al.; Recent reports that myelin basic protein (MBP) can be ADP-ribosylated and contains specific sites that bind GTP and GM1 ganglioside, have suggested an analogy to the properties of cholera toxin . Comparisons of pairs of sequences between these two proteins yielded two regions of homology between MBP and the cholera toxin B (chol B) subunit, and one region of homology with the cholera toxin A (chol A) subunit . The matching sites within chol B consisted of a 17 amino acid residue sequence (residues 30-46 in chol B and residues 102-118 in human-MBP, hMBP, p less than 0.0007) and an 11 residue span (residues 31-41 in chol B and sequence 29-39 in hMBP, p less than 0.0004) . The homologous site within chol A corresponded to an 11 residue span (residues 130-140 in chol A and 67-77 in hMBP sequence, p less than 0.00007) . Since portions of the cholera toxin sequence are virtually identical to sections of the sequence in E . coli toxin, the homology is also valid for the same sequences in this toxin . The highly antigenic behavior of MBP that is related to the induction of experimental allergic encephalomyelitis may be paralleled by comparable neural pathology from the homologous regions of cholera toxin.

J Immunol, 1989 Jul 15, 143(2), 458 - 63
Cholera toxin-sensitive and insensitive signaling via surface Ig; Warner GL et al.; We have observed that a 2-h pretreatment of murine B cells with cholera toxin (CT) renders the B cell incapable of receiving an activation signal via surface Ig as measured by cell volume increase and entry into the S phase of the cell cycle . In contrast, CT pretreatment does not inhibit the delivery of a signal by IL-4, as measured by increase in cell volume . In fact, CT pretreated B cells are able to respond to anti-Ig in the presence of IL-4, as measured by both an increase in cell size and entry into S suggesting that IL-4 overcomes the effects of CT on normal B cell activation . Despite blocking the anti-Ig-mediated entry into the cell cycle, CT was not able to interfere with the induction of nonresponsiveness by anti-Ig in normal B cells or with the delivery of growth-inhibitory signal to the B cell lymphoma WEHI-231 . These results suggest that there are two signaling pathways mediated by cross-linking of surface Ig: one pathway sensitive and the other insensitive to modulation by CT.

J Immunol, 1989 Jul 15, 143(2), 484 - 90
Cholera toxin as a mucosal adjuvant . Glutaraldehyde treatment dissociates adjuvanticity from toxicity; Liang XP et al.; Cholera toxin (CT), either mixed with or conjugated to unrelated protein Ag, is known to enhance the intestinal IgA response of rodents toward the unrelated Ag . Although relatively low doses of CT exert this gut mucosal adjuvant effect, the inherent toxicity of CT is a hindrance to its use in humans . Our report demonstrates that CT treated with 20 mM glutaraldehyde retains adjuvant properties but exhibits more than 1000-fold lower toxicity than untreated toxin . Glutaraldehyde was also used in a one-stage conjugation procedure to couple CT covalently to Sendai virus . Again, toxicity was reduced more than 1000-fold . This drop in toxicity is consistent with an observed 100-fold loss in binding capacity of the CT B subunit and a 20- to 50-fold reduction in adenylate cyclase activation by the CT A subunit . Oral administration of this virus-toxoid conjugate resulted in increased gut antiviral IgA titers compared with oral administration of either virus alone or of virus mixed with glutaraldehyde-treated toxin . This marked decrease in toxicity may afford a practical approach for the use of CT as a mucosal adjuvant.

FEBS Lett, 1989 Jul 3, 250(2), 536 - 40
CD3/T-cell receptor coupling to a pertussis and cholera toxin-insensitive G-protein; Kvanta A et al.; We have analyzed the effect of CD3/T-cell receptor stimulation on GTP hydrolysis and GTP binding . We show that stimulation of Jurkat, T-cell, membranes with OKT3 results in a 50% increase in GTP hydrolysis which is specifically inhibited by GDP . Pretreatment of the membranes with neither pertussis toxin nor cholera toxin inhibited the GTP hydrolysis . We also show that stimulation with OKT3 increases the binding of GTP gamma S to Jurkat membranes . These data strongly implicate the involvement of a G-protein in CD3/T-cell receptor signalling.

J Membr Biol, 1989 Jul, 109(1), 21 - 8
Interaction of the B subunit of cholera toxin with endogenous ganglioside GM1 causes changes in membrane potential of rat thymocytes; Mulhern SA et al.; The fluorescent anionic dye, bisoxonol, and flow cytometry have been used to monitor changes in the membrane potential of rat thymocytes exposed to the B subunit of cholera toxin . The B subunit induced a rapid hyperpolarization, which was due to activation of a Ca2+-sensitive K+ channel . Reduction of extracellular Ca2+ to less than 1 microM by the addition of {ethylene-bis(oxyethylenenitrilo)}tetraacetic acid immediately abolished the hyperpolarization caused by the B subunit . Cells treated with quinine and tetraethylammonium lost their ability to respond to the B subunit, whereas 4-aminopyridine did not have any effect . Thus, calcium-sensitive and not voltage-gated K+ channels appeared to be responsible for the hyperpolarization . The results of ion substitution experiments indicated that extracellular Na+ was not essential for changes in membrane potential . Further studies with ouabain, amiloride and furosemide demonstrated that electrogenic Na+/K+ ATPase, Na+/H+ antiporter and Na+/K+/Cl- cotransporter, respectively, were not involved in the hyperpolarization process induced by the B subunit . Thus, crosslinking of several molecules of ganglioside GM1 on the cell surface of rat thymocytes by the pentavalent B subunit of cholera toxin modulated plasma membrane permeability to K+ by triggering the opening of Ca2+-sensitive K+ channels . A role for gangliosides in regulating ion permeability would have important implications for the function of gangliosides in various cellular phenomena.

Reg Immunol, 1989 Jul-Aug, 2(4), 244 - 8
Cholera toxin as a mucosal adjuvant for respiratory antibody responses in mice; Liang XP et al.; Cholera toxin was investigated as an adjuvant for anti-virus antibody responses in the respiratory mucosa of mice . Two methods of applying cholera toxin were evaluated: oral administration and intranasal administration . Oral immunization with Sendai virus in the presence of cholera toxin effectively primed for respiratory anti-viral antibody responses, whereas oral immunization with Sendai virus alone was ineffective in this respect . In nasal washes, IgA was the predominant anti-viral antibody enhanced by oral cholera toxin; in bronchoalveolar washes, the enhanced anti-viral antibodies included IgG, IgA, and IgM . Effects of direct administration of cholera toxin to the respiratory mucosa on respiratory anti-viral antibody responses depended on the method of anesthesia used during immunization . With inhalation anesthesia (ether), cholera toxin had no adjuvant effect on respiratory antibody responses to coadministered Sendai virus . In contrast, under parenteral anesthesia (i.e., intraperitoneal ketamine), mice which received cholera toxin and Sendai virus via the respiratory tract showed significantly higher anti-viral IgA and IgG antibody titers in nasal washes and IgG antibody in bronchoalveolar washes than mice which received the virus only.

Res Microbiol, 1989 Jul-Aug, 140(6), 393 - 404
Erythrocyte receptors for cholera and heat-labile enterotoxins of Escherichia coli; Ricci LC et al.; Many serological reactions using red blood cells (RBC) such as radial immune haemolysis (RIH) and indirect haemagglutination (IH) tests have often been used for the detection of cholera toxin (CT) and heat-labile (LT) enterotoxin produced by porcine and human Escherichia coli strains . In these tests, the enterotoxins bind to sheep, bovine and guinea-pig RBC without any ligand . We studied several factors which might interfere with such binding, as well as the nature of the receptors involved . Treatment of erythrocytes with different enzymes revealed that proteolytic enzymes had no effect on the adsorption of enterotoxins to RBC . Conversely, treatment with neuraminidase increased the adsorption . Experiments carried out with delipidized RBC revealed that none of the enterotoxins under study bound to the cells thus treated . Pre-incubation of ganglioside fractions with the enterotoxins blocked RIH and IH reactions and the biological effect of them on Vero cells . Assaying RBC ganglioside fractions by thin-layer chromatography revealed the presence of GM1 . Our results suggest that the receptors for GT and LT enterotoxins in sheep, bovine and guinea pig RBC are gangliosides: mainly GM1.

Virology, 1989 Jul, 171(1), 18 - 27
Hog cholera virus--characterization of specific antiserum and identification of cDNA clones; Rumenapf T et al.; A specific antiserum was raised against the pestivirus inducing hog cholera (hog cholera virus, HCV) . Using immunoprecipitation and SDS-PAGE, this antiserum served for comparison of HCV-induced proteins with those from a related and better characterized pestivirus, bovine viral diarrhea virus (BVDV) . In addition to immunological relationships, the apparent molecular weights of some proteins induced by both viruses were quite similar . HCV genomic RNA was found to be about 12 kb in length, comparable to BVDV RNA . cDNA was synthesized starting from RNA isolated from partially purified virions and cloned in lambda gt11 . Screening with the antiserum resulted in identification of several positive clones . Partial sequencing of one HCV-derived cDNA clone revealed a high degree of homology to a portion of the BVDV sequence.

Biochemistry, 1989 Jun 13, 28(12), 5019 - 24
Thermodynamics of intersubunit interactions in cholera toxin upon binding to the oligosaccharide portion of its cell surface receptor, ganglioside GM1; Schon A et al.; The binding and the energetics of the interaction of cholera toxin with the oligosaccharide portion of ganglioside GM1 (oligo-GM1), the toxin cell surface receptor, have been studied by high-sensitivity isothermal titration calorimetry and differential scanning calorimetry . Previously, we have shown that the association of cholera toxin to ganglioside GM1 enhances the cooperative interactions between subunits in the B-subunit pentamer {Goins, B., & Freire, E . (1988) Biochemistry 27, 2046-2052} . New experiments presented in this paper reveal that the oligosaccharide portion of the receptor is by itself able to enhance the intersubunit cooperative interactions within the B pentamer . This effect is seen in the protein unfolding transition as a shift from independent unfolding of the B promoters toward a cooperative unfolding . To identify the origin of this effect, the binding of cholera toxin to oligo-GM1 has been measured calorimetrically under isothermal conditions . The binding curve at 37 degrees C is sigmoidal, indicating cooperative binding . The binding data can be described in terms of a nearest-neighbor cooperative interaction binding model . In terms of this model, the association of a oligo-GM1 molecule to a B protomer affects the association to adjacent B promoters within the pentameric ring . The measured intrinsic binding enthalpy per protomer is -22 kcal/mol and the cooperative interaction enthalpy -11 kcal/mol . The intrinsic binding constant determined calorimetrically is 1.05 x 10(6) M-1 at 37 degrees C and the cooperative Gibbs free energy equal to -850 cal/mol.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochim Biophys Acta, 1989 Jun 6, 981(2), 200 - 6
Cholera toxin action on rabbit renal brush-border membranes inhibits phosphate transport; Al-Mahrouq HA et al.; Cholera toxin was used to enhance ADP-ribosylation of rabbit renal brush-border membranes . Treatment of brush-border membrane sheets with cholera toxin in the presence of NAD resulted in a specific inhibition of the initial phase of Na+-dependent Pi uptake, compared to controls incubated with NAD alone . The Pi uptake was determined after conversion of the membrane sheets to vesicles . The equilibrium uptake of Pi, the Na+-independent uptake of Pi, the Na+-dependent uptake of L-proline and the activities of several brush-border membrane enzymes were not changed . The inhibition of Pi transport was dependent on the presence of both NAD and cholera toxin . Incubation of membrane sheets with {3H}NAD produced acid-stable binding of radioactivity to the membranes and the binding was increased 5-fold by the presence of cholera toxin . The use of {32P}NAD and autoradiography confirmed that the bound radioactivity was associated with several different membrane proteins, and that cholera toxin increased binding to these proteins including three that were not labelled in the absence of the toxin . The specific inhibitory action of cholera toxin on Na+/Pi cotransport is probably mediated by ADP-ribosylation of membrane proteins, suggesting that the Pi transport system can be regulated by ADP-ribosylation, at least in vitro.

J Immunol, 1989 Jun 1, 142(11), 3781 - 7
Cholera toxin promotes B cell isotype differentiation; Lycke N et al.; Cholera toxin (CT) is a powerful oral immunogen and adjuvant that elicits strong IgG and IgA antibody responses . In our study we investigated whether this property of CT was associated with an effect on B cell isotype differentiation . Initially, we determined the effect of CT on normal LPS-induced Peyer's patch B cells and found that whereas CT is strongly inhibitory of IgM production, it increases by approximately three-fold the number and frequency of IgG- and IgA-producing cells . Subsequently, using cell sorting technology, we demonstrated that CT acts on membrane (m)IgM+, mIgG/mIgA- B cells rather than mIgG/mIgA+ B cells . In addition, we showed that CT does not cause selective inhibition of mIgM, or enhancement of mIgG/mIgA B cell proliferation . In parallel studies we determined the effect of CT on the differentiation of a clonal B cell population, CH12.LX cells, i.e., a population comprised mainly of mIgM+ cells (98%) admixed with a small subpopulation of mIgA+ cells (2%) . Here we found that CT (in the absence of LPS) causes a rapid decrease (24 h) in the intensity of mIgM expression as well as a marked increase in the size of the subpopulation expressing mIgA . In addition, we found that CT (in the presence of LPS), inhibits CH12.LX IgM production while increasing the absolute number and frequency of IgA-producing cells . In contrast, CT inhibits IgA production by CH12.LX.A2 cells, a subclone of CH12.LX cells that bears only IgA . Finally, we demonstrated that CT is equally inhibitory of the proliferation of CH12.LX cells and CH12.LX.A2 cells . Taken together, these effects of CT on normal B cells and a clonal B cell line indicate that CT induces substantial numbers of mIgM+ cells to undergo isotype differentiation into mIgG+ or mIgA+ B cells . In a final series of studies we showed that the effect of CT on isotype differentiation was mimicked by the B subunit of CT, i.e., the subunit that does not activate intracellular adenylate cyclase; thus the induction of isotype differentiation by CT is not mediated by a perturbation in cAMP level.

Vaccine, 1989 Jun, 7(3), 257 - 62
Enhancement of protective antibody responses by cholera toxin B subunit inoculated intranasally with influenza vaccine; Tamura SI et al.; Effects of the B subunit of cholera toxin (CTB) on the primary antibody responses to influenza virus A/PR/8/34 (PR-8) (H1N1) HA vaccine and on protection against viral challenge were investigated in Balb/c mice which were immunized intranasally with both the vaccine and CTB . The dose of CTB (greater than or equal to 1 microgram) inoculated with the vaccine (greater than or equal to 0.15 microgram) induced high responses of both antiviral IgA antibodies in the nasal wash and haemagglutinin-inhibiting (HI) antibody in the serum, enough to provide complete protection against viral challenge four weeks after immunization . High levels of antibody were maintained for more than 16 weeks after inoculation, affording complete protection during this interval . The inoculation of HA vaccine prepared from influenza viruses A/Yamagata/120/86 (H1N1) or A/Fukuoka/C29/85 (H3N2) together with CTB provided partial protection against PR-8 infection, with production of antiviral IgA antibodies which were cross-reactive to PR-8 antigens whereas immunization with CTB and HA vaccine prepared from a different type of influenza virus (B/Ibaraki/2/85) failed to protect against PR-8 infection . These results indicate that CTB can produce an augmented and persistent antibody response to PR-8 HA vaccine, which is cross-protective to other A-type virus infections . The mechanisms by which CTB enhances the protective antibody responses to the nasally inoculated vaccine were investigated . The ability of CTB to augment antibody responses was lost, either when CTB was inoculated via the intravenous or subcutaneous route, or when CTB was introduced into nasal site one day before or after the vaccine inoculation.(ABSTRACT TRUNCATED AT 250 WORDS)

Naunyn Schmiedebergs Arch Pharmacol, 1989 Jun, 339(6), 704 - 5
Inhibition of cholera toxin-induced intestinal secretion by the 5-HT3 receptor antagonist ICS 205-930; Buchheit KH; The secretory effect of cholera toxin on the gut has been ascribed to the activation of submucosal 5-hydroxytryptamine receptors by 5-hydroxytryptamine released from the enterochromaffin cells . The hypothesis that neuronally located 5-HT3 receptors are involved in cholera toxin-induced diarrhoea has now been tested . Intestinal secretion was stimulated in mice by oral administration of pure cholera toxin and the effects of ICS 205-930, a potent and selective antagonist at 5-HT3 receptors, and of ketanserin, a compound that blocks 5-HT2 receptors, were investigated . Oral administration of cholera toxin resulted in a significant accumulation of fluid in the intestine . Pretreatment of the animals with ICS 205-930 partly prevented this effect and a maximal reduction of about 50% was achieved with doses of 0.3 mg/kg i.p . and higher . Ketanserin had only minimal effects up to a high dose of 1 mg/kg i.p . when a 20% reduction of fluid accumulation was seen . The data support the view that activation of 5-HT3 receptors plays a major role in the secretory effect of cholera toxin in the gut.

Scand J Immunol, 1989 Jun, 29(6), 739 - 45
Adjuvant effect of cholera toxin on the mucosal immune response to soluble proteins . Differences between mouse strains and protein antigens; Wilson AD et al.; We examined the effect of orally administered cholera toxin (CT) on the immune response to keyhole limpet haemocyanin (KLH) and ovalbumin (OVA) in high (C57Bl/6 H2b), medium (CBA H2k), and low (BALB/c H2d) responder I-A haplotypes to CT . Mice were fed OVA or KLH on three occasions at 10-day intervals and the effect of simultaneous feeding of CT determined . Isotype-specific antibody levels were assayed in serum samples collected 7 days after the last immunization . Antibody was also measured in the supernatant of gut explant cultures incubated at either 4 or 37 degrees C . Increased antibody levels in cultures kept at 37 degrees C indicated release of local intracellular antibody . Cholera toxin exerted an adjuvant effect on the mucosal response of all three strains to KLH and OVA . Overall, the responses to CT and the second protein were not correlated; we interpret these findings to indicate that while CT had an effect on the mucosal immune system which enhances the immune response to itself and other protein antigens, the final outcome of the response to the second antigen is dependent on differences in the ability of the strains to process, recognize, and respond to a particular antigen.

J Comp Neurol, 1989 May 8, 283(2), 285 - 302
Monoaminergic, peptidergic, and cholinergic afferents to the cat facial nucleus as evidenced by a double immunostaining method with unconjugated cholera toxin as a retrograde tracer; Fort P et al.; Using a sensitive double immunostaining technique with unconjugated cholera-toxin B subunit as a retrograde tracer, the authors determined the nuclei of origin of monoaminergic, peptidergic, and cholinergic afferent projections to the cat facial nucleus (FN) . The FN as a whole receives substantial afferent projections, with relative subnuclear differences, from the following areas: 1) the perioculomotor areas, the contralateral paralemniscal region, and the mesencephalic reticular formation dorsal to the red nucleus; 2) the ipsilateral parabrachial region and the nucleus reticularis pontis, pars ventralis; and 3) the nuclei reticularis parvicellularis, magnocellularis, ventralis, and dorsalis of the medulla . In addition, the present study demonstrated that the lateral portion of the FN receives specific projections from the contralateral medial and olivary pretectal nuclei and the ipsilateral reticular formation of the pons . It was also found that the FN receives: 1) serotoninergic inputs mainly from the nuclei raphe obscurus, pallidus, magnus, and the caudal ventrolateral bulbar reticular formation; 2) catecholaminergic afferent projections from the A7 noradrenaline cell group located in the Kolliker-Fuse, parabrachialis lateralis, and locus subcoeruleus nuclei; 3) methionin-enkephalin-like inputs originating in the pretectal complex, the nucleus paragigantocellularis lateralis and the caudal raphe nuclei; 4) substance P-like afferent projections mainly from the Edinger-Westphal complex and the caudal raphe nuclei; and 5) cholinergic afferents from 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 physiological significance of FN inputs relevant to tonic and phasic events occurring at the level of the facial musculature during the period of paradoxical sleep in the cat.

Biochem J, 1989 May 1, 259(3), 679 - 84
The effect of cholera toxin on the inhibition of vasopressin-stimulated inositol phospholipid hydrolysis is a cyclic AMP-mediated event at the level of receptor binding; Gardner SD et al.; Incubation of L6 skeletal myoblasts for 16 h with cholera toxin but not with pertussis toxin, led to the inhibition of inositol phosphate generation induced by subsequent exposure to vasopressin . The effects of the toxin on inositol lipid metabolism were accompanied by the total ADP-ribosylation of the available cholera-toxin substrates within the cells . Immunological analysis demonstrated that the two polypeptides modified in vivo by cholera toxin were different forms of Gs alpha (alpha subunit of Gs) . No novel cholera-toxin substrate(s) were detected . The cholera-toxin-mediated inhibition of vasopressin-stimulated inositol phosphate generation could be mimicked by both forskolin and dibutyryl cyclic AMP, but not by the separated subunits of the toxin . Receptor-binding studies demonstrated that the inhibition of agonist-stimulated inositol phosphate generation was accompanied by a decrease in cell-surface vasopressin-binding sites, with no effect on the affinity of these for the hormone . We suggest that the effect of cholera toxin and agents which increase intracellular cyclic AMP on vasopressin-stimulated inositol lipid hydrolysis is an effect on receptor number, and that there is no requirement to postulate a role for a novel G-protein, which is a substrate for cholera toxin, in the regulation of inositol phospholipid metabolism.

Oncogene, 1989 May, 4(5), 659 - 63
Identification of the pertussis and cholera toxin substrates in normal and N-ras transformed NIH3T3 fibroblasts and an assessment of their involvement in bombesin-stimulation of inositol phospholipid metabolism; Milligan G et al.; Cholera and pertussis toxin-sensitive G-proteins were examined using specific immunological probes in wild type NIH3T3 cells and in clones of these cells containing the N-ras gene attached to a promotor where expression either was (T15+) or was not (T15-) induced . The major pertussis toxin sensitive-polypeptide had the immunological characteristics of Gi2 . Two distinct forms of Gs alpha (45 and 42 kDa) were identified . Long term over-expression of p21N-ras (T15+ cells) did not alter the levels of Gi2 alpha or of Gs alpha . Pretreatment of NIH3T3 or T15 cells with either pertussis toxin or cholera toxin led to the complete in situ ADP-ribosylation of the respective G-proteins . Modification of Gi2 by pertussis toxin, however, had no inhibitory effect on the ability of bombesin to stimulate the production of inositol phosphates in any of these cells lines . Treatment of these cells with cholera toxin elicited a potent inhibition of the bombesin-stimulated production of inositol phosphates . This could be mimicked, however, by other agents which increase intracellular cyclic AMP concentrations . Cholera toxin treatment did not produce a significant alteration in the number of bombesin receptors on the cell surface . These results suggest that, in the T15 cell line, enhanced coupling of bombesin receptors to a phospholipase C-mediated hydrolysis of inositol phospholipids is either produced directly by p21N-ras or that overexpression of this gene product leads to the enhanced expression or function of a cholera and pertussis toxin-insensitive G-protein which then mediates the effect.

J Immunol, 1989 May 1, 142(9), 3206 - 12
Subcellular distribution and membrane association of human neutrophil substrates for ADP-ribosylation by pertussis toxin and cholera toxin; Volpp BD et al.; Neutrophil guanine nucleotide-binding proteins are important components of receptor-mediated cellular responses such as degranulation, chemotaxis, and superoxide production . Because the cytoplasmic granules of neutrophils serve as an intracellular store of receptors and NADPH oxidase components, we investigated the subcellular distribution of substrates for ADP-ribosylation by both pertussis and cholera toxins . Cholera toxin substrates of Mr 43 and 52 kDa were present only in the plasma membrane fraction . A 39-kDa pertussis toxin substrate was present in the plasma membrane, cytosol, and a specific granule-enriched fraction . There were no substrates for either toxin in the primary granules . Quantitative GTP-gamma-5 binding was localized predominantly to the plasma membrane fraction (47%), but significant portions were found in the specific granule-enriched fractions (13%) and cytosol (34%) as well . Two-dimensional gel electrophoresis and chymotryptic digests of the pertussis toxin substrate from these three subcellular fractions suggested that they are highly homologous . Triton X-114 phase partitioning was used to investigate the hydrophobicity of the toxin substrates . The pertussis toxin substrates in the plasma membrane and granule fractions behaved like integral membrane proteins, whereas the cytosolic substrate partitioned into both lipophilic and aqueous fractions . ADP-ribosylation converted the substrates to a somewhat less lipophilic form . These data suggest that the specific granules or an organelle of similar density serve as an intracellular store of a G protein with a 39-kDa alpha-subunit and that the cytosolic fraction of neutrophils contains free alpha-subunits of the same size.

Biochemistry, 1989 Apr 18, 28(8), 3360 - 5
Interactions of antibody aromatic residues with a peptide of cholera toxin observed by two-dimensional transferred nuclear Overhauser effect difference spectroscopy; Anglister J et al.; The interactions between a peptide of cholera toxin and the aromatic amino acids of the TE33 antipeptide antibody, cross-reactive with the toxin, have been studied by NOESY difference spectroscopy . The 2D difference between the NOESY spectrum of the Fab with a 4-fold excess of the peptide and that of the peptide-saturated Fab reveals cross-peaks growing with excess of the peptide . These cross-peaks are due to magnetization transfer between the Fab and neighboring bound peptide protons, and a further transfer to the free peptide protons by exchange between bound and free peptide (transferred NOE) . Additional cross-peaks appearing in the difference spectrum are due to a combination of intramolecular interactions between bound peptide protons and exchange between bound and free peptide . Assignment of cross-peaks is attained by specific deuteration of antibody aromatic amino acids using also the resonance assignment of the free peptide, deduced from the COSY spectrum of the peptide solution . The antibody combining site is found to be highly aromatic . We have identified one or two histidine, two tyrosine, and two tryptophan residues and one phenylalanine residue of the antibody interacting with valine-3, proline-4, glycine-5, glutamine-7, histidine-8, and aspartate-10 of the peptide . The 2D TRNOE difference spectroscopy can be used to study protein-ligand interactions, given that the ligand off rate is fast relative to the spin-lattice relaxation time of the protein and ligand protons (about 1 s) . The resolution obtained in the difference spectra implies that the technique is equally applicable for studying proteins having a molecular weight larger than 50,000.(ABSTRACT TRUNCATED AT 250 WORDS)

J Biol Chem, 1989 Apr 5, 264(10), 5352 - 7
Cholera toxin induces cAMP-independent degradation of Gs; Chang FH et al.; Cholera toxin stimulates adenylyl cyclase by catalyzing ADP-ribosylation of the alpha chain (alpha s) of Gs, a guanine nucleotide binding regulatory protein . In a rat pituitary cell line, GH3, the toxin-induced increase in GTP-dependent adenylyl cyclase activity is maximal at 1 h; adenylyl cyclase remains elevated for at least 32 h . Surprisingly, cholera toxin also induces a 74-95% decrease in the amount of immunoreactive alpha s in the same cells, as assessed on immunoblots probed with either of two antisera directed against separate alpha s peptide sequences . The decrease in immunoreactive alpha s, which begins after 1 h of toxin treatment and is complete by 8 h, is accompanied by a comparable decrease in the amount of biochemically active alpha s, as assessed by its ability to complement the biochemical defect of alpha s-deficient S49 cyc- membranes . Cholera toxin induces similar decreases in alpha s in wild type S49 lymphoma cells, in S49 kin- mutants, which lack cAMP-dependent protein kinase, and in S49 H21 a mutants, in which alpha s is unable to assume an active conformation upon binding GTP . The toxin-induced decrease in alpha s is somewhat temperature-dependent, but is not blocked by agents that increase lysosomal pH or by colchicine, which promotes breakdown of microtubules . alpha s in detergent-solubilized GH3 membranes is susceptible to proteolysis by an endogenous protease; this susceptibility is markedly increased in membranes from cells previously exposed to cholera toxin for 1 h . Taken together, these results suggest that cholera toxin-induced covalent modification of alpha s marks the protein for accelerated degradation . In addition, the persistence of elevated GTP-dependent adenylyl cyclase activity despite loss of a substantial fraction of alpha s suggests that the amount of alpha s membranes is greater than the amount necessary for maximal activation of cAMP synthesis by cholera toxin.

J Pharmacobiodyn, 1989 Apr, 12(4), 216 - 9
Noradrenaline release enhanced by cholera toxin and pertussis toxin in rat cerebral cortical slices; Qi SB et al.; The effects of cholera and pertussis toxin on the release of noradrenaline (NA) were studied in the rat cerebral cortex . The cerebral cortical slices were incubated with cholera or pertussis toxin for 2 h, subsequently loaded with 1-{3H}NA and superfused continuously . The pretreatment with cholera toxin significantly (p less than 0.05) enhanced the 20 mM KCl-evoked {3H}NA release . In contrast to a significant (p less than 0.05) increase in {3H}NA release by pertussis toxin, neither A-protomer nor B-oligomer of the toxin could affect the release . These results suggest that cholera and pertussis toxin-substrates, probably guanosine triphosphate-binding proteins, could be involved in the NA release in the central nervous system.

Clin Exp Immunol, 1989 Apr, 76(1), 111 - 6
Systemic delayed-type hypersensitivity to cholera toxin and a detoxified derivative; Kay RA et al.; The delayed-type hypersensitivity (DTH) response to cholera toxin (CT) and an immunopurified formalinized toxoid (TD) was studied in adult BALB/c mice . Intradermal injection of CT in non-immune animals produced substantial footpad oedema which interfered with the measurement of specific DTH but TD proved satisfactory as a challenge antigen . DTH to CT was induced by doses ranging from 0.1 to 10 micrograms, in complete Freund's adjuvant (CFA) . The optimal reaction was primed for by 1 microgram CT . Doses of TD with an equivalent B subunit content were equally capable of inducing DTH as the holotoxin . The time-course of the footpad swelling revealed that the DTH response was biphasic . Histological examination showed that the initial swelling was due to tissue oedema and the subsequent increase in footpad thickness was associated with a mononuclear cell infiltrate at the site of antigen challenge . Adoptive transfer experiments were used to demonstrate that the DTH was antigen-specific and could be passively transferred by immune effector T cells . This is the first study to demonstrate an effector T cell response to CT . A role for T cell reactions in the intestinal mucosa must now be examined as a potential contributory mechanism in the prevention of choleraic diarrhoea.

Infect Immun, 1989 Apr, 57(4), 1137 - 41
Adoptive transfer of gut mucosal antitoxin memory by isolated B cells 1 year after oral immunization with cholera toxin; Lycke N et al.; A protocol was elaborated for the adoptive transfer of lymphocytes from mice which were orally immunized with cholera toxin (CT) to enable the study of long-term gut mucosal immunological memory at the single-cell level . Mesenteric lymph node (MLN) cells were transferred 1 year after priming immunizations, and recipient animals were challenged perorally on days 1 and 2 with CT before sacrifice on day 6 to 7 following transfer of cells . Strong antitoxin ELISPOT spot-forming cell (SFC) responses were recorded in spleens, MLN, and laminae propriae (LP) of recipient mice . In contrast, no SFC were found in Peyer's patches . The magnitude of the response equaled that of the acute response seen after optimal oral CT immunization and was directly dependent on the number of transferred cells . The memory antitoxin response in MLN and LP required oral challenge with CT as opposed to the spleen SFC response, which could also be triggered by intravenous challenge with antigen . Spleen cells from mice immunized perorally with CT were as effective as MLN cells in transferring immunological memory detectable in the gut immune system . Irrespective of the tissue source of transferring immunological memory detectable in the gut immune system . Irrespective of the tissue source of the memory cells, the isotype distribution of the antitoxin SFC response in recipient mice was similar with predominantly immunoglobulin A (96%) in LP and immunoglobulin G (66%) in MLN and spleen . Transfer of antitoxic memory was completely abrogated by treatment of the cells with J11d monoclonal antibody and complement prior to their injection into recipient mice by was unaffected by treatment with anti-Thy-1.2 antibody and complement, suggesting that long-term gut mucosal memory is carried by B cells . Antitoxin B memory cells might help explain the long-term protection against recurrent disease seen in convalescents from cholera in cholera-endemic areas.

J Dairy Sci, 1989 Apr, 72(4), 892 - 9
Estrogen and progesterone augment growth responsiveness of mammary tissue to cholera toxin; Sheffield LG; Second (thoracic) mammary glands of endocrine intact mice were removed intact and incubated in Dulbecco's Modified Eagle's Medium supplemented with insulin, aldosterone, and cholera toxin . Insulin and aldosterone resulted in relatively little mammary development . However, insulin, aldosterone, and cholera toxin substantially increased mammary development, as assessed by development scores and DNA after 6 d of culture . Ovariectomy abolished the ability of cholera toxin to augment mammary development in vitro . Estradiol and progesterone injections for 3 d partly restored responsiveness of mammary tissue to cholera toxin, whereas responsiveness was greater after 6 d of injection than in endocrine-intact mice . Additionally, cyclic AMP-dependent protein kinase (kinase A) activity of fourth (inguinal) mammae was increased after as little as 3 d of estradiol and progesterone treatment . Cholera toxin induced phosphorylation of at least one protein was also increased by estradiol and progesterone . Because cholera toxin is a potent activator of adenylate cyclase, these findings suggest that estradiol and progesterone interact with cyclic AMP active agents to promote mammary development . This interaction may be mediated, at least in part, by increased kinase A activity and increased kinase A substrate availability.

Gastroenterol Clin Biol, 1989 Apr, 13(4), 383 - 7
{Response to cholera toxin of 2 epithelial intestinal cell lines . Effect of Saccharomyces boulardii}; Czerucka D et al.; Cholera toxin acts in vivo by activating intestinal adenylate cyclase . This study was designed to determine (1) whether normal rat epithelial intestinal cell lines (IRD 98 and IEC 17) respond to cholera toxin (CT) by an increased concentration of cyclic AMP and (2) whether the yeast Saccharomyces boulardii, which reduced CT-induced secretion of water and electrolytes using the isolated jejunal loop technique, has an effect on these models . The cAMP concentration evaluated in cells exposed to Saccharomyces boulardii and to cholera toxin (1 microgram/ml for 90 min) was compared to the concentration of cAMP obtained in control cells without yeast . Prior exposure of IRD 98 and IEC 17 cells to Saccharomyces boulardii, reduced CT-induced cAMP by 50 p . 100 . This effect disappeared after destruction of the yeast by heating . Results show that the IRD 98 and IEC 17 cells are good models for in vitro investigation of the effects of cholera toxin . Our results suggests that Saccharomyces boulardii prevents the water and electrolyte secretion induced by cholera toxin.

J Cell Biochem, 1989 Apr, 39(4), 391 - 400
Cholera toxin inhibits the increase in cytoplasmic free calcium induced via the CD2 pathway of human T-lymphocyte activation; Nunes J et al.; We investigated the action of cholera toxin on the intracellular ionized calcium {Ca2+}i increase induced by anti-CD2 and anti-CD3 monoclonal antibodies in the leukemic human T-cell line Jurkat . Cholera toxin inhibits in a dose-dependent manner these two pathways of human T-lymphocyte activation but with different half maximal inhibition doses (75 ng/ml for CD3, 30 ng/ml for CD2) . This effect cannot be accounted for only by the increase in cAMP induced by cholera toxin because forskolin, which raises cellular cyclic adenosine monophosphate (cAMP) to the same levels, induced only a small inhibition of the {Ca2+}i increase in similar conditions . Cholera toxin induced a decrease in the surface expression of the CD3 molecule, suggesting a down-regulation of the CD3 molecules . On the other hand, the expression of CD2 remained unchanged . Cell surface disappearance of the CD3 molecule cannot account for all the inhibitory effects of cholera toxin because CD2 molecule expression was not affected (no modifications in the half maximal binding of anti-CD2 monoclonal antibodies) . All together, these results suggest that cholera toxin acts on substrates, possibly G proteins, that could regulate the {Ca2+}i increase induced by anti-CD2 and anti-CD3 mAbs in Jurkat cells . In addition, the present study demonstrated that the rise in cellular cAMP partially inhibits the {Ca2+}i increase induced by anti-CD2 and anti-CD3 mAbs.

Leukemia, 1989 Apr, 3(4), 289 - 93
Cholera toxin resistance associated with deficient adenylate-cyclase activity in a subclone of the rat promyelocytic leukemia (BNML); Hermouet S et al.; The rat promyelocytic leukemia cell line BNML is highly sensitive to cAMP elevating agents, and to cholera toxin (CT) in particular: 99.9% of the cells are killed in less than 48 hr of toxin treatment . We described here a subclone of the same leukemia, which, in contrast, is completely resistant to CT but still sensitive to other cAMP inducers . This locates the defect responsible for CT resistance at the membrane, somewhere between surface CT receptors and adenylate cyclase . CT-resistant BNML cells (CTR-BNML) do have surface CT receptors (several thousands per cell) . Adenylate cyclase activity in CTR-BNML cells is not stimulated by cholera toxin . Other GS mediated stimulation of adenylate cyclase (by PGE, isoproterenol, histamine, NaF) remains relatively high, though 25-60% lower than in CTS-BNML cells . These results suggest that a specific adenylate cyclase defect is involved in the resistance of CTR-BNML cells to cholera toxin.

Biochem Biophys Res Commun, 1989 Mar 15, 159(2), 707 - 12
Neutralization of cholera toxin by rat IgA secretory antibodies induced by a free synthetic peptide; Delmas A et al.; Secretory immunoglobulin A (sIgA) is the major immunoglobulin in the bile of several species . They contribute to local immune defences of the gut . The protection against cholera toxin (CT) is due to the presence of specific sIgA in the bile and in the gut . We have already reported that oral administration of the peptide corresponding to the sequence 50-75 of cholera toxin B subunit elicits serum antibodies neutralizing CT activity, and that IgA and local protection are observed in the intestine of P50-75 orally immunized mice . In this study, we demonstrate the potential of this synthetic peptide as immunogen without carrier or adjuvant, not only in a strain known to be sensitive to CT, but also in an outbred one . Furthermore, this peptide stimulates the mucosal immunity, since we show that P50-75 induced-sIgA purified from rats bile and serum, are capable of neutralizing CT activity in the in vivo intestinal ligated loop test.

Am J Trop Med Hyg, 1989 Mar, 40(3), 320 - 2
Human depredation by vampire bats (Desmodus rotundus) following a hog cholera campaign; McCarthy TJ; Hog cholera control efforts in Belize in 1975 included the slaughter of village pigs, a primary food source for the vampire bat (Desmodus rotundus) . The bats then fed on secondary food sources, including humans . In 1 village, 22% of the families interviewed were exposed to attacks: 17 children and 2 adults were bit . Human depredation was not continuous as Desmodus located other hosts.

Pediatr Res, 1989 Mar, 25(3), 225 - 7
Development of intestinal host defense: an increased sensitivity in the adenylate cyclase response to cholera toxin in suckling rats; Seo JK et al.; To determine if developmental variations existed in the second messenger system that mediates cholera toxin (CT) action, the adenylate cyclase (AC) response was studied in 2-wk-old suckling and 6-wk-old weaned rats . AC was assayed in the proximal small intestine 4 h after intraduodenal administration of various doses of CT . Dose-effect analysis showed a 9-fold increase in the sensitivity of the CT-activated cyclase response in suckling rats when measured by the ED50, expressed as microgram CT/g body wt (0.03 for 2 wk, 0.27 for 6 wk) . When the CT dose was expressed as microgram/animal, suckling rats were 50 times more sensitive than 6-wk-old rats . In addition, the CT-induced fluid secretion was closely correlated with the elevated cyclase activities (correlation coefficient: 0.83 for 2 wk, 0.93 for 6 wk) . Furthermore, more fluid seemed to be secreted/unit wt of gut in the sucklings, even when the same level of enzyme activity was compared . A maximum of 3- to 4-fold rise in AC activation occurred at 0.5 microgram CT/g body wt, but both the basal and the maximal stimulated levels of AC were not developmentally different . This study demonstrates an in vivo increase in AC responsiveness to CT that may be in part responsible for the increased incidence of toxigenic diarrhea in neonates.

Immunology, 1989 Mar, 66(3), 410 - 5
The immunological consequences of feeding cholera toxin . I . Feeding cholera toxin suppresses the induction of systemic delayed-type hypersensitivity but not humoral immunity; Kay RA et al.; Immunization of adult BALB/c mice with 1 microgram cholera toxin (CT) in complete Freund's adjuvant (CFA) induced both humoral (IgG and IgA) and cell-mediated (DTH) immunity . Although an immunopurified, formalinized, cholera toxoid (TD) in CFA was inferior to the native holotoxin at inducing antitoxin antibodies, both cholera-derived antigens were equally immunogenic for specific DTH . When mice were fed either 1 microgram CT or 5 microgram TD 1 week before immunization, the induction of DTH was inhibited but the development of specific antibody was the same as in sham-fed controls . A feed of 10 micrograms CT not only suppressed the induction of DTH but also enhanced the IgG antitoxin responses measured 1 week after immunization . A dose of TD (50 micrograms), with a similar cholera toxin B subunit content, also induced oral tolerance for DTH but had no effect on the subsequent development of humoral immunity . The smallest doses of CT or TD fed (0.1 microgram and 0.5 microgram, respectively) failed to affect the development of either limb of the systemic immune response . These results suggest that oral tolerance for DTH is not consequent upon the metabolic actions of CT but that stimulation of systemic antibodies after enteric administration may be . Pretreating mice with cyclophosphamide (Cy) (100 mg/kg) before feeding CT abrogated the induction of oral tolerance for DTH but had no effect on humoral immunity, suggesting that suppressor T cells may be responsible for the induction of oral tolerance in these animals.

J Virol Methods, 1989 Mar, 23(3), 253 - 61
Application of peroxidase labelled antibody assays for detection of porcine IgG antibodies to hog cholera and bovine viral diarrhea viruses; Afshar A et al.; Rapid, sensitive peroxidase labelled antibody (PLA) assays using microtiter systems, were developed for detection of hog cholera virus (HCV) and cross-reacting bovine viral diarrhea virus (BVDV) antibodies in pig sera . HCV-infected pig kidney cell line (PK 15) prepared in microtiter plates were fixed and used in PLA assays . After inoculation with test serum, bound antibodies (HCV/BVDV) were reacted with either horseradish peroxidase (HRP) conjugated anti-porcine immunoglobulin (H & L) or biotinylated protein A (BPA) and subsequent HRP labelled avidin (A) . Positive reactions were easily visualized under an inverted light microscope as foci of brown colored cells after enzyme degradation of hydrogen peroxidase in the presence of amino-ethylcarbazole (AEC) . The PLA assays were superior to the indirect fluorescent antibody (IFA) test in detecting anti-HCV antibodies in porcine sera collected early after inoculation of pigs with a lapinized HCV vaccine . The performances of the PLA, IFA and FA neutralization (FAN) tests in measuring the immune response in the vaccinated pigs were comparable . Cross-reacting anti-BVDV antibody, as measured by a microtiter serum neutralization (MTSN) test, was not demonstrable in vaccinated pigs until they were challenged with a virulent HCV, 13 weeks later . The PLA assays relative to the IFA test detected more reactive samples among porcine field sera collected from HC-free pigs in Canada . Of 795 samples, 24 (3.01%) were reactive in the PLA employing HRP anti-porcine IgG, and 21 (2.6%) in the PLA, using BPA-HRP-A . When 324 of these sera were screened by the IFA test (using HC antigen), only one sample (0.30%) was found reactive.(ABSTRACT TRUNCATED AT 250 WORDS)

Immunology, 1989 Mar, 66(3), 416 - 21
The immunological consequences of feeding cholera toxin . II . Mechanisms responsible for the induction of oral tolerance for DTH; Kay RA et al.; The mechanisms behind the induction of oral tolerance after feeding cholera toxin (CT) were examined using cell and serum transfer protocols . The feeding of CT or cholera toxoid (TD) induced a splenic cell capable of inhibiting the induction of systemic delayed-type hypersensitivity (DTH) but not humoral immunity . Depletion studies showed that this cell was Thy-1.2 positive . Transfer experiments suggested that suppressor cell activity was present in the mesenteric lymph nodes (MLN) and spleens of donor mice 1 week but not 3 days after feeding CT . When spleen cells were transferred to syngeneic recipients at various times after immunization, they were more effective at inhibiting systemic DTH when transferred within a short time of immunization . If the cells were transferred 6 days after immunization they no longer suppressed the development of DTH, which suggested that they inhibit the afferent limb of this immune response . This has been confirmed by the failure of a tolerogenic dose of CT, administered by gavage, to suppress the activity of mature effector TDTH cells . Serum collected 1 hr after feeding CT also suppressed the induction of systemic DTH . However, the tolerogenic activity of CT-fed serum was abrogated by the pretreatment of recipients with cyclophosphamide (Cy) (100 mg/kg), suggesting that this activity is mediated through the induction of suppressor cells . Transfer of fed serum, however, did not induce the splenic suppressor cell described above and we would suggest that several mechanisms may operate in the mucosal regulation of systemic DTH.

Trans R Soc Trop Med Hyg, 1989 Mar-Apr, 83(2), 279 - 81
An outbreak of nosocomial cholera in a 755-bed hospital; Swaddiwudhipong W et al.; From 30 October to 7 December 1984, an outbreak of nosocomial cholera involving 11 cases of biotype El Tor, serotype Inaba, took place in a 755-bed hospital in southern Thailand . The outbreak occurred primarily among patients admitted with severe illness . Of the 11 cases, 7 were children and 4 were adults . Most cases had mild symptoms of cholera and no case died in this outbreak . The first 2 cases occurred sporadically with a subsequent cluster of cases showing an explosive pattern . A case-control study found that a history of receiving liquid tube-fed diet was significantly more common among cholera cases than their matched controls, but it could not be determined how the diet was contaminated with cholera . Cases were also significantly more likely than controls to be on oral antacid medication which could increase risk of infection by neutralizing gastric acidity . No additional cases occurred after extensive implementation of control measures.

Biochemistry, 1989 Feb 7, 28(3), 1333 - 40
Crystallization of isoelectrically homogeneous cholera toxin; Spangler BD et al.; Past difficulty in growing good crystals of cholera toxin has prevented the study of the crystal structure of this important protein . We have determined that failure of cholera toxin to crystallize well has been due to its heterogeneity . We have now succeeded in overcoming the problem by isolating a single isoelectric variant of this oligomeric protein (one A subunit and five B subunits) . Cholera toxin purified by our procedure readily forms large single crystals . The crystal form (space group P2(1), a = 73.0 A, b = 92.2 A, c = 60.6 A, beta = 106.4 degrees, one molecule in the asymmetric unit) has been described previously {Sigler et al . (1977) Science (Washington, D.C.) 197, 1277-1278} . We have recorded data from native crystals of cholera toxin to 3.0-A resolution with our electronic area detectors . With these data, we have found the orientation of a 5-fold symmetry axis within these crystals, perpendicular to the screw dyad of the crystal . We are now determining the crystal structure of cholera toxin by a combination of multiple heavy-atom isomorphous replacement and density modification techniques, making use of rotational 5-fold averaging of the B subunits.

Brain Res, 1989 Feb 6, 479(1), 130 - 7
Pretectal and tectal projections to the homologue of the dorsal lateral geniculate nucleus in the pigeon: an anterograde and retrograde tracing study with cholera toxin conjugated to horseradish peroxidase; Wild JM; The possibility of a projection from the pretectal nucleus, lentiformis mesencephali (LM), to the principal optic nuclei (OPT) of the dorsolateral thalamus was investigated using cholera toxin conjugated to horseradish peroxidase (CT-HRP) . Injections of CT-HRP into LM produced massive anterograde labeling of the pars lateralis of the nucleus dorsolateralis anterior thalami (DLL), and sparse labeling of the pars magnocellularis (DLAmc) . Injections of CT-HRP into OPT in turn produced massive retrograde labeling of both parvocellular and magnocellular divisions of LM . These results relate to possible neural mechanisms underlying optokinetic nystagmus . OPT injections also retrogradely labeled small neurons throughout the rostrocaudal extent of the tectum which were confined to superficial laminae.

J Nutr, 1989 Feb, 119(2), 280 - 5
Short term neonatal starvation altered cholera toxin binding in rabbits; Baker SS et al.; Acute neonatal malnutrition alters lumenal glycoproteins as demonstrated by altered lectin binding . To determine the effect of a 72-h fast on lumenal glycolipids, specifically the monosialoganglioside GM1, we quantitated cholera toxin (CT) binding and adenylate cyclase activity . The calculated number of specific sites for CT binding to microvillus membrane (MVM) from newborn rabbits fasted for 72 h was decreased in MVM from proximal small bowel (7 +/- 0.8 x 10(8)/micrograms protein) compared to 72-h control neonatal rabbits (18 +/- 3.3 x 10(8) micrograms protein) . In distal small bowel there was no difference in the calculated receptor sites/micrograms MVM protein between fasted (8 +/- 1.7 x 10(8)) and fed (11 +/- 4 x 10(8)) groups . MVM prepared from proximal small bowel of fed animals bound significantly more CT than MVM prepared from distal small bowel of fed animals . The affinity for CT was the same in all MVM preparations . Neuraminidase treatment of MVM resulted in increased CT binding in fed and fasted rabbit proximal and distal MVM preparations, but the greatest increase occurred in MVM prepared from proximal small bowel from fasted animals . There was no difference in adenylate cyclase activity in fed, fasted, and proximal or distal small bowel crude membrane preparations . Refeeding (120 h) resulted in normalization of CT binding in MVM from proximal small bowel of fasted animals . We conclude a 72-h fast in neonatal rabbits resulted in decreased regional CT binding in MVM prepared from proximal small bowel of fasted animals, but no change in adenylate cyclase activity . Refeeding reverses CT binding abnormalities.

In Vitro Cell Dev Biol, 1989 Feb, 25(2), 205 - 12
Rat kidney proximal tubule cells in defined medium: the roles of cholera toxin, extracellular calcium and serum in cell growth and expression of gamma-glutamyltransferase; Hatzinger PB et al.; A culture system is described in which rat kidney proximal tubule epithelial cells (RPTE) can be prepared with good yield and high viability and grown in culture under serum-free conditions . The cells require EGF, insulin, cholera toxin and either 1% dialyzed serum or a complex of bovine serum albumin with oleic acid (BSA/OA) . The cells can be maintained for long periods of time and express several markers for RPTE . The cells have both alkaline phosphatase and gamma-glutamyltransferase activity and respond to parathyroid hormone but not vasopressin . The specific activity of gamma-glutamyltransferase decreases when the cells begin to grow, but increases when they reach confluence . Extracellular calcium plays a role in the induction of gamma-glutamyltransferase in confluent cells . Cells grown in media containing low calcium, i.e . less than 0.4 mM, have reduced specific activity of gamma-glutamyltransferase . Extracellular calcium also alters the morphology of the cells in that cells grown in low calcium are single cells or loose clusters suggesting poor cell-cell contact . When the calcium is raised to 1.0 mM, the cells change their shape and organization to adopt the morphology of cells maintained continuously in 1.0 mM calcium . The cells can be passaged onto plastic surfaces which have been coated with collagen but cannot be subcultured on uncoated or serum coated plastic . This culture system will be a useful model for the investigation of renal carcinogenesis and the role of cell proliferation in that process.

J Pediatr Gastroenterol Nutr, 1989 Feb, 8(2), 252 - 8
Inhibition of cholera-toxin-stimulated intestinal secretion by CGS 9343B in rats: a specific calmodulin inhibitor; Fedorak RN et al.; In this study, the effect of CGS 9343B on cholera-toxin-stimulated intestinal secretion in rats was determined using in vivo isolated loops . This recently developed compound is a potent and specific inhibitor of calmodulin, but not of protein kinase C . At a luminal dose of 15 mg/kg, CGS 9343B has little effect on basal intestinal absorption but completely inhibited the secretory effects of cholera toxin . The less specific calmodulin inhibitor, trifluoperazine, has a similar antisecretory effect, but unlike with CGS 9343B, severe toxicity was noted at luminal doses of 7.5 mg/kg . In animals where two intestinal loops were created, one with cholera toxin alone and the other with CGS 9343B and cholera toxin, significant inhibition of secretion was observed in both loops, consistent with a systemic effect of this compound . Finally, both CGS 9343B and trifluoperazine inhibited choleratoxin stimulated increases in mucosal cyclic AMP content, whereas basal levels were unaffected . We conclude that CGS 9343B significantly inhibits choleratoxin-stimulated intestinal secretions, possibly by inhibition of calmodulin-dependent adenylate cyclase activity . Its lack of major toxicity at therapeutic doses makes this compound potentially useful for the treatment of enterotoxigenic diarrheal diseases.

Zentralbl Veterinarmed B, 1989 Feb, 36(1), 76 - 80
Differentiation of hog cholera and bovine virus diarrhoea viruses in pigs using monoclonal antibodies; Zhou Y et al.; Monoclonal antibodies against hog cholera and bovine viral diarrhoea viruses were assayed on organ tissue sections of experimentally infected animals . The animals had been infected simultaneously with both viruses . The antibodies were tested using an indirect immunofluorescence test and an indirect enzyme immunoassay with a biotin/streptavidin/peroxidase detection system . A polyclonal hyperimmune serum was used as a control in direct immunofluorescence tests . Both techniques based on monoclonal antibodies were more sensitive and more specific than the conventional test, the enzyme immunoassay being more sensitive than the immunofluorescence test . Small amounts of BVD viral antigen were demonstrable with monoclonal antibodies in most organ tissues.

Am J Physiol, 1989 Feb, 256(2 Pt 1), G335 - 41
Emergence of Na+-glucose cotransport in an epithelial secretory cell line sensitive to cholera toxin; Nath SK et al.; Epithelial properties and effects of cholera toxin (CT) and glucose were investigated in human rectal tumor cell line HRT-18 . Addition of 10(-3) M dibutyryl adenosine 3',5'-cyclic monophosphate (DbcAMP), 10(-8) M vasoactive intestinal peptide, 10(-5) M epinephrine, and 10(-5) M forskolin to the serosal side and of 3.5 micrograms/ml CT to the mucosal side and of 2 micrograms/ml A23187 to both the serosal and mucosal sides raised short-circuit current (Isc) . This rise was reversed by serosal addition of 5 x 10(-5) M bumetanide or 10(-4) M ouabain . In filters treated with CT, Isc and net chloride flux (JClnet) increased after 60 min from 0.05 +/- 0.008 and -0.04 in the Ringer to 0.32 +/- 0.05 and -0.33 mueq.h-1.cm-2, respectively . Addition of 10(-2) M glucose further raised Isc by stimulating net sodium flux (JNanet) (0.70 +/- 0.08 and + 0.58 mueq.h-1.cm-2, respectively) . This additional augmentation of Isc was reversed by 0.5 mM phlorizin and was mimicked by 3-O-methyl-D-glucose . When the filters were stimulated by cAMP for 15 min, Isc was also enhanced by addition of glucose . In untreated filters, Isc, JNanet, and JClnet did not differ significantly before and after addition of glucose . It is concluded that HRT-18 cells in basal state do not display absorptive properties but secretory properties stimulated by CT . However they exhibit Na+-glucose cotransport once stimulated by either CT or cAMP.

Gastroenterology, 1989 Feb, 96(2 Pt 1), 368 - 76
Involvement of 5-hydroxytryptamine, prostaglandin E2, and cyclic adenosine monophosphate in cholera toxin-induced fluid secretion in the small intestine of the rat in vivo; Beubler E et al.; The diarrhea of cholera is considered to rely solely on a cyclic adenosine monophosphate-mediated secretory mechanism . However, both 5-hydroxytryptamine and prostaglandin E2 have been proposed to be involved in the pathogenesis of cholera . In vivo experiments were performed, therefore, in the rat jejunum to investigate the influence of purified cholera toxin on fluid secretion, luminal release of 5-hydroxytryptamine and prostaglandin E2, and formation of mucosal cyclic adenosine monophosphate . Also the effects of ketanserin, indomethacin, verapamil, and nifedipine on the named parameters were studied . Cholera toxin dose-dependently (0.1-0.5 microgram/ml) and time-dependently (1-5 h) increased mean net fluid secretion with a maximum response at 4 h . It also caused a significant (p less than 0.01) rise in release of 5-hydroxytryptamine and prostaglandin E2, in addition to formation of cyclic adenosine monophosphate . The dose-response curve for cholera toxin-induced fluid secretion was shifted to the right by indomethacin (10 mg/kg s.c.) and ketanserin (200 micrograms/kg s.c.), none of which caused a change in cholera toxin-induced release of 5-hydroxytryptamine . However, both agents significantly decreased the release of prostaglandin E2 . Verapamil (0.2-9.5 micrograms/min i.a.) and nifedipine (0.05-0.5 microgram/min i.a.) dose-dependently reduced cholera toxin-induced fluid secretion . The estimated local concentrations at half-maximal inhibition were 5 x 10(-7) M verapamil and 5 x 10(-8) M nifedipine, respectively . The cholera toxin-induced increase in release of 5-hydroxytryptamine and prostaglandin E2 and formation of cyclic adenosine monophosphate was unaffected by verapamil . These results support the concept that cholera toxin-induced fluid secretion in vivo is caused, in part, by release of 5-hydroxytryptamine, which in turn stimulates formation of prostaglandin E2.

J Mol Cell Cardiol, 1989 Feb, 21 Suppl 1, 97 - 102
Participation of a guanine nucleotide-binding protein cascade in cholera toxin activation of adenylate cyclase; Vaughan M et al.; Guanine nucleotide-binding (G) proteins are involved in several transmembrane signaling systems . Choleragen (cholera toxin) activates adenylate cyclase by catalyzing the ADP-ribosylation of Gs alpha, the stimulatory G protein of the cyclase system . This reaction is enhanced by another guanine nucleotide-binding protein termed ADP-ribosylation factor or ARF that was purified from bovine brain membranes {R . A . Kahn and A . G . Gilman, Journal of Biological Chemistry (1986) 261, 7906-7911} . It was recently found that this ARF also increases the NAD:agmatine and NAD:protein ADP-ribosyltransferase, NAD glycohydrolase and auto-ADP-ribosylation activities of the toxin . We have purified and characterized two soluble proteins from bovine brain that act in a similar fashion to enhance choleragen activity in each of these reactions . The membrane and soluble factors are all proteins of approximately 19 kDa that require GTP or GTP analogues for activity and are ADP-ribosylated by the toxin . The ARF proteins apparently interact directly with choleragen in a GTP-dependent fashion to increase its catalytic activity and thus are part of a G protein cascade through which the toxin activates adenylate cyclase . The physiological function of the ARF proteins, as well as their possible relationships to the ras oncogene products and/or the family of G proteins that includes Gs alpha, remains to be determined.

J Comp Neurol, 1989 Jan 8, 279(2), 327 - 39
Morphological types of spinomesencephalic neurons in the marginal zone (lamina I) of the rat spinal cord, as shown after retrograde labelling with cholera toxin subunit B; Lima D et al.; Retrogradely labelled lamina I neurons were studied after intramesencephalic injections of subunit B of cholera toxin . The tracer was visualized with a mixture of two monoclonal antibodies followed by the peroxidase-antiperoxidase technique that produced Golgi-like staining of the labelled cells . A morphological and morphometric analysis in the three anatomical viewing planes disclosed two structural neuronal types which were recognized as the fusiform and pyramidal cells of our Golgi-based classification of rat marginal cells . Fusiform cells had a bipolar longitudinally elongated dendritic arbor, were located in the lateral third of lamina I, and appeared to project mainly to the contralateral parabrachial nuclei . It is asserted that these cells may convey the spinal input which elicits visceral responses generated in that area . Pyramidal cells were longitudinally oriented pyramids with the triangular base straddling the dorsal horn/white matter border and a dendritic arbor extending lateromedially and mainly rostrocaudally throughout superficial lamina I and the dorsal funiculus . These cells occurred along the entire mediolateral extent of lamina I and seemed to have a prevalent projection to the contralateral caudal ventrolateral periaqueductal gray . They may represent the ascending branch of the spinal-midbrain loop centered in that zone which controls nociceptive transmission postsynaptically in the dorsal horn.

FEBS Lett, 1989 Jan 2, 242(2), 309 - 13
Identification of cholera toxin-binding sites in the nucleus of intestinal epithelial cells; Parkinson ME et al.; Post-embedding immunogold electron microscopy shows several binding sites for cholera toxin in mouse intestinal epithelial cells, particularly in the heterochromatin of the nucleus as well as in the plasma membrane . Anti-ganglioside GM1 antibodies also bound to the nucleus, but did not interfere with the binding of toxin . 125I-labelled toxin bound specifically to a nuclear preparation from rabbit intestinal cells.

Scand J Gastroenterol, 1989 Jan, 24(1), 1 - 8
Effect of bicarbonate, acetate, and citrate on water and sodium movement in normal and cholera toxin-treated rat small intestine; Rolston DD et al.; Bicarbonate, citrate, or acetate are commonly included in oral rehydration solutions to correct acidosis and possibly because of their ability to promote water and sodium absorption . We have investigated the effect of these anions on water and sodium transport in normal and also in secreting (cholera toxin-treated) rat small intestine using a single-pass perfusion technique . In normal jejunum bicarbonate and acetate produced net absorption, and citrate net secretion of both water and sodium . In normal ileum all anions produced net absorption of water and sodium . In the secreting jejunum, however, bicarbonate had no effect on water and sodium secretion, whereas acetate and citrate actually enhanced the secretory state for both water and sodium . None of these anions had any effect on water and sodium secretion in the ileum . These observations suggest that normal and secreting intestine are qualitatively different with regard to handling of these organic anions . The addition, therefore, of bicarbonate, acetate, or citrate to oral rehydration solutions may have no beneficial effect with regard to the promotion of water and sodium absorption in the secreting intestine during acute diarrhoeal states and could actually be deleterious.

Mol Immunol, 1989 Jan, 26(1), 95 - 100
Studies of the antigenic structure of two cross-reacting proteins, pertussis and cholera toxins, using synthetic peptides; Presentini R et al.; Peptide fragments of pertussis toxin subunit 1 (PT-S1) have been synthesized in order to investigate their antigenic and immunogenic activity, and to evaluate their possible use as components of a new vaccine . Two peptides (sequence 73-82, EAERAGRGTG and sequence 107-116, YVDTYGDNAG) were selected for their predictable exposure on the surface of the molecule, and a third (8-18, YRYDSRPPEDV) for its homology with the sequence 6-16 of cholera toxin subunit A (CT-A 6-16) (YRADSRPPDEI) . Antipeptide polyclonal antibodies produced in rabbits, were tested in different immunoassays for their ability to interact with toxin proteins . All of them proved interactive with recombinant PT-S1 (rPT-S1); CT-A interact not only, as expected, with anti 8-18 antibodies, due to the high homology between the two toxins in this region, but also, unexpectedly, with anti 107-116 antibodies, in spite of the lack of homology of this peptide with the entire CT . We also found a direct cross-reactivity between the two toxins: anti PT and anti rPT-S1 antibodies interacted with CT-A, whereas anti CT antibodies did not recognize PT . Antipertussis antibodies also recognized the peptide 8-18, which therefore represents at least a part of an antigenic determinant of the toxin, while no interaction could be evidenced between anti-cholera antibodies and any of the peptides, thus demonstrating important differences in the antigenic structures of the two toxins . None of the antipeptide antibodies examined showed protective activity against the toxins in a Chinese hamster ovary (CHO) cell test.

J Dairy Sci, 1989 Jan, 72(1), 41 - 8
Influence of cholera toxin (an adenylate cyclase activator) on deoxyribonucleic acid synthesis of bovine mammary tissue in vitro and in athymic nude mice; Sheffield LG; Bovine mammary tissue from midpregnant heifers was placed in explant culture to which chlorea toxin (a potent adenylate cyclase activator) was added (0 to 100 ng/ml) . Chlorea toxin increased incorporation of thymidine into DNA in the cultures; response was maximum with 10 ng/ml chlorea toxin and approximately 24 h after addition of cholera toxin . In other studies, bovine mammary tissue was transplanted subcutaneously to ovariectomized athymic nude mice . Subsequent treatment of the mice with cholera toxin alone did not affect growth of bovine mammary tissue . However, treatment with estradiol plus progesterone increased DNA synthesis in epithelial cells . In estradiol plus progesterone-treated mice, cholera toxin injections further increased DNA synthesis . In addition, estradiol plus progesterone treatment in vivo (in athymic nude mice) increased DNA synthesis of bovine mammary tissue in response to cholera toxin in vitro . This synergism between cholera toxin and ovarian steroids may have been mediated, at least in part, by estradiol plus progesterone induction of cyclic AMP dependent protein kinase, as the activity of this enzyme was increased by estradiol plus progesterone.

J Virol Methods, 1989 Jan, 23(1), 77 - 9
A rapid serum neutralization test in microplates for the detection of antibodies to hog cholera virus; Buonavoglia C et al.; The fluorescent antibody serum neutralization (FASN) test for the detection of antibodies to hog cholera virus was developed utilizing 96-well and Terasaki microplates . This microtechnique, especially when performed in Terasaki plates, offers some advantage if compared with conventional FASN in coverslip cell cultures, being easier and more rapid, saving of reagents and allowing simple microscopic observation.

Nat Immun Cell Growth Regul, 1989, 8(4), 231 - 44
Immunomodulatory effects of cholera toxin in mice; Hussain A et al.; Immunomodulatory effects of cholera toxin (CT) were investigated in a murine model using various immunological parameters . C3H/HeN mice were injected with 2 micrograms of CT at various intervals (from 6 h to 21 days) before the immunological assays . Thymocytes were markedly decreased in their absolute number, and the phenotypes in such cells were clearly shifted from Thy1.2high+ PNAhigh+ to Thy1.2low+ PNAlow+ 2-4 days after the CT treatment . Spleen T cells were relatively increased, while surface IgM positive B cells were rather decreased . Natural killer activity and in vivo and in vitro cytotoxic T lymphocyte activity were markedly suppressed during the early stages after the CT treatment but recovered completely within 21 days . Mixed lymphocyte reaction was profoundly suppressed at least for the 1st week after the CT treatment . Furthermore, EL-4 tumor of C57BL/6 origin grew progressively and killed the recipient C3H mice when such tumor cells were inoculated 6 h after the CT treatment . On the contrary, a marked augmentation of direct (IgM) and indirect (IgG) plaque-forming cell responses to sheep red blood cells was seen after CT treatment . Delayed footpad reaction to SRBC was also augmented after CT treatment . As the mechanisms, both direct augmentation of CD4+ T cells and direct suppression of CD8+ T cells appeared to occur at a time due to the CT treatment . An indirect effect of CT through the release of the endogenous steroids was dismissed in the present study . Taken together, CT appears to have differential immunomodulatory effects on various immune effector cells through various mechanisms.

Int Arch Allergy Appl Immunol, 1989, 88(3), 273 - 9
Role of local IgA antitoxin-producing cells for intestinal protection against cholera toxin challenge; Lycke N et al.; We examined in mice, perorally immunized with cholera toxin (CT) or cholera B subunit (CTB), the association between protection against intestinal toxin challenge and frequency and function of gut mucosal IgA antitoxin-forming cells . The in vitro production of IgA antitoxin by isolated cells and the toxin-neutralizing ability of culture supernatants were determined . Repeated oral immunizations with CT gave rise to high numbers of IgA antitoxin 'spot-forming' cells (SFC) in the lamina propria as well as to protection against challenge with CT in ligated intestinal loops . In contrast, mice immunized with purified CTB, gave poor IgA antitoxin SFC responses in the lamina propria and little or no protection . When a small amount of CT was used to adjuvant the response to CTB, many IgA antitoxin SFC were found; however, protection in intestinal loops remained poor . This discrepancy was explained by the predominant localization of antitoxin SFC in the proximal small intestine following oral CTB/CT-adjuvant immunization, whereas relatively few SFC were found further down in the intestine where the loop-protection test was performed . Thus, when lamina propria plasma cells were isolated from challenged loops and cultured in vitro, they released only low titers of IgA antitoxin and CT-neutralizing antibodies in culture supernatants; this was in contrast to cells from optimally immunized mice which gave supernatants with high IgA antitoxin and toxin-neutralizing antibody titers . Increasing the dose of CT, added as adjuvant to the CTB, resulted in better protection and higher numbers of IgA antitoxin SFC in more distal parts of the intestine.(ABSTRACT TRUNCATED AT 250 WORDS)

J Immunol, 1989 Jan 1, 142(1), 20 - 7
Cellular basis of immunomodulation by cholera toxin in vitro with possible association to the adjuvant function in vivo; Lycke N et al.; Cholera toxin (CT) is a potent oral immunogen that also acts as a strong mucosal adjuvant for immune responses to related as well as unrelated Ag . To elucidate the immunomodulating effects of CT at the cellular level we have examined interactions of CT with APC and with B and T lymphocytes in vitro . CT markedly stimulated the production of IL-1 from APC (mouse peritoneal macrophages or macrophage cell line P388D1) but did not induce Ia-Ag and had marginal, if any, effect in potentiating Ia Ag expression stimulated by rIFN-gamma on these cells . CT had differential effect on T cell proliferation in vitro, usually strongly inhibitory but on Con A-stimulated spleen cells during prolonged (greater than or equal to 5 days) culture or when added on day 4 or later to these cultures up to a two- to three-fold enhancement of proliferation was seen . CT-induced inhibition of T cell proliferation was associated with decreased production of IL-2 and anergy to exogenously added IL-2 despite apparently normal expression of IL-2R . Similar to what was found with T cells LPS-stimulated spleen B cells demonstrated both inhibition and enhancement of proliferation in the presence of CT: in high concentrations (greater than or equal to 10(-8) M) and early in culture (day 3) CT had a strong inhibitory effect on the proliferation of B cells, whereas later (day 6) and/or at lower CT concentrations (10(-9) to 10(-11) M) the proliferation was increased up to 10-fold . The net effect of CT treatment on Ig-production by LPS-stimulated spleen B cells was seen as an enhanced level of IgA and IgG but not IgM in culture supernatants . The differential effects of CT on the cells of the immune system observed in vitro may, singly or in combination, explain the immunostimulatory function of CT.

Curr Top Microbiol Immunol, 1989, 146, 29 - 33
Cholera toxin and its subunits as potential oral adjuvants; Elson CO; Cholera toxin has been shown to have adjuvant effects in multiple different systems . The dose, timing and genetic background of the recipient all seem to be important variables . The role of the two subunits in both the immunogenicity and the adjuvanticity of this molecule remain unclear . The mechanisms of the adjuvant effect likely involves effects on regulatory T cells; there is evidence that the adjuvant effect is due at least in part to inhibition of suppressor T cells . When KLH is used as a model antigen, the adjuvanticity of cholera toxin appears to be related to its immunogenicity in that both properties occur mainly in mouse strains that are high responders to cholera toxin . The genetic engineering of chimeric neoantigens consisting of cholera toxin subunits coupled to antigens of interest has been shown to be technically possible and is an attractive future approach for the generation of effective oral vaccines.

Biotherapy, 1989, 1(2), 97 - 102
Combined treatment of autoimmune MRL/Mp-lpr/lpr mice with cholera toxin plus irradiation . Combined treatment of autoimmune MRL/l mice; Fan JL et al.; MRL/Mp-lpr/lpr (MRL/1) mice spontaneously develop autoimmune diseases like systemic lupus erythematosus (SLE) from 2 months of age, accompanied by massive lymphadenopathy . Such mice of 2 months of age were treated with 1 microgram cholera toxin (CT) every 7 days and/or with 400 rad of one-shot 60Co irradiation . CT treatment alone markedly improved nephritis as evaluated by proteinuria and moderately suppressed lymphadenopathy and anti-DNA antibody production, while irradiation alone prominently improved lymphadenopathy but showed little effect on both nephritis and anti-DNA antibody production . On the other hand, when mice were treated with the combination of CT plus irradiation, autoimmune nephritis as well as anti-DNA production and lymphadenopathy were almost completely inhibited . Taken together, each agent exerts the improvement effect at the different points from each other in an abnormal immunological circuit displayed in MRL/1 mice . This kind of combined treatment may be applicable to the clinical use for autoimmune diseases.

Digestion, 1989, 44(1), 14 - 9
The effect of opiates on the intestinal immune response to cholera toxin in mice; Dinari G et al.; We studied the effect of opiates on the intestinal immunoglobulin A response in mice . C57BL mice were orally immunized by two doses of 10 micrograms of cholera toxin, 2 weeks apart . Experimental groups received subcutaneous injections of morphine, either 10 or 20 mg/kg/day, in two divided doses . Morphine was given for 4 days, starting 1 day prior to each cholera toxin dose . Intestinal secretions were collected by lavage 1 week after the last cholera toxin dose, and assayed for specific anticholera toxin antibody and total immunoglobulin A . Results were expressed as units of anticholera toxin per nanogram immunoglobulin A . It was found that morphine, 20 mg/kg/day, reduced the response from 30.9 +/- 3.11 to 9.78 +2- 1.42 units/ng (M +/- SEM; p less than 0.0001) . 10 mg/kg/day of morphine slightly reduced the immune response to 21.38 +/- 3.51 units/ng (M +/- SEM), but failed to achieve statistical significance . Naloxone administration prior to morphine injections abolished the inhibitory effects of morphine . Morphine administration had no effect on the response to a booster dose of cholera toxin 3 months after the initial cholera toxin immunization and morphine administration . It is concluded that morphine has a significant inhibitory effect on the intestinal immune response, but does not effect long-term mucosal immunological memory . The effect is probably mediated by a specific opiate receptor, as it is blocked by naloxone . This effect may have clinical implications.

Vestn Akad Med Nauk SSSR, 1989, (9), 60 - 7
{Lessons of cholera pandemic VII}; Pokrovskii VI et al.; Basic epidemiological regularities of El-Tor Cholera during pandemic VII are described with differentiation of the four periods of its global spread and evaluation of the efficacy of therapeutic and preventive measures . The findings of studies into the ecology of the causative organism in the surface water basins testify to the possibility of its long-lasting existence in the saprophytic phase, which requires differentiation of the anti-cholera actions . The necessity of improvement of the global epidemiological control is substantiated.

Indian J Pediatr, 1989 Jan-Feb, 56(1), 93 - 6
Cholera gastroenteritis amongst children in Delhi; Aggarwal P et al.; Cholera gastroenteritis amongst 3595 children under twelve years suffering from acute watery diarrhea was studied for a period of five years (1982-86) . V . cholerae 01 could be isolated from 31.7% of total specimens studied . Distribution in different age groups out of total gastroenteritis cases was 7.5% in less than 2 years, 13.1% in 2-5 years and 11.1% in greater than 5-12 years . Out of total cholera cases (1141 isolate) 23.4% occurred in the age group less than 2 yrs., 41.4% in 2-5 yrs . and 35.1% in greater than 5-12 yrs . Infection occurred more often in males in all the age groups . Throughout the study, cholera was observed during summer monsoon season with Ogawa being predominant serotype.

Cell Signal, 1989, 1(5), 421 - 33
ADP-ribosylation of thylakoid membrane polypeptides by cholera toxin is correlated with inhibition of thylakoid GTPase activity and protein phosphorylation; Millner PA et al.; Incubation of pea thylakoid membranes with {32P}-NAD+ in the presence of cholera toxin resulted in the {32P}-ADP-ribosylation of a 60 kDa thylakoid membrane polypeptide . When ATP was included in the incubation mixture, a 29 kDa polypeptide was also labelled . In the absence of electron transfer cofactors or inhibitors, the extent of labelling depended on whether the membranes were preincubated in the light or dark and also on the developmental stage of the leaves used for thylakoid isolation . Irrespective of the latter, the strongest labelling was observed when DCMU was present in the light . After pretreatment of the thylakoid membranes with cholera toxin plus NAD+ under the same conditions, light-stimulated GTPase activity and protein phosphorylation were inhibited . The extent of inhibition for both processes appeared to be correlated with the amount of {32P}-ADP-ribosylation found when {32P}-NAD+ was included in the pretreatment mixture . The data presented are fully consistent with the 60 and 29 kDa polypeptides functioning as thylakoid membrane associated guanine nucleotide binding regulatory proteins.

Life Sci, 1989, 45(8), 679 - 83
Cholera toxin enhances ischemia-induced arrhythmias in the isolated rat heart--involvement of a guanine nucleotide binding protein (Gs); Huang XD et al.; Cholera toxin (CTX) at a dose, which disturbed the intestinal functions, was administered into the rat via the tail vein . At 3 hr after injection, the heart was removed and perfused or subject to global ischemia in the Langendorff isolated heart preparation . Electrocardiogram (ECG) was recorded throughout the experiment . The myocardial cAMP content was measured in the intact non-ischemic heart, and in the isolated ischemic heart at 2.5, 5 and 10 min after ischemia . It was found that the incidence and severity of malignant ventricular arrhythmias including ventricular tachycardia (VT) and ventricular fibrillation (VF) was significantly increased during ischemia in the CTX treated group . The cAMP content was also significantly increased in the CTX treated group in both intact non-ischemic and ischemic hearts, indicating an activation of the guanine nucleotide regulatory protein (Gs) . The results of the present study provide evidence that activation of Gs during ischemia may also contribute to the genesis of arrhythmia.

J Wildl Dis, 1989 Jan, 25(1), 61 - 5
A survey of wild swine in the United States for evidence of hog cholera; Nettles VF et al.; The results of surveillance for hog cholera (HC) in wild swine (Sus scrofa) collected from throughout the United States from 1979 to 1987 are presented . Sera collected from 1,218 wild swine and tissues from 637 were evaluated for HC antibodies and virus, respectively . Included within this surveillance were samples from Santa Cruz and Santa Rosa Islands, California, where HC virus had been deliberately introduced into wild swine during the 1950's in attempts to eradicate these animals . All evaluations were considered negative for HC . It appears that the HC virus does not maintain itself in dispersed swine populations and that wild swine have not remained a reservoir of HC since its eradication in domestic swine in the United States.

Am J Physiol, 1989 Jan, 256(1 Pt 1), G220 - 5
Age and cortisone alter host responsiveness to cholera toxin in the developing gut; Chu SH et al.; To assess the influence of immaturity on the responsiveness of enterocytes to specific pathogens, a dose-response curve for cholera toxin (CT)-induced fluid secretion was determined in the proximal small intestine of rats at 2 and 4 wk of age . The suckling rat was approximately 50 times more sensitive to CT in triggering the secretory response than the weaned rat, when estimated by the medium-effective dose (ED50, 0.8 vs . 38.9 nM) . Cortisone, known to promote enterocyte maturation, when injected into suckling rats, decreased host sensitivity approximately 1,000 times . Neither age nor cortisone decreased the receptor binding of 125I-labeled CT to intestinal microvillus membranes . In contrast, cortisone treatment caused a threefold increase in receptor density from 14.5 to 43.0 pmol/mg protein . The enzyme responsible for the sodium pump, Na+-K+-ATPase, showed a threefold increase in activity both after weaning and after a cortisone treatment . These data indicate that the immature gut exhibited an increased host sensitivity to CT stimulation that was not correlated with initial receptor binding but was related to a lowered Na+-K+-ATPase activity, suggesting that an underdeveloped sodium pump may be partially responsible for the high incidence of secretory diarrhea in neonates.

Sudhoffs Arch Z Wissenschaftsgesch, 1989, 73(2), 176 - 89
{Cholera in 1831 . Challenges for science and the federal government}; Stamm-Kuhlmann T; The peak of the first great cholera pandemic in 1831 fomented the controversy among contagionists and non-contagionists . In the following year the public debate centered around the correct interpretation of the recent experiences with cholera . The central government of the bureaucratic-absolutist monarchy in Prussia adhered to a firmly contagionist interpretation of the disease and reacted accordingly . Local authorities in Konigsberg and Berlin and the bourgeoisie in the merchant city of Danzig, however, stressed the destructive consequences of the cordon system . They considered the results of an interruption in trade and industry to be worse than the damage inflicted by the epidemic . The summer of 1831 demonstrated that cholera could not be stopped by the cordons, but the King's medical advisors nevertheless remained contagionists . Non-contagionists put forward several hypotheses to explain the origin and the spreading of cholera, mainly "miasma" theory and the Hippocratic paradigm of "epidemic constitution" . The correlation between poverty and disease, however, was widely noticed . Physicians in the city of Bremen pointed to the necessity of sanitary precautions to be taken in cholera-free periods . On the other hand, many "honest" citizens believed that individuals with a "dissolute" conduct of life were more at risk to contract cholera than others . Instead of costly sanitary policies, the well-to-do classes preferred to identify the defense against cholera with the segregation of unwelcome elements of society . The article is based on hitherto unpublished sources from the former Prussian State Archives at Merseburg, GDR, and the State Archive of the Hanseatic City of Bremen.

Cell Signal, 1989, 1(1), 65 - 74
Foetal calf serum enhances cholera toxin-catalysed ADP-ribosylation of the pertussis toxin-sensitive guanine nucleotide binding protein, Gi2, in rat glioma C6BU1 cells; Milligan G; The major G-protein of rat glioma C6BU1 cells corresponds immunologically to Gi2 . In the absence of guanine nucleotides, this protein is shown to be a substrate for ADP-ribosylation catalysed by both cholera and pertussis toxins . Under these conditions, a receptor for a growth factor, which has previously been shown to be activated by foetal calf serum, modulated the effects of both cholera and pertussis toxins on the G-protein . These ligand-mediated alterations of cholera and pertussis toxin-catalysed ADP ribosylation demonstrate that, in this system, the growth factor receptor interacts functionally with Gi2.

Arch Virol, 1989, 105(1-2), 113 - 8
High titre Hog Cholera virus production on Cytodex 3 microcarrier cultures; Caij A et al.; In an attempt to produce Hog Cholera virus (HCV) preparations of high titre, optimal growth and trypsinization conditions of PK-15 microcarrier cell cultures were defined . Infecting a PK-15 Cytodex 3 microcarrier culture with HCV increased the yield of virus more than 10 times compared with conventional monolayer culture in Roux flasks.

J Biol Chem, 1988 Dec 25, 263(36), 19626 - 32
Cholera toxin increases the rate of antigen-stimulated calcium influx in rat basophilic leukemia cells; Narasimhan V et al.; Cholera toxin pretreatment has been found to cause a 3-fold increase in the initial rate of antigen-stimulated secretion of serotonin from rat basophilic leukemia (RBL) cells . Under similar conditions, cholera toxin enhances the antigen-stimulated rise in cytoplasmic free ionized calcium levels and causes a 2-3-fold increase in the rate of antigen-stimulated influx of 45Ca . In intact RBL cells cholera toxin pretreatment potentiates the antigen-stimulated production of inositol phosphates, but in permeabilized cells, with strongly buffered free calcium levels, no effect of cholera toxin pretreatment on the antigen-stimulated activation of cellular phospholipase activities is observed . In addition, pretreatment of cells with tetradecanoylphorbol acetate inhibits antigen-stimulated production of inositol phosphates by greater than 95%, while the stimulated influx of 45Ca remains unaffected . These data indicate that the antigen-stimulated influx of calcium into RBL cells can be dissociated from the production of inositol phosphates in these cells . The observed effects of cholera toxin on exocytosis and Ca2+ influx in RBL cells are not due to the elevation of cellular cyclic AMP levels since a variety of agents capable of elevating cellular cyclic AMP levels do not mimic these effects . Together, these data suggest that a cholera toxin-sensitive guanine nucleotide-binding protein is involved in the pathway responsible for the antigen-stimulated influx of calcium into RBL cells.






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