Biochem Biophys Res Commun, 1988 Dec 15, 157(2), 605 - 10 Regulation of proliferation by the cholera toxin B subunit in FRTL-5 cells may involve a mechanism independent from the modulation of membrane receptor function; Tetsumoto T et al.; In quiescent rat thyroid (FRTL-5) cells, the B subunit of cholera toxin, which binds to cell surface ganglioside GM1 specifically, alone induced DNA synthesis and markedly enhanced that induced by insulin in serum-free medium . On the other hand, the B subunit inhibited DNA synthesis induced by thyrotropin (TSH) . The B subunit did not activate adenylate cyclase and had no effect on the TSH-induced cyclic adenosine 3',5'-monophosphate (cAMP) production . Moreover, the B subunit inhibited DNA synthesis induced by dibutyryl cAMP (Bt2cAMP) or phorbol-12-myristate-13-acetate (PMA) . These data demonstrate that the B subunit has both stimulatory and inhibitory effects on DNA synthesis in FRTL-5 cells depending on the presence of other growth factors and that these effects on cell proliferation by the interaction of the B subunit, possibly with cell surface ganglioside GM1, may involve a mechanism independent from the modulation of membrane receptor function through interaction with growth factor receptor. Pediatr Res, 1988 Dec, 24(6), 751 - 5 Luminal antisecretory effects of a beta-casomorphin analogue on rabbit ileum treated with cholera toxin; Ben Mansour A et al.; Because the physiologic significance of the presence of the opioid peptides beta-casomorphins (beta-CMs) in enzymatic digestion of milk proteins is still undetermined, the effect of the nonmetabolized beta-CM analogue beta-{DAla2,4,Tyr5}CM-5-NH2 on water and electrolyte movements was studied in vivo and in vitro in rabbit ileum untreated or treated with cholera toxin (CT) . When this analogue was introduced in vivo at a concentration of 10(-3) M into the lumen of rabbit ileal loops, it significantly stimulated net water absorption in untreated loops and reduced net water secretion in CT-treated loops . In vitro addition of this analogue (10(-4) M) to the serosal compartment of untreated ileum in an Ussing chamber reduced Isc (delta Isc = 0.44 +/- 0.05 muEq.h-1/cm2) and stimulated net Na and Cl absorption to the same extent . In CT-treated ileum, both serosal (10(-4) M) and mucosal (5.10(-4) M) addition of the analogue did not further modify the rise in Isc caused by CT but also stimulated net Na and Cl absorption . On the mucosal side, the effect of the analogue was accompanied by its transfer from the luminal to the blood side of the tissue . The transferred analogue was intact as shown by HPLC (Jm----s = 2.4 +/- 0.8 nmol.h-1/cm2) . These results demonstrated that the beta-CM analogue stimulates intestinal absorption of electrolytes in rabbit ileum both in the basal state and after its stimulation by CT.(ABSTRACT TRUNCATED AT 250 WORDS) Am J Med, 1988 Dec, 85(6), 811 - 6 Orally administered bovine colostral anti-cholera toxin antibodies: results of two clinical trials; McClead RE Jr et al.; PURPOSE: Previous reports have suggested that binding of luminal toxin by orally administered antitoxin antibodies might be a feasible treatment for cholera . We therefore conducted two randomized, controlled clinical trials to test the therapeutic efficacy of orally administered anti-cholera toxin bovine colostral immunoglobulins in patients with cholera diarrhea . PATIENTS AND METHODS: In Trial I, 45 patients with cholera were randomly assigned to receive two doses (2 g in 100 ml of water each) of anti-cholera toxin bovine colostral immunoglobulins, non-immune bovine colostral immunoglobulins, or 100 ml of water alone . In Trial II, 20 patients were randomly assigned to receive either 2 g of anti-cholera toxin bovine colostral immunoglobulins or non-immune bovine colostral immunoglobulins or water every two hours for a total of eight doses . The mean stool volumes in each of two sequential eight-hour periods following initiation of therapy were not significantly different among study groups in either trial . RESULTS: Measurement of immunoglobulin in stools showed that bovine IgG was detected in 19 of 25 patients given bovine colostral immunoglobulins in both trials . Cholera toxin neutralizing activity, as assessed with the rabbit ileal loop assay, was found in the stools of three of seven patients given anti-cholera toxin bovine colostral immunoglobulins in Trail I, and in all six patients given anti-cholera toxin bovine colostral immunoglobulins in Trail II . CONCLUSION: We conclude that oral anti-cholera toxin bovine colostral immunoglobulins are not effective in the treatment of patients with active cholera diarrhea . The prophylactic benefit of oral antitoxin antibody remains to be determined. Biochem J, 1988 Dec 1, 256(2), 383 - 90 Cholera toxin partially inhibits the T-cell response to phytohaemagglutinin through the ADP-ribosylation of a 45 kDa membrane protein; Nel AE et al.; This study examines the influence of cholera toxin (CT) on T lymphocyte activation by the mitogenic lectin phytohaemagglutinin (PHA) . CT suppressed lectin-induced {3H}thymidine uptake in a dose-dependent fashion and acted synergistically with PHA in the generation of intracellular cyclic AMP . The toxin was assumed to act on Gs, because it also stimulated ADP-ribosylation of a 45 kDa membrane protein in vitro; no additional substrates were seen . The inhibitory effect of the adenylate cyclase/cyclic AMP pathway was shown to be directed at a concomitant stimulatory pathway, namely inositol phospholipid turnover . Lectin-stimulated 32P incorporation into both phosphatidylinositol as well as its 4,5-biphosphate derivative was depressed in the presence of CT or exogenous dibutyryl cyclic AMP . This, in turn, was associated with reduced activation of C-kinase as determined by decreased lectin-induced translocation from the cytosol to the surface membrane . These results indicate that Gs probably acts as a transducer between the PHA receptor and adenylate cyclase and may give rise to an exaggerated adenylate cyclase response in the presence of CT . It would seem as if reduction in inositol phospholipid turnover is related to the elevation of cyclic AMP rather than a CT effect on a putative transducer which acts directly on phospholipase C . Our study does not exclude the existence of non-CT-sensitive transducers in this capacity. Biochim Biophys Acta, 1988 Nov 18, 972(2), 232 - 8 Evidence for a cholera-toxin-sensitive G-protein involved in the regulation of phosphatidylinositol 4-phosphate kinase of rat liver membranes; Urumow T et al.; Studies on the phosphorylation of inositol phospholipids of rat liver membranes have shown that {gamma S}pppG stimulates 32P incorporation from {gamma-32P}ATP into PI and PIP . This effect appeared specific for stable GTP analogues and could not be reproduced by other compounds . ADP-ribosylation of the membranes with cholera toxin resulted in a large decrease of PIP2 without changes in the level of PIP . Since an activation of phospholipase C can be ruled out, the lowering of PIP2 is explained on the basis of an inhibition of PIP kinase (EC 2.7.1.68) . From these results it appears that a novel cholera-toxin-sensitive G-protein is involved in the regulation of PIP kinase. Biochem Biophys Res Commun, 1988 Nov 15, 156(3), 1160 - 5 ADP-ribosylation of bovine S-antigen by cholera toxin; Komori N et al.; The S-antigen (alias 48K protein or arrestin) of bovine rod photoreceptors contains two stretches of amino acid sequence homologous to the ADP-ribosylation sites of the alpha subunit of transducin (Ta) . We have found that cholera toxin transfers the ADP-ribosyl group from NAD to purified bovine S-antigen as well as to S-antigen in rod outer segment membranes, while Bordetella pertussis toxin is unable to catalyze the transfer reaction efficiently . Under the same conditions, both toxins catalyzed ADP-ribosylation of Ta in rod outer segments . The ADP-ribosylation of S-antigen by cholera toxin indicates that S-antigen not only exhibits sequence homology with the ADP-ribosylation sites of Ta, but it must also resemble Ta in the tertiary structure of the domain which determines the susceptibility of S-antigen to the catalytic action of cholera toxin . These results suggest that S-antigen may function as a competitor of Ta in some stage of the cGMP cascade of visual transduction. J Immunol, 1988 Nov 15, 141(10), 3429 - 37 Cholera toxin discriminates between murine T lymphocyte proliferation stimulated by activators of protein kinase C and proliferation stimulated by IL-2 . Possible role for intracellular cAMP; Kim DK et al.; The regulation of the activation of T lymphocyte proliferation is not well understood . It is known that the tumor promoter, PMA, which activates protein kinase C (PKC), can induce the proliferation of several murine CTL clones; in combination with calcium ionophores, which raise the level of intracellular Ca2+, PMA can also stimulate the proliferation of several HTL clones . Activation of the TCR is believed to result in the liberation of diacylglycerol, which is an activator of PKC, and inositol 1,4,5-trisphosphate, which stimulates an increase in intracellular levels of calcium . We now report that pretreatment with cholera toxin (CT) inhibits the proliferation of murine T cell clones stimulated through the TCR/CD3 complex . In addition, CT-pretreatment blocks the proliferation of CTL clones activated with PMA or of HTL clones activated with PMA + calcium ionophore . In contrast, CT-pre-treatment inhibits much less effectively (100- to 1000-fold) the proliferation of these T cell clones stimulated with IL-2 . Furthermore, activators of PKC, but not IL-2, potentiate the CT-induced cAMP elevation in T cell clones . The ability of CT to inhibit much more effectively the proliferation triggered by putative activators of PKC than that induced by IL-2 may be mediated by cAMP-dependent mechanisms. Virus Res, 1988 Nov, 11(4), 281 - 91 Hog cholera virus: identification and characterization of the viral RNA and the virus-specific RNA synthesized in infected swine kidney cells; Moormann RJ et al.; Virus-specific RNA in swine kidney cells infected with hog cholera virus and the RNA of gradient-purified virions were identified and characterized . The time curve for virus replication in SK-6 cells was established . The logarithmic phase of virus growth was between 4 and 12 h after virus adsorption . Virus production was maximum at 20 h after virus adsorption . Analysis of cytoplasmic RNA in a neutral agarose gel showed that the time curve for synthesis of a high molecular weight RNA in infected cells closely paralleled that for virus growth . Electrophoresis in an agarose-formaldehyde gel of {3H}uridine-labeled RNA, synthesized with actinomycin-D, showed that this high molecular weight RNA species was approximately 12 kilobases (kb) in length . RNA, extracted from virions purified in a glycerol-tartrate gradient, co-migrated in a neutral agarose gel with the 12 kb RNA species; moreover, it hybridized specifically with a cDNA clone prepared against the intracellular 12 kb RNA . These results confirm the virus-specific nature of the 12 kb RNA . Since no prominent subgenomic virus-specific RNA was identified in infected cells, an RNA species of this size may also act as a messenger RNA. J Comp Neurol, 1988 Nov 1, 277(1), 1 - 20 The nuclei of origin of monoaminergic, peptidergic, and cholinergic afferents to the cat nucleus reticularis magnocellularis: a double-labeling study with cholera toxin as a retrograde tracer; Luppi PH et al.; Using a sensitive double-immunostaining technique with nonconjugated cholera toxin B subunit (CT) as a retrograde tracer, we examined the cells of origin and the histochemical nature of afferents to the cat nucleus reticularis magnocellularis (Mc) of the medulla oblongata . After injections of CT confined to the Mc, we found that the major afferents to the Mc arise from: (1) the lateral part of the bed nucleus of the stria terminalis, the nucleus of the anterior commissure, the preoptic area, the central nucleus of the amygdala, the posterior hypothalamus, and the nucleus of the fields of Forel; (2) the Edinger-Westphal nucleus, the mesencephalic reticular formation, and the ventrolateral part of the periaqueductal grey; (3) the nuclei locus coeruleus alpha (LC alpha), peri-LC alpha, locus subcoeruleus, and reticularis pontis oralis and caudalis; (4) the caudal raphe nuclei; and (5) the nucleus reticularis ventralis of the medulla.(ABSTRACT TRUNCATED AT 400 WORDS) Infect Immun, 1988 Nov, 56(11), 2871 - 5 Cholera enterotoxin-induced mucus secretion and increase in the mucus blanket of the rabbit ileum in vivo; Leitch GJ; The in vivo rabbit ileum was used to study the relationship of cholera enterotoxin-induced water and electrolyte secretion and mucus secretion and to determine whether the enterotoxin influenced the intestinal mucus blanket . In experiments in which luminal fluid viscosity was used to assess mucus secretion, it was found that while cholera enterotoxin induced a sustained secretion of water and electrolytes, the toxin-induced mucus hypersecretion was short lived (3 to 5 h) and subsequent exposure of the mucosa to cholera enterotoxin or prostaglandin E1 did not stimulate mucus secretion further . Dibutyryl cyclic AMP and theophylline caused a modest mucus secretion in ileal loops which differed from that of cholera enterotoxin in both magnitude and in the fact that the mucus secretion occurred 2 to 3 h after the onset of water and electrolyte secretion . An oral replacement solution was used in the ileum to reduce the enterotoxin-induced loss of water and electrolytes into the lumen . While such a solution slowed the appearance of acidic glycoprotein in the intestinal lumen, it did not change the amount of glycoprotein secreted over a 7-h period, suggesting that toxin-induced mucus secretion was not simply due to a flushing action of the experimentally caused diarrhea . To assess mucus blanket thickness, neutral glycoprotein was recovered from the blanket of rabbit ileal loops 7 h after exposure to cholera enterotoxin and the thickness of the mucus blanket was measured directly 4 and 18 h after toxin exposure . Both methods indicated that even though cholera enterotoxin-induced mucus hypersecretion had subsided and there was histological evidence of goblet cell mucin depletion, there was a sustained increase in mucus blanket thickness that was detectable for at least 18 h after mucosal enterotoxin exposure. Endocrinology, 1988 Nov, 123(5), 2367 - 73 Autoregulation of acute progesterone and adenosine 3',5'-monophosphate responses to follicle-stimulating hormone (FSH) in porcine granulosa cells: effects of FSH, cholera toxin, forskolin, and pertussis toxin; Ford KA et al.; The purpose of these studies was to determine how exposure to FSH affects subsequent responsiveness of adenylyl cyclase and progesterone production to FSH in immature porcine granulosa cells in vitro . Acute cAMP and progesterone responses to FSH and the postreceptor activators of cyclase, forskolin and cholera toxin, were determined after a 24-h preincubation with FSH . Pretreatment with FSH (1-1000 ng/ml) resulted in an increase in subsequent basal progesterone production which was dependent on preincubation FSH concentration . The cAMP response to FSH, on the other hand, was reduced after preincubation with FSH in a manner dependent on preincubation FSH concentration . Removal of FSH with acidified medium and subsequent incubation in FSH-free medium resulted in recovery of the cAMP response to FSH, indicating that attenuation of the response is reversible . Attenuation of the cAMP response to FSH does not appear to be due to 1) a loss of activity of the catalytic moiety of cyclase, since the response to forskolin and cholera toxin was not decreased by FSH; 2) a decrease in coupling of the stimulatory guanine nucleotide regulatory protein with the catalytic moiety of cyclase, since the response to cholera toxin was not reduced by FSH; 3) inhibitory signals, since preincubation with pertussis toxin did not affect the subsequent response to FSH; or 4) cAMP itself, since neither cholera toxin nor the cAMP analog 8-(4-chlorophenyl-thio)cAMP affected the response to FSH . It appears, instead, that FSH regulates FSH responsiveness by regulating the interaction of the FSH receptor with stimulatory guanine nucleotide regulatory protein. Brain Res, 1988 Oct 18, 462(2), 301 - 12 Retinohypothalamic projections in the hamster and rat demonstrated using cholera toxin; Johnson RF et al.; The organization of retinohypothalamic tract (RHT) projections in the rat and hamster was studied using anterograde transport of cholera toxin conjugated to HRP (CT-HRP) . In both species the major RHT projections lead to the suprachiasmatic nuclei (SCN) . This projection begins in the rostral SCN as a loose plexus in the hamster and a a dense aggregation of terminals along the chiasmal border in the rat . Through the remainder of the SCN there is a very dense terminal plexus in the ventral and lateral part of the nucleus with fewer terminals present medially . The RHT projection to the SCN is greater contralaterally in the rat whereas in the hamster the contralateral and ipsilateral projections are approximately equal . In addition to projections to the SCN, the RHT projects to the anterior hypothalamic area, the retrochiasmatic area and lateral hypothalamic area in both species . The anterior hypothalamic projections are more extensive in the hamster than in the rat and extend into the perifornical region, the dorsal hypothalamus and zona incerta . The SCN and anterior hypothalamic projections are continuous with a projection to the retrochiasmatic area and, in the hamster, with a projection extending into the subparaventricular zone with some axons and terminals continuing into the paraventricular nucleus . In contrast to these, the lateral hypothalamic projection in the rat is more extensive than in the hamster . Albino and pigmented rats show identical projections . In addition to the hypothalamic projections, there is in the hamster a small projection along the base of the telencephalon to the anterior amygdaloid area and cortical amygdaloid nucleus and a very sparse projection to the anterior thalamic nuclei.(ABSTRACT TRUNCATED AT 250 WORDS) Biochem J, 1988 Oct 15, 255(2), 705 - 13 Fluoroaluminates mimic muscarinic- and oxytocin-receptor-mediated generation of inositol phosphates and contraction in the intact guinea-pig myometrium . Role for a pertussis/cholera-toxin-insensitive G protein; Marc S et al.; 1 . In the intact guinea-pig myometrium, carbachol and oxytocin stimulated a specific receptor-mediated phospholipase C activation, catalysing the breakdown of PtdIns(4,5)P2 with the sequential generation of InsP3, InsP2 and InsP . Stimulation of muscarinic receptors also triggered an inhibition of cyclic AMP accumulation caused by prostacyclin . 2 . NaF plus AlCl3 mimicked the effects of carbachol and oxytocin by inducing, in a dose-dependent manner, the generation of all three inositol phosphates as well as uterine contractions . AlCl3 enhanced the fluoride effect, supporting the concept that A1F4- was the active species . Under similar conditions, fluoroaluminates activated the guanine nucleotide regulatory protein Gi, reproducing the inhibitory effect of carbachol on cyclic AMP concentrations . 3 . Both carbachol- and oxytocin-mediated increases in inositol phosphates, as well as contractions, were insensitive to pertussis toxin, under conditions where the expression of Gi was totally prevented . Cholera toxin, which activates Gs and enhances cyclic AMP accumulation, failed to affect basal or oxytocin-evoked inositol phosphate generation, but induced a slight, though consistent, attenuation of the muscarinic inositol phosphate response, which was similarly evoked by forskolin . 4 . The data provide evidence that, in the myometrium, (a) a G protein mediates the generation of inositol phosphates and the Ca2+-dependent contractile event, (b) the relevant G protein that most probably couples muscarinic and oxytocin receptors to phospholipase C is different from Gi and Gs, the proteins that couple receptors to adenylate cyclase, and (c) cyclic AMP does not seem to control the phosphoinositide cycle, but rather exerts a negative regulation at the muscarinic-receptor level. Avian Dis, 1988 Oct-Dec, 32(4), 718 - 21 Estimate of economic impact of fowl cholera in turkeys in Georgia in 1986; Morris MP et al.; Information gathered from cases of fowl cholera (FC) in commercial turkey flocks through case records, flock records, and telephone and mail surveys was used to estimate disease costs . The cost to the Georgia commercial turkey industry in 1986 from preventive measures, treatment of outbreaks, and production losses from the disease was estimated at $634,545 . The cost of FC per kg of live production was estimated to be $0.015. Virology, 1988 Oct, 166(2), 583 - 5 Characterization of the phage-specific transfer RNA molecules coded by cholera phage phi 149; Mandal N et al.; Aminoacylation of tRNA isolated from choleraphage phi 149-infected cells with individual 3H-labeled L-amino acids followed by hybridization with phage DNA revealed that the phage encodes tRNAs specific for arginine, proline, glycine, isoleucine, serine, valine, tyrosine, histidine, lysine, leucine, tryptophan, and aspartic acid . Aminoacylation of phage-coded tRNAs isolated from phage DNA-RNA hybrids also confirmed this observation except for tryptophan. Microbiologica, 1988 Oct, 11(4), 371 - 8 A study on the susceptibility of minipig kidney (MPK) and rabbit kidney (RK13) cell line cultures to the lapinized Chinese strain of hog cholera virus; Rivero VB et al.; The susceptibility of two established cell lines of pig (MPK = minipig kidney) and rabbit (RK13 = rabbit kidney) origin to the lapinized Chinese (LC) strain of hog cholera virus (HCV) was studied . Spleen cells from rabbits infected with the virus under study were inoculated to cell cultures of either MPK and RK13 cells and subsequent passages were made by culturing the trypsinized infected cells with the normal cells . Only the MPK cell line appeared to be susceptible to virus replication . Since no cytopathic effects (CPE) were observed, the presence of the viral antigen in the inoculated cultures was detected by immunofluorescence tests . The virulence of the virus for rabbits was enhanced after its cultivation in MPK cell cultures . When the MPK cell culture system adapted virus was tested in neutralization trials in the presence of an HCV reference immune serum it was found that the virus did not modify its antigenic structure in any extent . Finally, the culture adapted virus appeared to be more immunogenic for rabbits than the original rabbits adapted virus . Based on these results, it seems reasonable to suggest the use of MPK cell line for the propagation of the LC strain of HCV as an alternative to the use of rabbits for the preparation of HCV vaccine. Vaccine, 1988 Oct, 6(5), 409 - 13 Protection against influenza virus infection by vaccine inoculated intranasally with cholera toxin B subunit; Tamura S et al.; Secretory IgA antibodies in mucosa are known to play an essential role in protection against various infectious agents . To enhance the induction of protective mucosal antibodies, influenza HA vaccine was inoculated intranasally into mice with the B subunit of cholera toxin (CTB), which is known to be an excellent mucosal self-adjuvanting molecule . This combination resulted in high levels of antiviral IgA antibodies in nasal secretions and enhanced serum haemagglutinin-inhibiting (HI) antibodies 4 weeks after inoculation, compared with the inoculation of vaccine alone which induced only a low level of HI serum antibodies and no local IgA antibodies . (Subcutaneous or intraperitoneal inoculation of the vaccine with CTB failed to induce detectable nasal antiviral IgA antibodies) . Levels of nasal IgA and serum HI antibodies increased in a dose-dependent fashion with increasing nasal doses of both vaccine and CTB, and correlated with the degree of protection against viral challenge . A greater protective effect was seen with cholera toxin than with its B subunit . Moreover, a second administration of vaccine alone, 4 weeks after the inoculation of the vaccine with CTB, elevated the level of the antiviral IgA nasal antibodies to 10-100 times higher than that of the primary response . These results suggest that either CT or CTB could be used as a potent adjuvant to induce protective secretory antibodies by nasal vaccination against pathogens impinging on respiratory mucosa. Proc Natl Acad Sci U S A, 1988 Oct, 85(19), 7260 - 4 Cholera toxin potentiates IgE-coupled inositol phospholipid hydrolysis and mediator secretion by RBL-2H3 cells; McCloskey MA; Binding of polyvalent antigens to IgE present in Fc epsilon receptors on the surface of mast cells and the RBL-2H3 cell line triggers the exocytotic release of allergic mediators . Preincubation of RBL-2H3 cells with cholera toxin (CT) was found to potentiate greater than or equal to 2- to 3-fold the rate and final amount of antigen-induced secretion of {3H}serotonin and N-acetyl beta-D-glucosaminidase . This was accompanied by a more variable increase in the initial rate of antigen-triggered formation of inositol phosphates . The holotoxin was required for potentiation, as neither the A nor the B subunit was effective when added separately . Four observations indicate that cAMP was not the primary effector of the augmentation of secretion caused by CT: (i) culture conditions were found in which CT caused large increases in secretion but very modest (or no) increases in cAMP; (ii) under other conditions, progressive increase in {CT} caused a maximum 2.5- to 3-fold increase in cAMP followed by a return to basal levels, whereas the secretory response saturated and remained stable; (iii) permeant cAMP analogs consistently enhanced secretion at low doses and inhibited at higher doses, but the peak enhancement was always much less than that achieved by an optimal dose of CT; (iv) the selective phosphodiesterase inhibitor Ro 20-1724 exhibited similar biphasic dose-response curves, the maximum enhancement again being small compared to that caused by CT itself . Both in vitro and in vivo, CT catalyzed transfer of ADP-ribose from NAD to two membrane proteins that comigrated on NaDodSO4/polyacrylamide gel electrophoresis with two CT substrates in other cell types, and these were identified by immunoblotting as Gs alpha . These results suggest that ADP-ribosylation of a cholera toxin substrate potentiates IgE-mediated secretion from RBL-2H3 cells by a largely cAMP-independent route. Biochem Biophys Res Commun, 1988 Sep 15, 155(2), 1051 - 9 Photoaffinity labeling with GTP-gamma-azidoanilide of a cholera toxin-sensitive 40 kDa protein from pancreatic acinar cells; Schafer R et al.; In isolated pancreatic acinar plasma membranes a 40 kDa protein was labeled with the photoreactive GTP-analogue {alpha 32P} GTP-gamma-azidoanilide . Increased incorporation of the photolabel into the 40 kDa protein was obtained in the presence of increasing concentrations of cholecystokinin-octapeptide (10(-8) - 10(-5) M) but not with carbachol . Adenylyl cyclase activating hormones such as vasoactive intestinal polypeptide and secretin had no effect . Pretreatment of plasma membranes with cholera toxin reduced incorporation of GTP-gamma-azidoanilide into the 40 kDa protein by about 30% . This reduction was reversed if ADP-ribosylation by cholera toxin was performed in the presence of cholecystokinin, whereas carbachol had no effect . The data indicate that a cholera toxin-sensitive 40 kDa GTP-binding protein is involved in functionally coupling cholecystokinin- but not muscarinic acetylcholine-receptors to phospholipase C. Mol Cell Endocrinol, 1988 Sep, 59(1-2), 125 - 33 Modulation of steroidogenesis in choriocarcinoma cells by cholera toxin, phorbol ester, epidermal growth factor and insulin-like growth factor I; Ritvos O; The effects of cholera toxin (CT), which stimulates adenylate cyclase, 12-O-tetradecanoylphorbol 13-acetate (TPA), a protein kinase C activator, epidermal growth factor (EGF) and insulin-like growth factor I (IGF-I) on progesterone (P) and estradiol (E2) secretion by human choriocarcinoma JEG-3 cells were studied . During a 48 h incubation, CT, TPA and EGF stimulated P production in a concentration-dependent manner, whereas IGF-I was without effect . CT (1.0 ng/ml), TPA (10 ng/ml) and EGF (10 ng/ml) stimulated P production maximally 4.3-, 3.3- and 2.3-fold over basal, respectively . When added together with CT, TPA and EGF stimulated P production 10.0- and 5.0-fold over basal production showing that the effects of CT plus TPA were more than additive but those of CT plus EGF less than additive . Time-course studies indicated that the effects were detectable at 12 h, and continued to increase up to 48 h . The conversion of added dehydroepiandrosterone sulfate (DHEAS) to E2 was stimulated by CT and TPA and inhibited by IGF-I in a concentration-dependent manner . By contrast, EGF had no effect . The maximal responses in E2 production were 3.2- and 2.0-fold over unstimulated cells by CT (1.0 ng/ml) and TPA (10 ng/ml), respectively . When both agents were added together, their effects on E2 production were additive with 5.5-fold increase over unstimulated cells . IGF-I (30 ng/ml) inhibited maximally basal and CT-stimulated E2 production by 33% and 42%, respectively.(ABSTRACT TRUNCATED AT 250 WORDS) Anal Biochem, 1988 Sep, 173(2), 368 - 75 Quantification of gangliotetraose gangliosides with cholera toxin; Wu GS et al.; A procedure is described for assay of GM1 and other gangliotetraose-type gangliosides at the picomole level . The gangliosides are absorbed onto polystyrene microwells and treated with neuraminidase and then with cholera toxin B subunits conjugated to horseradish peroxidase . Color is developed and quantified spectrophotometrically . Omission of neuraminidase gives a measure of GM1 alone . Linearity was obtained between 0.5 and 3 pmol . This procedure was applied to ganglioside mixtures isolated fron neuro-2A neuroblastoma and PC12 pheochromocytoma cells . For the latter, an additional step involving reaction with fucosidase increased the yield of GM1 due to the presence of fucosylated gangliosides . Application of the same reagents as a TLC overlay procedure to the gangliosides from neuro-2A cells revealed the presence of GD1a, GD1b, and GT1b in addition to GM1, thus confirming the presence of a family of gangliotetraose gangliosides. Jpn J Pharmacol, 1988 Sep, 48(1), 23 - 30 Developmental changes in the levels of substrates for cholera toxin-catalyzed and pertussis toxin-catalyzed ADP-ribosylation in rat cardiac cell membranes; Kojima M et al.; Developmental changes in the substrates for cholera toxin (CTX)- and pertussis toxin (PTX)-catalyzed ADP-ribosylation in cardiac (ventricular) cell membranes were studied in fetal (16- to 20-day), neonatal (0- to 20-day) and adult (2- to 3-month) rats . The CTX and PTX substrates were determined by the method of CTX-catalyzed and PTX-catalyzed ADP-ribosylation of the alpha-subunit of GTP-binding (G) proteins, respectively . As early as fetal day 16, three substrates (45-, 47- and 52-kDa proteins) were identified for CTX-catalyzed ADP-ribosylation and one substrate (41-kDa protein) for PTX-catalyzed ADP-ribosylation . The levels of the three CTX substrates (fmol/mg tissue) increased with development between fetal day 16 and neonatal day 16, and then they decreased to their adult levels . The level of the one PTX substrate (fmol/mg tissue) changed as follows: the substrate decreased between fetal day 16 and the day of birth, increased abruptly for 4 days neonatal and increased slowly thereafter until neonatal day 16, and then decreased to the final adult level . The PTX substrate seems to reach a nearly maximum level earlier than the CTX substrates . This information is essential for understanding the developmental changes in the transmembrane signaling system between membrane receptors and their effectors which are coupled with the stimulatory and inhibitory G proteins. J Immunol, 1988 Sep 1, 141(5), 1495 - 501 Oral administration of cholera toxin-Sendai virus conjugate potentiates gut and respiratory immunity against Sendai virus; Liang XP et al.; Successful oral immunization to prevent infectious diseases in the gastrointestinal tract as well as distant mucosal tissues may depend on the effectiveness of an Ag to induce gut immune responses . We and others have previously reported that cholera toxin possesses strong adjuvant effects on the gut immune response to co-administered Ag . To explore further adjuvant effects of cholera toxin, the holotoxin or its B subunit was chemically cross-linked to Sendai virus . The resulting conjugates, which were not infectious, were evaluated for their capacity to induce gut immune responses against Sendai virus after oral administration to mice . Conjugating cholera toxin to virus significantly enhanced the adjuvant activity of cholera toxin compared to simple mixing . Cholera toxin B subunit, however, did not show an adjuvant effect either by itself or conjugated with the virus . Oral administration of the Sendai virus-cholera toxin conjugate was also able to prime for protective anti-viral responses in the respiratory tract . Mice that were orally immunized with the conjugate and intra-nasally boosted with inactivated virus alone showed virus-specific IgA titers in nasal secretions that correlated with protection against direct nasal challenge with live Sendai virus . For comparison, s.c . immunization was also studied . Systemic immunization with the virus-cholera toxin conjugate induced virus-specific antibody responses in serum as well as in the respiratory tract but failed to protect the upper respiratory tract against virus challenge . Systemic immunization plus an intra-nasal boost did, however, confer a variable degree of protection to the upper respiratory tract, which correlated primarily with bronchoalveolar lavage (lung) antibody titers. Dig Dis Sci, 1988 Sep, 33(9), 1153 - 8 Discrepancy between effects of cholera toxin on net fluid movement and cAMP levels in rat jejunum, ileum, and colon; Farack UM et al.; Regional differences in the response to cholera toxin were evaluated in rat jejunum, ileum, and colon in vivo . Ligated intestinal loops were exposed to a supramaximal concentration of cholera toxin for 5 hr, and net fluid transport, adenosine 3',5'-monophosphate (cAMP) concentrations, and adenylate cyclase and phosphodiesterase activities of mucosal homogenates were determined . The fluid transport response and the specific activities of adenylate cyclase (with and without cholera toxin) and phosphodiesterase declined progressively from the jejunum to the colon . In contrast, cAMP concentrations (with and without cholera toxin) were lowest in the jejunum and highest in the colon . These results demonstrate that cAMP concentrations of the total mucosal homogenate do not parallel cholera toxin-induced fluid secretion in the three intestinal segments . Rather, the activities of adenylate cyclase and phosphodiesterase suggest a relation between fluid secretion and the turnover of cAMP. Immunology, 1988 Aug, 64(4), 691 - 5 Mucosal immune response to cholera toxin in ageing rats . I . Antibody and antibody-containing cell response; Schmucker DL et al.; Although ageing is accompanied by systemic immunodeficiencies, the status of the mucosal immune system in the elderly remains unresolved . The gastrointestinal mucosal immune response was evaluated in young, mature and old male rats subjected to intra-intestinal immunization with cholera toxin (CTx) . Five days following secondary immunization, the alpha-CTx-IgA titre in the bile of immunized rats was markedly reduced, i.e . the values measured in young rats were approximately five-fold higher than those of old animals . alpha-CTx-IgA levels in non-immunized rats were negligible and age-related shifts in other antibody titres (alpha-CTx IgG and IgM) were not significant . The antibody response to CTx was not reflected in the total IgA content of the samples . The number of alpha-CTx antibody-containing cells (ACCs) in the small intestinal lamina propria was significantly reduced in old immunized rats in comparison with the young or mature animals . These data suggest that ageing compromises both non-immune cell (antibody transport by hepatocytes) and immune cell (number of ACCs in the gut wall) functions in response to cholera toxin immunization in this animal model. Proc Natl Acad Sci U S A, 1988 Aug, 85(16), 5899 - 902 Platelet cytosolic 44-kDa protein is a substrate of cholera toxin-induced ADP-ribosylation and is not recognized by antisera against the alpha subunit of the stimulatory guanine nucleotide-binding regulatory protein; Molina y Vedia LM et al.; ADP-ribosylation induced by cholera toxin and pertussis toxin was studied in particulate and cytosolic fractions of human platelets . Platelets were disrupted by a cycle of freezing and thawing in the presence of a hyposmotic buffer containing protease inhibitors . In both fractions, the A subunit of cholera toxin ADP-ribosylates two proteins with molecular masses of 42 and 44 kDa, whereas pertussis toxin ADP-ribosylates a 41-kDa polypeptide . Two antisera against the alpha subunit of the stimulatory guanine nucleotide-binding regulatory protein recognize only the 42-kDa polypeptide . Cholera toxin-induced ADP-ribosylation of the 42- and 44-kDa proteins is reduced by pretreatment of platelets with iloprost, a prostacyclin analog . The 44-kDa protein, which is substrate of cholera toxin, could be extracted completely from the membrane and recovered in the cytosolic fraction when the cells were disrupted by Dounce homogenization and the pellet was extensively washed . A 44-kDa protein can also be labeled with 8-azidoguanosine 5'-{alpha-32P}triphosphate in the cytosol and membranes . These findings indicate that cholera and pertussis toxins produced covalent modifications of proteins present in particulate and cytosolic platelet fractions . Moreover, the 44-kDa protein might be an alpha subunit of a guanine nucleotide-binding regulatory protein that is not recognized by available antisera. J Infect Dis, 1988 Aug, 158(2), 372 - 7 Cross-protection by B subunit-whole cell cholera vaccine against diarrhea associated with heat-labile toxin-producing enterotoxigenic Escherichia coli: results of a large-scale field trial; Clemens JD et al.; The B subunit (BS) of cholera toxin and that of the heat-labile enterotoxin (LT) of enterotoxigenic Escherichia coli (ETEC) are antigenically similar . We therefore assessed whether a combined cholera toxin BS/whole-cell (BS-WC) oral vaccine against cholera conferred cross-protection against LT-producing ETEC (LT-ETEC) diarrhea in a randomized, double-blind field trial among rural Bangladeshi children and women . The 24,770 persons who ingested two or more doses of BS-WC vaccine were compared with 24,842 controls who took two or more doses of killed whole-cell (WC) oral cholera vaccine . Sixty-seven percent fewer episodes of LT-ETEC diarrhea were noted in the BS-WC group than in the WC group during short-term (three-month) follow-up (P less than .01), but no reduction was evident during the ensuing nine months . Short-term protection was particularly notable against LT-ETEC diarrhea causing life-threatening dehydration (protective efficacy, 86%; P less than .05). Acta Physiol Scand, 1988 Aug, 133(4), 551 - 7 Somatostatin and methionine-enkephalin inhibit cholera toxin-induced jejunal net fluid secretion and release of vasoactive intestinal polypeptide in the cat in vivo; Eklund S et al.; A major part of the net fluid secretion that is elicited by cholera toxin in the small intestine of the cat has been shown to be mediated by intramural nervous reflex(es) . The release of vasoactive intestinal polypeptide (VIP) from the small intestine is increased by cholera toxin . We report that close intra-arterial infusions of methionine-enkephalin (met-enk) and somatostatin cause a parallel reduction in cholera toxin-induced net fluid secretion and in VIP release from the small intestine of the cat . Intestinal blood flow was slightly, but significantly increased by met-enk and not influenced by somatostatin . These results strengthen the hypothesis that VIP is involved as a neurotransmitter in the nervous reflex mediating cholera toxin-induced secretion. Biochem J, 1988 Aug 1, 253(3), 765 - 75 Cholera-toxin and corticotropin modulation of inositol phosphate accumulation induced by vasopressin and angiotensin II in rat glomerulosa cells; Guillon G et al.; Vasopressin (VP) and angiotensin II (AT II) stimulate the production of inositol phosphates (IP) in rat glomerulosa cells . Guanosine 5'-{gamma-thio}triphosphate (GTP{S}), but not VP or AT II, stimulates IP production in a myo-{3H}inositol-prelabelled glomerulosa-cell membrane preparation . In combination with GTP{S}, these hormones potentiate the response to GTP{S}, indicating the existence of a G-protein involved in the coupling of the VP and AT II receptor with the phospholipase C . ADP-ribosylation with pertussis toxin (IAP) revealed the specific labelling of a single molecule of 41 kDa . No significant inhibition of VP- or AT II-stimulated IP accumulation was detected in intact cells when the whole 41 kDa molecule was endogenously ADP-ribosylated by IAP treatment . On the contrary, when glomerulosa cells were infected with cholera toxin (CT), both the VP- and AT II-stimulated IP accumulations were inhibited in a dose-dependent manner . Yet these effects were partial even at high concentrations of CT, and could not be related to the ADP-ribosylation of 'alpha s' molecules . Similarly, when the cells were infected with 1 microgram of CT/ml, the specific binding of VP and AT II decreased by 50-60% . Such results may signify that the treatment primarily affects the densities of the hormone receptors . When glomerulosa cells were incubated for 15 h in the presence of 10 nM-corticotropin (ACTH), a condition in which the intracellular concentration of cyclic AMP was increased 3-fold, the maximum IP response to 0.1 microM-VP or -AT II was decreased by 50% . When similar experiments were carried out only after a 15 min incubation period with the same concentration of ACTH, the increase in cyclic AMP was more pronounced, but no inhibition of hormone-induced IP accumulation was observed . Altogether, these results may suggest that CT exerts its action on the VP- or AT II-sensitive phospholipase C systems via a prolonged increase in intracellular cyclic AMP. Biochem J, 1988 Aug 1, 253(3), 735 - 43 Interactions of cholera toxin with isolated hepatocytes . Effects of low pH, chloroquine and monensin on toxin internalization, processing and action; Janicot M et al.; The major steps in cholera-toxin action, i.e . binding, internalization, generation of A1 peptide and activation of adenylate cyclase, were examined in isolated hepatocytes . The binding of toxin involves a single class of high-affinity sites (KD congruent to 0.1 nM; Bmax . congruent to 10(7) sites/cell) . At 37 degrees C, cell-associated toxin is progressively internalized, as judged by the loss of its accessibility to antibodies against whole toxin, A and B subunits (about 50, 75 and 30% of initially bound toxin after 40 min respectively) . Two distinct pathways are involved in this process: endocytosis of the whole toxin, and selective penetration of the A subunit into the plasma membrane . Exposure of hepatocytes to an acidic medium (pH 5) results in a rapid and marked disappearance of the A subunit from the cell surface . Generation of A1 peptide and activation of adenylate cyclase by the toxin occur after a lag phase (10 min at 37 degrees C), and increase with time in a parallel manner up to 2-3% A1 peptide generated; they are unaffected by exposure of cells to an acidic medium . Chloroquine and monensin, which elevate the pH in acidic organelles, inhibit by 2-4-fold both the generation of A1 peptide and the activation of adenylate cyclase . Unexpectedly, these drugs also inhibit the internalization of the toxin . These results suggest that an acidic pH facilitates the penetration of A subunit into the plasma membrane and presumably the endosomal membrane as well, and that endocytosis of cholera toxin is required for generation of A1 peptide and activation of adenylate cyclase. J Invest Dermatol, 1988 Aug, 91(2), 154 - 7 Increased cholera toxin-, and forskolin-induced cyclic AMP accumulations in psoriatic involved versus uninvolved or normal human epidermis; Iizuka H et al.; Psoriatic involved epidermis reveals variously altered receptor-adenylate cyclase responses; among them the most prominent is defective beta-adrenergic adenylate cyclase response, which is normally the major receptor-adenylate cyclase system of human epidermis . It is known that activation of hormone-stimulated adenylate cyclase, a membrane-bound enzyme complex, requires functional coupling of at least 3 distinct subunits: 1) receptor subunit (R), 2) guanine nucleotide binding protein (G), and 3) catalytic subunit (C) . The precise nature of the beta-adrenergic defect in the psoriatic epidermis, however, remains to be determined, especially in terms of G and C function . Using the involved and uninvolved skin from psoriatic patients, we investigated effects of cholera toxin (which monitors G-C interaction) and forskolin (which monitors C function) on the adenylate cyclase system of epidermis, which were compared with those of normal human epidermis . Both agents increased cyclic AMP levels of involved, uninvolved, and normal human epidermis . Marked accumulations were observed in the presence of cyclic nucleotide phosphodiesterase inhibitor, isobutyl-methylxanthine (IBMX); without the phosphodiesterase inhibitor, the effect of each agent was minimal . Comparison of the effects of cholera toxin revealed that the psoriatic involved epidermis accumulates much more cyclic AMP than the uninvolved epidermis (involved: 193 +/- 65; uninvolved: 117 +/- 54 pmoles/mg protein/5 h) . Similarly forskolin-induced cyclic AMP accumulations of the involved epidermis were much more than those of uninvolved epidermis (involved: 374 +/- 152; uninvolved: 101 +/- 41 pmoles/mg protein/2 h) . Those of normal human epidermis were not significantly different from those of uninvolved epidermis (cholera toxin: 99 +/- 36 pmoles/mg protein/5 h; forskolin: 84 +/- 22 pmoles/mg protein/2 h) . Our results indicate that G and C function and their interaction is not defective (but rather increased) in the psoriatic involved epidermis . This suggests that the defective beta-adrenergic response of psoriatic involved epidermis reflects defective R or R-G interaction of the epidermal adenylate cyclase system. Biochim Biophys Acta, 1988 Jul 29, 970(3), 355 - 62 Cholera toxin and pertussis toxin substrates and endogenous ADP-ribosyltransferase activity in Drosophila melanogaster; Hopkins RS et al.; Cholera toxin- and pertussis toxin-catalyzed ADP-ribosylation were used to identify and localize G protein substrates in Drosophila melanogaster and in Manduca sexta . Cholera toxin catalyzes ADP-ribosylation of 37 kDa and 50 kDa polypeptides, but these polypeptides are also substrates for an ADP-ribosyltransferase (EC 2.4.2.30) activity endogenous to the Drosophila extracts . Pertussis toxin modifies 37 kDa and 39 kDa polypeptides in Drosophila homogenates . The pattern of proteolysis of the 39 kDa pertussis toxin substrate is similar to that of mammalian Go and is influenced by guanyl nucleotide binding . The 39 kDa Go-like Drosophila and Manduca pertussis toxin substrates are found primarily in neural tissues . These studies provide further evidence that G proteins are present in Drosophila and that this organism can therefore be used to investigate the physiological roles of these enzymes using advanced genetic manipulations. J Biol Chem, 1988 Jul 5, 263(19), 9499 - 501 Crystallization and preliminary x-ray diffraction study of cholera toxin B-subunit; Maulik PR et al.; Cholera toxin binds to its ganglioside GM1 receptor via its B-subunit, a pentameric assembly of identical subunits (Mr = 11,600) . Diffraction quality crystals of cholera toxin B-subunit have been obtained at room temperature by vapor diffusion with polyethylene glycol in the presence of the nonionic detergent beta-octyl glucoside . The crystals have been characterized with x-radiation as monoclinic, space group P21, with unit cell dimensions a = 39.0 A, b = 94.3 A, c = 67.5 A, beta = 96.0 degrees . There are two molecules per unit cell, with one molecule (Mr = 58,000) in each asymmetric unit . Precession photographs (micron = 13 degrees) show that crystals diffract beyond 3.3-A resolution and are stable in the x-ray beam at room temperature for at least 40 h; thus, they can be used to collect three-dimensional crystallographic data. J Infect Dis, 1988 Jul, 158(1), 60 - 9 Field trial of oral cholera vaccines in Bangladesh: results of one year of follow-up; Clemens JD et al.; We assessed the protective efficacy (PE) of three doses of B subunit-killed whole cell (BS-WC) and killed whole cell-only (WC) oral cholera vaccines in a randomized, double-blind trial among 62,285 children and women residing in rural Bangladesh . After one complete year of surveillance, 110 cases of cholera were detected in the placebo group, 52 in the WC group (PE, 53%; P less than .0001), and 41 in the BS-WC group (PE, 62%; P less than .0001) . Protection was greater for BS-WC recipients than for WC recipients only during the initial eight months of observation . Both vaccines conferred equivalent protection against cholera associated with life-threatening dehydration and against less severe cholera . High-grade, sustained protection was observed in persons vaccinated when older than five years; in younger persons protection was transient . We conclude that BS-WC and WC vaccines confer significant protection against cholera, particularly in persons vaccinated when older than five years. Endocrinology, 1988 Jul, 123(1), 438 - 44 Modulation of prolactin-stimulated Nb2 lymphoma cell mitogenesis by cholera toxin and pertussis toxin; Larsen JL et al.; We have investigated whether cholera toxin (CT)- or pertussis toxin (IAP)-sensitive G proteins are involved in ovine (o) PRL-stimulated mitogenesis in the lactogen-dependent rat Nb2 node lymphoma cell line . Addition of IAP to medium caused a biphasic effect on oPRL-stimulated cell number . Low doses (10(-3) ng/ml) enhanced (mean +/- SEM, 15 +/- 3%) whereas higher doses (greater than or equal to 10 ng/ml) inhibited (24 +/- 3%) mitogenesis stimulated by a submaximal dose of oPRL (0.1 ng/ml) compared to control values . The cAMP analog 8-bromo-cAMP also had a biphasic effect on cell division stimulated by submaximal doses of PRL . Low doses (10(-5) M) enhanced whereas higher doses (10(-3) M) inhibited Nb2 cell growth in response to PRL . Incubation with CT only inhibited oPRL-stimulated mitogenesis in a dose-dependent manner . Maximal inhibition (63 +/- 7%) occurred at a concentration of 10 ng/ml or more . Phorbol myristate acetate (PMA) enhanced mitogenesis stimulated by PRL alone and in the presence of either stimulatory or inhibitory doses of IAP, but PMA did not block IAP inhibition . In contrast, PMA had no effect on cells incubated with CT; the inhibition of PRL-stimulated cell division by CT remained unchanged . Lactogenic receptor-binding sites per cell and affinity were not significantly affected by PMA, IAP, or CT, suggesting a postreceptor mechanism of action . In summary, these data demonstrate that cAMP modifies PRL-stimulated Nb2 cell mitogenesis . The differences between IAP and CT (i.e . biphasic effect, degree of inhibition, and differential effect of PMA) suggest that these agents could also modulate PRL actions in the Nb2 cell through different mechanisms, including a cAMP-independent pathway. Reg Immunol, 1988 Jul-Aug, 1(1), 32 - 40 Intraduodenal application of cholera holotoxin increases the potential of clones from Peyer's patch B cells of relevant and unrelated specificities to secrete IgG and IgA; Lebman DA et al.; Although it has been established that cholera toxin is both an effective mucosal immunogen and adjuvant, the mechanism by which it acts has not been determined . The relative contributions of the pharmocologic and binding capacities of holotoxin have been assessed by comparing holotoxin, acid dissociated B subunit, and B subunit from the Texas Star variant, which is deficient in production of the A subunit, for their ability to generate toxin-specific precursors in vivo that give rise to clones secreting exclusively IgA in vitro . In this way we demonstrated that although B subunit is as immunogenic as the holotoxin, pharmacologic activity appears to play a role in the generation of IgA-committed precursors . In addition, intraduodenal (i.d.) application of holotoxin can act to alter the isotype display of previously primed B cells in Peyer's patches (PP) specific for a chemically unrelated hapten resulting in an increase in the proportion of their clones that secrete IgA or IgG and a decrease in the proportion that secrete IgM following antigen-dependent in vitro clonal expansion . Intraduodenal application of cholera toxin did not appear to nonspecifically increase the proportion of IgA-committed precursors as judged by staining for sIgA and mRNA alpha levels . However, following i.d . application of cholera toxin, an overall downward shift in the levels of sIgD and s kappa was observed in the PP B cell population . We suggest that the holotoxin can nonspecifically affect the isotype display of PP B cells by altering their responsiveness to the stimuli present in the in vitro cultures. Acta Physiol Scand, 1988 Jul, 133(3), 289 - 95 The effect of splanchnic nerve stimulation and neuropeptide Y on cholera secretion and release of vasoactive intestinal polypeptide in the feline small intestine; Sjoqvist A et al.; The effect of sympathetic nerve stimulation and intra-arterial infusion of neuropeptide Y (NPY) on net fluid secretion and release of vasoactive intestinal polypeptide (VIP) was studied in the cat small intestine during a secretion due to cholera toxin . Activation of the splanchnic nerves (4 Hz, 5 ms, 5 V) decreased net fluid secretion to 57 +/- 10% of control . Concomitantly, the release of VIP was reduced to less than 50% . Furthermore, close i.a . infusion of NPY (estimated increase in plasma concentration 75 nmol l-1) reduced the net fluid secretion and VIP release to 27 +/- 5 and 28 +/- 4% of the pre-stimulatory value . The correlation between the decrease in net fluid secretion and reduction in VIP release showed a strong positive correlation (r = 0.83) . These results strongly indicate that the antisecretory effect of sympathetic nerve stimulation during cholera diarrhoea is mediated by inhibition of secretory VIP neurons in the intestinal mucosa . A similar mechanism is also proposed for the intravascularly administered NPY. Microbiologica, 1988 Jul, 11(3), 263 - 4 Susceptibility of a minipig kidney cell line (MPK) to hog cholera virus; Buonavoglia C et al.; A comparitive study on the different susceptibility of MPK cells (Minipig Kidney cell line) and PK15 cells (Pig Kidney cell line) to the Hog Cholera Virus (HCV) was conducted . Higher HCV titres (3 log10) were reached on MPK cells compared with PK15 cells. Immunopharmacology, 1988 Jul-Aug, 16(1), 53 - 60 The effect of single dose, intravenous cyclophosphamide on the mouse intestinal IgA response to cholera toxin; Karacic JJ et al.; The IgA response of gut associated lymphoid tissue (GALT) to enteric pathogens is a vital component of the mucosal barrier . This study describes the effect of cyclophosphamide (Cy) on the IgA anti-CT response of Peyer's patch (PP) and lamina propria cells derived from mice previously challenged enterically with two weekly doses of 10 micrograms CT . Under normal circumstances, both PP and lamina propria responses peaked 7 days after the second dose of CT . To evaluate the effect of a single dose of Cy on this response, mice were given Cy (50 mg/kg) intravenously on days -2, 0, 2 or 7 relative to the initial dose of CT . Cultures of PP and jejunal segments were established 7 days after the booster dose of CT (time or normal peak response) . A single dose of Cy suppressed the IgA anti-CT response of PP and, to a lesser extent, jejunal segment cultures only if the drug was given 2 days before the primary dose of CT . This suppression of the anti-CT response was overcome when Cy was given 2 days before CT priming, and CT was administered three times, on days 0, 7 and 14; thus, the effect of Cy was brief and did not appear to promote tolerance to CT . These data show that a single, moderate dose of Cy, given before enteric priming of the GALT, can inhibit the mucosal IgA response to CT . The effect of Cy is relatively brief and dependent upon the time at which the drug is given relative to the induction of the mucosal immune response. In Vitro Cell Dev Biol, 1988 Jul, 24(7), 677 - 82 Characteristics of the porcine kidney cell line IB-RS-2 clone D10 (IB-RS-2 D10) which is free of hog cholera virus; House JA et al.; A Brazilian stock of clone C17 of the IB-RS-2 porcine kidney cell line which was contaminated with hog cholera virus (HCV) was cloned . One clone designated IB-RS-2 D10 was determined to be free of HCV, 20 other viruses, and Mycoplasma . IB-RS-2 D10 cells possessed the same viral susceptibility pattern as the contaminated parent cells to the viruses of foot-and-mouth disease, swine vesicular disease, vesicular exanthema of swine, transmissible gastroenteritis, and several other viruses . The IB-RS-2 D10 cells had a median chromosome count of 34, were morphologically epithelioid cells, and were resistant to HCV infection . Freedom from HCV affords advantages for vaccine production and avoids laboratory contamination. J Neurosci Methods, 1988 Jul, 24(3), 225 - 35 Tracing of neuronal connections with cholera toxin subunit B: light and electron microscopic immunohistochemistry using monoclonal antibodies; Ericson H et al.; Subunit B of cholera toxin was used as a tracer substance in the central nervous system after being injected into various brain regions, mainly somatosensory relay structures . The tracer was localized with an immunoperoxidase technique, using monoclonal antibodies raised in mouse hybridomas . This method, which is applicable in both light and electron microscopic studies, is characterized by high contrast between specific labeling and unspecific background activity . It yields excellent retrograde labeling of the dendritic tree and is thus suitable for studying the neuronal cytoarchitecture and, on the ultrastructural level, the synaptic organization of identified projection neurons. Biochem Biophys Res Commun, 1988 Jun 30, 153(3), 967 - 72 Excretion of cholera toxin from Escherichia coli: a potential oral vaccine for cholera; Dasgupta U et al.; Escherichia coli strain N100 has been mutagenized by transposon mutagenesis and mutants with a cell surface leaky phenotype have been isolated . The mutant designated as E . coli N100::Tn5 excreted periplasmic proteins like ribonuclease and alkaline phosphatase . When this mutant strain was transformed with plasmids containing cloned cholera toxin genes, the toxin protein synthesized in the cells were excreted . The potentiality of this strain as a live oral vaccine for cholera has been discussed. Lancet, 1988 Jun 18, 1(8599), 1375 - 9 Impact of B subunit killed whole-cell and killed whole-cell-only oral vaccines against cholera upon treated diarrhoeal illness and mortality in an area endemic for cholera; Clemens JD et al.; The impact of B subunit killed whole-cell (BS-WC) and killed whole-cell-only (WC) oral cholera vaccines was assessed in a randomised double-blind trial in rural Bangladesh . 62,285 children aged 2-15 years and women aged over 15 ingested three doses of one of the vaccines or placebo . During the first year of follow-up there was a 26% reduction of all visits for treatment of diarrhoea in the BS-WC group and a 22% reduction in the WC group . The reduction of all admissions for fatal or severely dehydrating diarrhoea was 48% in the BS-WC group and 33% in the WC group . Overall mortality rates were 26% lower in the BS-WC group and 23% lower in the WC group during the first year, and reductions of mortality were observed only in women vaccinated at ages over 15 years . However, no differences in cumulative mortality were evident at the end of the second year of surveillance. Eur J Epidemiol, 1988 Jun, 4(2), 227 - 30 An outbreak of cholera in a refugee camp in Africa; Djeddah C et al.; A total of 541 cases of cholera were observed between May 7 and July 19, 1985 among the 9,929 displaced persons present in a refugee camp in Africa . In spite of malnutrition and other diseases affecting this population, only 12 deaths occurred . Antiepidemic measures consisted of preparation of isolation-wards, treatment of contaminated materials, training of refugees and patient care . Mass prophylaxis, initially considered, was dropped before the end of the epidemic. Biochem J, 1988 Jun 1, 252(2), 369 - 73 Opioid peptides promote cholera-toxin-catalysed ADP-ribosylation of the inhibitory guanine-nucleotide-binding protein (Gi) in membranes of neuroblastoma x glioma hybrid cells; Milligan G et al.; NG108-15 neuroblastoma x glioma hybrid cells express a major 45 kDa substrate for cholera toxin and a 40 kDa substrate(s) for pertussis toxin when ADP-ribosylation is performed in the presence of GTP . In the absence of exogenous GTP, however, cholera toxin was shown to catalyse incorporation of radioactivity into a 40 kDa protein as well as into the 45 kDa polypeptide . In membranes of cells which had been pretreated in vivo with pertussis toxin, the 40 kDa band was no longer a substrate for either pertussis or cholera toxin in vitro, whereas in membranes from cholera-toxin-pretreated cells the 40 kDa band was still a substrate for fresh cholera toxin in vitro and for pertussis toxin . In this cell line, opioid peptides have been shown to inhibit adenylate cyclase exclusively by interacting with Gi (inhibitory G-protein) and with no other pertussis-toxin-sensitive G-protein . Opioid agonists, but not antagonists, promoted the cholera-toxin-catalysed ADP-ribosylation of the 40 kDa polypeptide, hence demonstrating that this cholera-toxin substrate was indeed the alpha-subunit of Gi . These results demonstrate that Gi can be a substrate for either cholera or pertussis toxin under appropriate conditions. Eur J Biochem, 1988 Jun 1, 174(2), 317 - 21 Variations in guanine-binding proteins (Gs, Gi) in cultured bovine adrenal cells . Consequences on the effects of phorbol ester and angiotensin II on adrenocorticotropin-induced and cholera-toxin-induced cAMP production; Begeot M et al.; The corticotropin (ACTH) or cholera-toxin-induced cAMP production by cultured bovine adrenal cells increased progressively between days 0 and 7 of culture . Angiotensin II (A-II), which inhibited both basal and ACTH-stimulated adenylate cyclase of crude adrenal membranes, had no effect on ACTH-induced or cholera-toxin-induced cAMP production by fresh isolated cells (day 0) but progressively potentiated the stimulatory action of both effectors from day 0----1 to day 7 of culture . In contrast, phorbol ester had a potentiating effect on fresh isolated cells . Pretreatment of cells with pertussis toxin enhanced the potentiating effect of A-II on cells between 0 and 3 days of culture, but not after 7 days . ADP-ribosylation by cholera toxin (ribosylating alpha s proteins) or pertussis toxin (alpha i proteins), of adrenal membranes prepared from fresh isolated or cultured cells revealed an increase in alpha s and a dramatic decrease in alpha i, the ratios alpha i/alpha s on days 0, 3 and 7 of culture were 4, 0.6 and 0.1 respectively . These results indicate that (a) A-II had a double effect on ACTH-induced or cholera-toxin-induced cAMP production: one inhibitory mediated by Gi, the other stimulatory mediated by protein kinase C activation; this could explain the lack of apparent effect of A-II on fresh cells; (b) the progressive decrease of alpha i might be responsible for the appearance of the potentiating effect of A-II whereas the progressive increase of alpha s could explain the enhanced responsiveness to ACTH or cholera toxin of cultured cells. Mol Pharmacol, 1988 Jun, 33(6), 592 - 7 Effects of cholera toxin on the coupling of thyrotropin-releasing hormone to a guanine nucleotide-binding protein in cultured GH3 cells; Yajima Y et al.; The effects of cholera toxin on the coupling of the thyrotropin-releasing hormone (TRH) receptor to a guanine nucleotide-binding (G) protein were examined in a GH3 clonal strain of rat pituitary tumor cells . Incubation of the cells with cholera toxin (50 ng/ml) for 16 hr caused a decrease in {3H}methyl-TRH binding to 59% of the control level and in TRH-stimulated low Km GTPase activity from 143 to 107% of the control level in the membrane-containing fraction . The effects of cholera toxin were time dependent; TRH-stimulated GTPase activity was reduced after a 3-hr incubation, whereas cholera toxin decreased {3H}methyl-TRH binding in the membrane-containing fraction after a 5-hr incubation . These results suggest that the inhibition of TRH-stimulated GTPase activity by cholera toxin treatment is not due to the decrease of receptor binding caused by this toxin . On the other hand, incubation of GH3 cell membranes with preactivated cholera toxin and NAD+ did not substantially alter the binding of {3H}methyl-TRH . In contrast, the cholera toxin-treated membranes demonstrated a partial reduction in the activity of TRH-induced low Km GTPase activity and a 10-fold increase in the concentration of guanine nucleotide required for a half-maximal effect in regulating the TRH receptor affinity for {3H}methyl-TRH . These data suggest that cholera toxin may act directly on a G protein that is associated with TRH-receptors. J Surg Oncol, 1988 Jun, 38(2), 108 - 12 Adenylate cyclase activity and cyclic adenosine monophosphate levels in colon cancer lines and dermal fibroblasts and the effects of cholera toxin and epidermal growth factor; Nelson RL et al.; The intracellular concentration and rate of cyclic adenosine monophosphate (cAMP) synthesis, as measured by adenylate cyclase (AC) activity, were measured in dermal fibroblast cultures, colon cancer lines, and cells cultured from colonic epithelium and colonic adenomas . Dermal fibroblasts had higher AC activity and intracellular cAMP levels than the colon cancer lines (p less than 0.05) . Benign colonic epithelial cultures (mucosa and adenomas) had AC levels similar to those found in dermal fibroblasts . To characterize further these observed differences, similar measurements were made in cultures incubated in cholera toxin (CT) or epidermal growth factor (EGF) . CT stimulated AC activity and cAMP accumulation in both cancers and fibroblasts . EGF had no effect on AC activity in cancers or fibroblasts, and no effect on cAMP concentration in cancer, although EGF incubation did increase intracellular cAMP in fibroblasts . Dermal fibroblasts from colon cancer-prone patients had AC activity and cAMP concentration not significantly different, though greater, than fibroblasts from healthy individuals . Therefore, although the product of the oncogene associated with colon cancer has been shown to be an activator of AC in yeast, in human colon cancer, AC activity and intracellular cAMP concentration were much lower than in dermal fibroblasts . This difference was so great that AC activity and intracellular concentration of cAMP might be biochemical markers that can be used to differentiate colon cancer from benign cells in tissue culture. J Neurochem, 1988 Jun, 50(6), 1791 - 7 ADP-ribosylation by cholera toxin of membranes derived from brain modifies the interaction of adenylate cyclase with guanine nucleotides and NaF; Tamir A et al.; We have developed a method to ADP-ribosylate the stimulatory guanine nucleotide-binding protein of adenylate cyclase (GS) in brain membranes by using cholera toxin . In particular, we used isonicotinic acid hydrazide and 3-acetylpyridine adenine dinucleotide to inhibit the potent NAD-glycohydrolase activity of brain membranes, and we used the detergent Triton X-100 (at 0.1%) to improve the accessibility of the toxin and guanine nucleotides used for supporting the ADP-ribosylation . This method reveals that GS is a very abundant protein in membranes derived from calf brain (approximately 30 pmol/mg of protein) . In brain, GS exists in large excess over the previously reported amount of the adenylate cyclase catalytic subunit . The modification of GS with an ADP-ribosyl residue (a) elicits a four- to fivefold activation of adenylate cyclase by GTP, (b) increases the stabilization of adenylate cyclase by GTP, and (c) reduces adenylate cyclase activation by fluoride but does not change basal activity, activation by guanosine 5'-(beta, gamma-imido)triphosphate, or the sensitivity of adenylate cyclase to heat-induced denaturation . A correlation between ADP-ribosylation and the alterations in the activation of adenylate cyclase by guanine nucleotides and by fluoride is presented. In Vitro Cell Dev Biol, 1988 Jun, 24(6), 537 - 44 Reinitiation of DNA synthesis in quiescent mouse keratinocytes; regulation by polypeptide hormones, cholera toxin, dexamethasone, and retinoic acid; Reiss M et al.; Cloned mouse keratinocytes (MK-1 cells) display density-dependent growth arrest when reaching confluency in a serum-free medium with a calcium concentration less than 0.1 mM, supplemented only with insulin and transferrin . In this quiescent state, greater than 95% of the cell population is in the Go/1 phase of the cell cycle . Treatment of quiescent MK-1 cells with 1 to 10 ng/ml epidermal growth factor (EGF) resulted in a sharp burst of DNA synthetic activity . Both insulin and cholera toxin potentiated the mitogenic effect of EGF, but neither agent was necessary or sufficient to induce thymidine incorporation into DNA . Dexamethasone abolished the effect of insulin, but not the mitogenic effect of EGF alone . In contrast, retinoic acid (RA) did not possess any mitogenic effect for quiescent MK-1 cells, nor did it modulate the actions of EGF or dexamethasone . A number of commercially available crude extracts of bovine brain and pituitary were also capable of initiating DNA synthesis in resting MK-1 cells . Finally, transforming growth factor type beta (TGF beta) proved to be a potent inhibitor of the mitogen-induced DNA synthesis in MK-1 cells (IC50:10 pM) . This defined culture system is eminently suited to study the regulation of DNA synthesis of epidermal cells . In addition, it can be used as a sensitive bioassay for the detection of epidermal mitogens, as well as inhibitors of DNA synthesis such as TGF beta. Biochim Biophys Acta, 1988 May 13, 969(3), 249 - 56 Insertion of ganglioside GM1 into rat glioma C6 cells renders them susceptible to growth inhibition by the B subunit of cholera toxin; Spiegel S; The B subunit of cholera toxin does not affect the growth of rat glioma C6 cells which are deficient of its receptor, ganglioside GM1 . Insertion of ganglioside GM1 into the plasma membrane of C6 cells renders them susceptible to inhibition of DNA synthesis by the B subunit . Exposure of C6 cells to butyrate induces an elevation of ganglioside GM1 as measured by an increase in binding of iodinated cholera toxin and also results in an inhibition of DNA synthesis by the B subunit . The extent of inhibition of DNA synthesis correlated with the binding of B subunit and was independent of adenylate cyclase activation or increases in intracellular cAMP levels. Immunol Lett, 1988 May, 18(1), 51 - 5 Cholera toxin neutralization: a comparison of purified serum IgG and biliary secretory IgA antibodies; Pierre P et al.; Rats were immunized three times with cholera toxin via the intraintestinal or intravenous route, and their respective biliary secretory IgA (sIgA) or serum IgG antibodies were affinity-purified on a cholera toxin immunoabsorbent . On a molar basis, the sIgA antibodies were roughly seven-fold more efficient than IgG antibodies in neutralizing cholera toxin in the ligated intestinal loop assay . Various explanations for this difference in neutralizing capacity are proposed. J Cell Biochem, 1988 May, 37(1), 119 - 29 Inhibition of the proliferation of Nb2 cells by femtomolar concentrations of cholera toxin and partial reversal of the effect by 12-O-tetradecanoyl-phorbol-13-acetate; Pines M et al.; One hour of exposure to cholera toxin is sufficient to elicit a significant delay in the initiation of DNA synthesis and cell division in lactogenic hormone-dependent Nb2-11C lymphoma cells . The inhibitory effect occurs already at very low concentrations of cholera toxin (5-50 fM), at which it is not accompanied by a detectable increase in intracellular cAMP, or ADP-ribosylation of the alpha subunit of Gs, the stimulatory guanine nucleotide binding protein of adenylate cyclase; IBMX, the phosphodiesterase inhibitor, acts synergistically to cholera toxin, indicating that a minute increase in cAMP may be sufficient for the inhibition . This indication is substantiated by the finding that dibutyryl cAMP also inhibits cell proliferation . Phorbol diester reverses partially the inhibitory activity of cholera toxin . It is most likely that this effect does not result from blocking the increase in cAMP, but rather from some subsequent, yet unidentified, events . The inhibitory effect of cholera toxin is not dependent on the concentration of the proliferation-stimulating lactogenic hormone and cannot be abolished or reduced by excess of the hormone . Cholera toxin also inhibits the autonomous proliferation of a lactogenic hormone-independent cell line (Nb2-SP); however, in this case the inhibition is not affected by TPA. Cell Immunol, 1988 May, 113(2), 235 - 50 The effects of pertussis toxin and cholera toxin on mitogen-induced interleukin-2 production: evidence for G protein involvement in signal transduction; Gilmore W et al.; GTP-binding proteins, known as G proteins, play important roles in transducing signals generated by the binding of specific ligands to cell surface receptors . We examined the possibility that a G protein is involved in transducing the concanavalin A (Con A) signal for IL-2 production using a T-cell hybridoma, FS6-14.13, and the bacterial toxins, pertussis toxin (PTX) and cholera toxin (CTX) . These toxins are known to interact with and modify the functions of G proteins . High concentrations of PTX (25-50 micrograms/ml) stimulated IL-2 production in the FS-6 cells in the absence of Con A, presumably due to the ability of its B subunit to crosslink membrane proteins . However, in the presence of Con A, PTX inhibited IL-2 production at concentrations ranging from 0.05 to 50 micrograms/ml . It is unlikely that this inhibition was due to a competitive interaction between Con A and PTX for binding sites at the cell surface, since high concentrations of PTX only minimally reduced Con A-FITC binding, evaluated by FACS analysis . In addition, concentrations of PTX which were not able to stimulate IL-2 production in the absence of Con A, retained their ability to inhibit IL-2 production in the presence of Con A . These data suggest the involvement of the PTX A subunit in this activity . In support of this possibility, PTX catalyzed ADP-ribosylation of a Mr = 41,000-Da protein in FS-6 membranes . This strongly suggests that a PTX substrate is involved in transducing the Con A signal for IL-2 production in FS-6 cells . CTX also inhibited Con A-induced IL-2 production, an effect mimicked by the addition of dibutyryl-cAMP . This suggests that a CTX substrate linked to the adenylyl cyclase-cAMP pathway is probably not involved in transducing the stimulatory Con A signal, but may play a role in downregulating T-cell activation. Biochem J, 1988 Apr 15, 251(2), 447 - 52 Insulin and glucagon attenuate the ability of cholera toxin to activate adenylate cyclase in intact hepatocytes; Irvine FJ et al.; Treatment of intact hepatocytes with cholera toxin at 37 degrees C caused a stable activation of adenylate cyclase activity after a lag period of around 10 min . The presence of either insulin (10 nM) or glucagon (10 nM) in the incubation medium had little effect on this lag period; however, these hormones markedly attenuated the maximal activation of adenylate cyclase activity that could be achieved by treatment with cholera toxin . Such actions of insulin and glucagon were dose-dependent, with EC50 values (concn . giving 50% inhibition) of 0.20 nM for insulin and 0.49 nM for glucagon, and were not additive . Treatment of intact hepatocytes with either glucagon or insulin did not affect the ability of cholera toxin to cause the ADP-ribosylation of the 45 kDa alpha-subunit of the stimulatory guanine nucleotide regulatory protein, Gs, in intact hepatocytes . It is suggested that treatment of intact hepatocytes with either insulin or glucagon attenuates the stimulatory action of ADP-ribosylated Gs on adenylate cyclase. Epidemiol Infect, 1988 Apr, 100(2), 279 - 89 Epidemic cholera in Mali: high mortality and multiple routes of transmission in a famine area; Tauxe RV et al.; During the 1984 cholera epidemic in Mali, 1793 cases and 406 deaths were reported, a death-to-case ratio of 23% . In four affected villages, the mean clinical attack rate was 1.5 and 29% of affected persons died . In 66% of cases the illness began more than 48 h after the village outbreak began, when supplies from outside the village were potentially available . Deaths occurred because patients failed to seek care or received only limited rehydration therapy when they did . Case-control studies identified two routes of transmission: drinking water from one well in a village outside the drought area, and eating left-over millet gruel in a drought-affected village . Drought-related scarcity of curdled milk may permit millet gruel to be a vehicle for cholera . Cholera mortality in the Sahel could be greatly reduced by rapid intervention in affected villages, wide distribution of effective rehydration materials, and educating the population to seek treatment quickly. Vet Microbiol, 1988 Apr, 16(4), 315 - 21 Identification of hog cholera viral isolates by use of monoclonal antibodies to pestiviruses; Hess RG et al.; A collection of 90 field isolates of hog cholera virus (HCV) was used to test the specificity of four hybridoma cell lines secreting monoclonal antibodies against pestiviruses . Reaction of virus isolates and monoclonal antibodies was controlled by an indirect immunofluorescence assay (IFA) . Two monoclonal antibodies which had been generated against HC virus strain "Alfort 187" were reactive only with HCV field isolates and an HCV reference strain but not with bovine viral diarrhoea virus (BVDV) reference strains . Two other monoclonal antibodies (generated against BVDV, strain NADL) reacted only with BVDV reference strains but not with HCV field isolates, although with 3 of these strains focal reactions involving only a few cells were detected . The ability to discriminate between both viruses is a diagnostic need which may be fulfilled by these monoclonal antibodies. Proc Natl Acad Sci U S A, 1988 Apr, 85(8), 2504 - 8 Effect of cholera toxin on histamine release from bone marrow-derived mouse mast cells; Saito H et al.; Bone marrow-derived mouse mast cells were sensitized with monoclonal mouse IgE antibody and treated with cholera toxin (CT), which ADP-ribosylated the alpha-subunit of the stimulatory guanine nucleotide-binding regulatory protein Gs, prior to challenge with either antigen or thrombin . The CT treatment increased intracellular cAMP levels, but neither enhanced nor inhibited antigen-induced histamine release or arachidonate release . The same treatment of the sensitized bone marrow-derived mouse mast cells with CT markedly enhanced thrombin-induced histamine release without affecting arachidonate release . The CT treatment failed to affect antigen-induced and thrombin-induced generation of inositol trisphosphate and of diacylglycerol or mobilization of intracellular Ca2+ . The results indicate that Gs in bone marrow-derived mouse mast cells is not involved in the transduction of the antigen-induced or thrombin-induced triggering signal to phospholipase C, which initiates the enhancement of phosphatidylinositol turnover . The enhancement of thrombin-induced histamine release by CT treatment with the observations that thrombin-induced histamine release was inhibited by pretreatment of the cells with pertussis toxin suggest that the involvement of a guanine nucleotide-binding regulatory protein in thrombin-induced biochemical events is an event distal to Ca2+ mobilization. Biochemistry, 1988 Mar 22, 27(6), 2046 - 52 Thermal stability and intersubunit interactions of cholera toxin in solution and in association with its cell-surface receptor ganglioside GM1; Goins B et al.; The thermal stability of cholera toxin free in solution and in association with its cell-surface receptor ganglioside GM1 has been studied by using high-sensitivity differential scanning calorimetry and differential solubility thermal gel analysis . In the absence of ganglioside GM1, cholera toxin undergoes two distinct thermally induced transitions centered at 51 and 74 degrees C, respectively . The low-temperature transition has been assigned to the irreversible thermal denaturation of the active A subunit . The second transition has been assigned to the reversible unfolding of the B subunit pentamer . The isolated B subunit pentamer exhibits a single transition also centered at 74 degrees C, suggesting that the attachment of the A subunit does not contribute to the stability of the pentamer . In the intact toxin, the A subunit dissociates from the B subunit pentamer at a temperature that coincides with the onset of the B subunit thermal unfolding . In aqueous solution, the denatured A subunit precipitates after dissociation from the B subunit pentamer . This phenomenon can be detected calorimetrically by the appearance of an exothermic heat effect . In the presence of ganglioside GM1, the B subunit is greatly stabilized as indicated by an increase of 20 degrees C in the transition temperature . In addition, ganglioside GM1 greatly enhances the cooperative interactions between B subunits . In the absence of ganglioside, each monomer within the B pentamer unfolds in an independent fashion whereas the fully ganglioside-bound pentamer behaves as a single cooperative unit . On the contrary, the thermotropic behavior of the A subunit is only slightly affected by the presence of increasing concentrations of ganglioside GM1.(ABSTRACT TRUNCATED AT 250 WORDS) Science, 1988 Mar 11, 239(4845), 1272 - 6 Three-dimensional structure of cholera toxin penetrating a lipid membrane; Ribi HO et al.; Two-dimensional crystals of cholera toxin bound to receptors in a lipid membrane give diffraction extending to 15 A resolution . Three-dimensional structure determination reveals a ring of five B subunits on the membrane surface, with one-third of the A subunit occupying the center of the ring . The remaining mass of the A subunit appears to penetrate the hydrophobic interior of the membrane . Cleavage of a disulfide bond in the A subunit, which activates the toxin, causes a major conformational change, with the A subunit mostly exiting from the B ring. Arkh Anat Gistol Embriol, 1988 Mar, 94(3), 69 - 72 {Morphological bases for the resistance of the intestines to the action of cholera toxoid}; Kalugina OP; Effect of antigen (cholera toxoid) has been studied on distribution of specific antibody-containing plasma cells in the lamina propria of the mucous membrane of the guinea pig small and large intestine . Since it is known that experimental cholera is easily reproduced in these animals, amount of plasmocytes is determined after primary intraperitoneal and secondary intraduodenal immunization with cholera toxoid . At the primary immunization maximal increase in amount of plasmocytes is noted in the jejunum on the 70th day; at the secondary--on the 19th day . At the primary parenteral immunization of the animals with the antigen, the intensity of the immune response in the lamina propria of the mucous membrane increases . At the secondary immunization the local immunity is more manifested in comparison with the primary one. Mol Immunol, 1988 Mar, 25(3), 223 - 30 Induction of polyclonal and monoclonal antibody responses to cholera toxin by the synthetic peptide approach; Ghose AC et al.; The induction of an antibody response to cholera toxin (CT) was studied by using the synthetic peptide approach . Two peptides, corresponding to the amino acid sequences from residues 57 to 69 (CTBP1) and 47 to 60 (CTBP2) of the cholera toxin B subunit, were synthesized by the solid-phase method . These peptides were primarily chosen on the basis of their hydrophilicity and sequence identity with the B subunit of E . coli toxin (LTh) . Synthesized peptides were coupled to carrier proteins through additional cysteine residues at the carboxyl (CTBP1) or amino terminal ends (CTBP2) . Rabbit antisera to the peptide-carrier conjugates were found to react with the free peptides as well as intact CT, its B subunit and LTh as determined by the conventional enzyme-linked immunosorbent assay (ELISA) . On the other hand, anti-peptide sera failed to react with CT and LTh in GM1 (ganglioside)--ELISA, thereby suggesting the possible involvement of CTBP1 and CTBP2 peptide regions of the toxin molecule in GM1 receptor binding . Both anti-peptide sera possessed rather weak toxin neutralizing activity in the rabbit ileal loop assay . However, such activity was statistically significant (0.02 less than P less than 0.05) only in the case of anti-CTBP2 serum . Similar results were also obtained with mouse polyclonal anti-peptide sera . Ten mouse monoclonal antibodies were obtained against the CTBP1 peptide, five of which reacted to CT, the B subunit and LTh in ELISA . Interestingly, one monoclonal showed strong reactivity against CT and LTh although it reacted very weakly against the immunizing peptide CTBP1 . It appears that the immunizing peptide probably exists in multiple conformers in the conjugated form, some of which may mimic more closely its structural features in the intact protein than in the free state . Results obtained in this study suggest that synthetic peptides can serve as useful probes for the structural analysis of CT or related toxins and may be useful in vaccine development. Exp Hematol, 1988 Mar, 16(3), 195 - 200 Cholera toxin and phorbol diesters synergistically modulate murine hematopoietic progenitor cell proliferation; Long MW et al.; Little information exists concerning the role of guanine nucleotide-binding proteins (GNBP) in hematopoietic progenitor cell proliferation . We hypothesized that GNBP-mediated activation of adenylate cyclase plays an important role in factor-driven hematopoietic cell proliferation . Using cholera toxin and other probes of the cyclase system, we observe that cyclase activation results in a lineage-specific amplification of megakaryocytic progenitor cell numbers, an intermediate effect on erythroid progenitors, and, conversely, an inhibition of granulocytic colony formation . The effect of GNBP activation is synergistic with, and dependent upon, concomitant activation of the cells with phorbol diesters . This suggests a role for both calcium-dependent mechanisms, such as protein kinase C activation, and for other processes mediated by GNBP and cyclic AMP . The use of an intracellular calcium antagonist (Quin-2) partially abrogates the GNBP-mediated response, confirming that the effects of GNBP activation involve both calcium-dependent and calcium-independent processes . We conclude that hematopoietic progenitor cells are influenced by lineage-specific alterations in GTP-binding protein function, which affects both adenylate cyclase activity and calcium homeostasis. Mol Cell Endocrinol, 1988 Mar, 56(1-2), 165 - 9 Differential regulation of hCG and progesterone secretion by cholera toxin and phorbol ester in human cytotrophoblasts; Ritvos O et al.; The secretion of hCG and progesterone (P) by human cytotrophoblasts was studied in response to cholera toxin (CT), which activates adenylate cyclase, and 12-O-tetradecanoylphorbol-13-acetate (TPA), a protein kinase C stimulator . During a 24 h incubation CT and TPA increased hCG and P secretion in a concentration-dependent manner . At maximal effective concentrations, CT (1.0 ng/ml), TPA (10 ng/ml) and CT plus TPA stimulated hCG production by 5.7-, 2.7- and 10.0-fold, respectively, and P production by 1.8-, 1.8- and 2.0-fold, respectively . Time-course studies indicated that these effects became detectable after 12 h and increased up to 48 h of incubation . During a 24 h culture TPA potentiated CT-induced cAMP formation by 1.4-fold indicating that its effects on hCG production may be, at least partly, mediated through cAMP . In conclusion, CT and TPA are potent stimulators of human cytotrophoblast hCG and P production . Simultaneous stimulation by these agents results in potentiation of hCG production whereas P production remains at the same level as with CT or TPA alone . The results suggest that the endocrine activity of human cytotrophoblasts is under multihormonal control and hCG and P secretion are differentially regulated. Biol Reprod, 1988 Mar, 38(2), 315 - 23 Inhibitory effect of analogues of cyclic nucleotides and cholera toxin on relaxin release from cultured porcine luteal cells; Taylor MJ et al.; The role of cyclic nucleotides (cyclic 3',5'-adenosine monophosphate {cAMP} and cyclic 3',5'-guanosine monophosphate {cGMP}) in the regulation of relaxin release from large porcine luteal cells was examined by use of a reverse hemolytic plaque assay . In this assay, luteal cells are cocultured in monolayers with protein-A-coupled ovine erythrocytes . In the presence of porcine relaxin antiserum and complement, a zone of hemolysis--a plaque--develops around relaxin-releasing luteal cells . The rate of development of plaques in time-course studies has been used as an index of the rate of relaxin release, and the size of plaques formed has been employed as a record of the cumulative amount of relaxin released by each cell . Treatment of monolayers with dibutyryl cAMP (dbcAMP, 60 mM) and dibutyryl GMP (dbcGMP, 15 mM resulted in a prompt inhibition in the rate of plaque formation . In addition, dbcAMP treatment reduced the average size of plaques formed . The stimulatory effect of prostaglandin F2 alpha (PGF2 alpha 10(-6) M) on relaxin release was significantly attenuated by combined treatment with dbcAMP (60 mM) . Cholera toxin treatment (500 ng/ml) effectively reduced the average size of plaques formed, but neither this agent nor the beta-adrenergic agonist, isoproterenol (up to 5 X 10(-3) M), influenced the rate of plaque formation . These results--which provide evidence to show that both basal and stimulated relaxin release by large porcine luteal cells can be inhibited by the cyclic nucleotide analogues, dbcAMP and dbcGMP--are consistent with the view that these compounds have the potential to act as a negative regulatory mechanism for relaxin release.(ABSTRACT TRUNCATED AT 250 WORDS) J Biol Chem, 1988 Feb 25, 263(6), 2808 - 16 The bombesin receptor is coupled to a guanine nucleotide-binding protein which is insensitive to pertussis and cholera toxins; Fischer JB et al.; The neuropeptide bombesin acts on a variety of target cells to stimulate the processes of secretion and cell proliferation . In this study we determined whether bombesin receptors interact with known guanine nucleotide-binding proteins in four different cell types: GH4C1 pituitary cells, HIT pancreatic islet cells, Swiss 3T3 fibroblasts, and rat brain tissue . Maximal concentrations of nonhydrolyzable GTP analogs decreased agonist binding to bombesin receptors in membranes from all four sources . In GH4C1 and HIT cell membranes GTP analogs inhibited bombesin receptor binding with IC50 values of about 0.1 microM, whereas GDP analogs were approximately 10-fold less potent . In contrast, GMP and the nonhydrolyzable ATP analog adenylyl-imidodiphosphate had no effect at 100 microM . Equilibrium binding experiments in GH4C1 and HIT cell membranes indicated a single class of binding sites with a dissociation constant (Kd) for {125I-Tyr4}bombesin of 24.4 +/- 7.0 pM and a binding capacity of 176 +/- 15 fmol/mg protein . Guanine nucleotides decreased the apparent affinity of the receptors without significantly changing receptor number . Consistent with this observation, guanine nucleotides also increased the rate of ligand dissociation . Pretreatment of GH4C1 or HIT cells with either pertussis toxin (100 ng/ml) or cholera toxin (500 ng/ml) for 18 h did not affect agonist binding to membrane bombesin receptors, its regulation by guanine nucleotides, or bombesin stimulation of hormone release . Although pertussis toxin pretreatment has been reported to block bombesin stimulation of DNA synthesis in Swiss 3T3 cells, it did not alter the binding properties of bombesin receptors in Swiss 3T3 membranes or inhibit the rapid increase in intracellular {Ca2+} produced by bombesin in these cells . In summary, our results indicate that the bombesin receptor interacts with a guanine nucleotide-binding protein which exhibits a different toxin sensitivity from those which regulate adenylate cyclase as well as those which couple some receptors to phospholipases. Eur J Biochem, 1988 Feb 1, 171(3), 509 - 13 Synergistic stimulation of S-adenosylmethionine decarboxylase activity by Ca2+ ionophore A23187, cholera toxin and 1-oleoyl-2-acetylglycerol; Otani S et al.; The Ca2+ ionophore A23187 induced S-adenosylmethionine decarboxylase in guinea-pig lymphocytes, and cholera toxin stimulated the induction synergistically . The activator of protein kinase C, 1-oleoyl-2-acetylglycerol, did not induce S-adenosylmethionine decarboxylase activity but potentiated the enzyme activity induced by A23187 or by A23187 and cholera toxin . The addition of both A23187 and cholera toxin induced S-adenosylmethionine decarboxylase, but the further addition of 1-oleoyl-2-acetylglycerol or 12-O-tetradecanoylphorbol 13-acetate did not potentiate the enzyme induction in protein kinase-C-down-regulated cells that had been treated with 12-O-tetradecanoylphorbol 13-acetate for 18 h . These results suggest that a Ca2+-dependent pathway, other than that for protein kinase C, is essential for the induction of S-adenosylmethionine decarboxylase and that a cAMP-dependent pathway and also protein kinase C are involved in the potentiation of the induction. Mol Endocrinol, 1988 Feb, 2(2), 148 - 58 Regulation of follicle-stimulating hormone binding to receptors on bovine calf testis membranes by cholera toxin-sensitive guanine nucleotide binding protein; Zhang SB et al.; In a previous study we reported that FSH receptors in bovine testes membranes are physically and functionally associated with a guanine nucleotide-binding protein (N protein) . In this study we examined the mechanism whereby GTP binding to N protein regulates FSH binding to its receptors . Binding of FSH to receptors decreased in the presence of GTP in a dose-dependent and noncompetitive manner . This effect did not require the presence of Mg+2 and is in contrast to the reported requirement for Mg+2 for GTP effects on human CG binding to ovarian receptors . Equilibrium binding experiments indicated that decreased hormone binding in the presence of GTP was not due to a decrease in the number of FSH receptors per se; rather, the altered binding isotherm was the result of a decrease in affinity of receptors for FSH . Moreover, the dissociation of {125I}human FSH from preformed FSH-receptor complex was rapid in onset and significantly accelerated in the presence of GTP . In a series of nucleotides, GTP was most effective in causing this effect . Evidently, occupancy of GTP binding sites on the N protein, including low affinity and high capacity sites, is necessary for GTP regulation of FSH binding to receptors . The fact that pretreatment of bovine testis membranes with cholera toxin plus NAD, but not pertussis toxin plus NAD, eliminates the GTP effect on FSH binding to its receptors suggests that the GTP regulatory binding protein mediating the GTP regulation of FSH binding is probably Ns and not Ni . Further characterization of FSH receptor sensitivity to GTP, however, indicated that the N protein involved does not exhibit all of the characteristics reported for Ns . For example, the affinity of GTP for N protein is relatively low even under conditions where GTP hydrolysis has a minimal effect in reducing the total concentration of GTP . Also, the absence of a requirement for Mg+2 in high affinity FSH receptor-N protein coupling is different from the requirement for Mg+2 seen with the beta-adrenergic receptor and Ns . Moreover, the N protein which mediates GTP regulation of FSH-receptor binding appears to be relatively insensitive to N-ethylmaleimide, unlike the N-ethylmaleimide sensitivity of the turkey erythrocyte Ns . These results suggest that differences may exist in the structure-function features of GTP regulatory binding protein associated with different types of hormone ligands and receptors. J Pharmacol Exp Ther, 1988 Feb, 244(2), 765 - 73 Cholera toxin and pertussis toxin stimulate prostaglandin E2 synthesis in a murine macrophage cell line; Burch RM et al.; When RAW264.7 murine macrophages were incubated with cholera toxin or pertussis toxin, prostaglandin E2 (PGE2) synthesis was enhanced markedly . Cholera toxin and pertussis toxin added together synergistically stimulated PGE2 synthesis . Cholera toxin and pertussis toxin also stimulated cyclic AMP (cAMP) accumulation . However, PGE2 synthesis was independent of increases in cAMP, as neither forskolin nor isoproterenol, which increased cAMP accumulation, nor dibutyryl-cAMP had any effect on PGE2 synthesis . In intact cells, cholera toxin and pertussis toxin stimulated phospholipase A2 to enhance metabolism of phosphatidylinositol to lysophosphatidylinositol and glycerophosphoinositol, with time courses similar to their stimulation of PGE2 synthesis . Cholera toxin catalyzed ADP-ribosylation of proteins of Mr 45,000 and 49,000 in intact cells, whereas an additional substrate of Mr 41,000 was observed in vitro . Preincubation of intact cells with pertussis toxin blocked subsequent in vitro labeling of the Mr 41,000 protein by cholera toxin, suggesting that the same protein was ADP-ribosylated by both toxins . Western blot analysis using specific antisera against Gi, Go and Gs revealed that the Mr 41,000 substrate was bound by the anti-Gi and anti-Go but not anti-Gs . The present data suggest that guanine nucleotide binding regulatory proteins are involved in the regulation of arachidonic acid metabolism to PGE2 in RAW264.7 cells . Furthermore, the possibility is raised that phospholipase A2 is regulated by both stimulatory and inhibitory guanine nucleotide binding proteins. Biochemistry, 1988 Jan 26, 27(2), 717 - 24 NMR study of the complexes between a synthetic peptide derived from the B subunit of cholera toxin and three monoclonal antibodies against it; Anglister J et al.; The contact interactions between a synthetic peptide and three different anti-peptide monoclonal antibodies have been studied by nuclear magnetic resonance (NMR) . The synthetic peptide is CTP3 (residues 50-64 of the B subunit of cholera toxin) suggested as a possible epitope for synthetic vaccine against cholera . The hybridoma cell lines TE33 and TE32 derived after immunization with CTP3 produce antibodies cross-reactive with the native toxin . The cell line TE34 produces anti-CTP3 antibodies that do not bind the toxin . Selective deuteriation of the antibodies has been used to simplify the proton NMR spectra and to assign resonances to specific types of amino acids . The difference spectra between the proton NMR spectrum of the peptide-Fab complex and that of Fab indicate that the combining site structures of TE32 and TE33 are very similar but differ considerably from the combining site structure of TE34 . By magnetization transfer experiments with selectively deuteriated Fab fragment of the antibody, we have found that in TE32 and TE33 the histidine residue of the peptide is buried in a hydrophobic pocket of the antibody combining site, formed by a tryptophan and two tyrosine residues . The hydrophobic nature of the pocket is further demonstrated by the lack of any pH titration effect on the chemical shift of the C4H of the bound peptide histidine . In contrast, for TE34 we have found only one tyrosine residue in contact with the histidine of the peptide.(ABSTRACT TRUNCATED AT 250 WORDS) J Biol Chem, 1988 Jan 25, 263(3), 1111 - 4 Epidermal growth factor stimulates formation of inositol phosphates in BALB/c/3T3 cells pretreated with cholera toxin and isobutylmethylxanthine; Olashaw NE et al.; Epidermal growth factor (EGF) stimulated the formation of inositol trisphosphate, inositol bisphosphate, and inositol phosphate in density-arrested BALB/c/3T3 cells pretreated for 1.5-4 h with cholera toxin, a potent activator of adenyl cyclase, and isobutylmethylxanthine (IBMX), a phosphodiesterase inhibitor . Concomitant addition of cholera toxin, IBMX, and EGF to cells did not increase inositol phosphate levels, and pretreatment with both agents was more effective than pretreatment with either alone . Pre-exposure of cells to cholera toxin and IBMX also enhanced the increase in inositol phosphates occurring in response to platelet-derived growth factor (PDGF) . Preincubation of cells with cholera toxin and IBMX in the presence of cycloheximide abolished the effects of these agents on EGF- and PDGF-stimulated inositol phosphate production as well as the lesser increase in inositol phosphate formation produced by cholera toxin and IBMX in the absence of hormone . Preincubation of cells with cycloheximide did not affect EGF binding or the ability of PDGF to stimulate inositol phosphate formation . Cycloheximide also precluded EGF-induced inositol phosphate production when presented to cells 3 h after addition of cholera toxin and IBMX . These findings show that, under the appropriate conditions, EGF is capable of stimulating inositol phosphate formation in a nontransformed cell line. Avian Dis, 1988 Jan-Mar, 32(1), 16 - 23 Epidemiology and financial impact of fowl cholera in turkeys: a retrospective analysis; Carpenter TE et al.; A retrospective study of the epidemiology and financial impact of fowl cholera (FC) in California meat turkeys during 1984 was performed . Data were collected from 64 flocks--23 FC-outbreak flocks and 41 controls (non-outbreak)--raised in the Central Valley of the state . Mean flock age at the time of the FC outbreak was 11.3 weeks . Flocks that reported a colibacillosis outbreak had increased odds (P = 0.11) of also having an FC outbreak . (This association may or may not indicate a cause-effect relationship.) There was no significant difference between FC-outbreak and control flocks in number of diseases reported, age at onset, or duration of diseases or syndromes except age at onset of roundheart disease . The relative mortality rates were 52% higher in FC-outbreak toms and 26% higher in FC-outbreak hens than in their controls . Medication costs were nearly tripled, and the relative condemnation rate was 60% higher in FC-outbreak flocks than in control flocks . The average costs of FC were nearly $0.40 per bird, or $18,750 per flock, in an outbreak flock of 50,000 birds, and $0.12 per bird, or $6000 per flock, in non-outbreak flocks vaccinated against FC. Int Arch Allergy Appl Immunol, 1988, 85(3), 358 - 63 IgG and IgA subclass distribution of antitoxin antibody responses after oral cholera vaccination or cholera disease; Jertborn M et al.; The immunoglobulin subclass distribution of cholera antitoxin antibody responses in serum was studied in Swedish volunteers after different routes of immunization with a cholera B subunit-whole cell vaccine (B + WCV) and in Bangladeshi patients convalescing from cholera disease . Both oral and parenteral immunization induced antitoxin antibodies of all the different IgG subclasses (IgG1, IgG2, IgG3 and IgG4) whilst the IgA antibodies were restricted to the IgA1 subclass . A single oral dose of B + WCV induced proportionally higher levels of IgG4 antitoxin in previously cholera-immunized volunteers than in a matched group who had not been cholera-vaccinated before, suggesting that repeated immunization preferentially stimulate formation of IgG4 antibodies . The IgG and IgA subclass distribution of antitoxin antibodies in orally vaccinated Swedes closely resembled that in Bangladeshi cholera patients. J Neurosci Methods, 1988 Jan, 22(3), 189 - 94 A simple and rapid method for the production of cholera B-chain coupled to horseradish peroxidase for neuronal tracing; McIlhinney RA et al.; A simple and rapid method is described for the production of cholera B-chain coupled to horseradish peroxidase suitable for neuronal tracing . Horseradish peroxidase was activated with a mild sodium periodate oxidation followed by incubation with the cholera B-chain, to yield the conjugates, samples of which were fractionated further by size exclusion chromatography . Both fractionated material and unfractionated conjugates were tested in vitro by a binding assay on rat synaptic membranes, and in vivo by their ability to undergo retrograde transport following injection into the adrenal medulla of rats . These assays showed that the unfractionated conjugates were equivalent to the fractionated conjugates in binding to the rat synaptic membranes, and that they both provided good retrograde labelling of neurones in the spinal cord after injection into the adrenal medulla, with extensive labelling of the distal dendrites. Infect Immun, 1988 Jan, 56(1), 230 - 3 Enterotoxigenic Escherichia coli diarrhea in an endemic area prepares the intestine for an anamnestic immunoglobulin A antitoxin response to oral cholera B subunit vaccination; Holmgren J et al.; We examined whether infection with enterotoxigenic Escherichia coli (ETEC) producing the heat-labile enterotoxin (LT) can prime the gut immune system to respond more efficiently to the immunologically related cholera B subunit component of a recently developed oral B subunit-whole-cell cholera vaccine (B-WCV) . Nine Bangladeshi adults who had been hospitalized for watery diarrhea caused by LT-producing ETEC were given a single oral immunization with B-WCV on day 28 after hospital admission . The vaccine preparation used was adjusted to contain a lower-than-usual dose of B subunit, which had been found in previous studies to elicit a significant gut mucosal immunoglobulin A antitoxin response mainly in individuals with previous toxin-specific primin