Am J Epidemiol, 1985 Jun, 121(6), 791 - 6 Predisposition for cholera of individuals with O blood group . Possible evolutionary significance; Glass RI et al.; At the Matlab Hospital of the International Centre for Diarrhoeal Disease Research, Bangladesh, the authors examined the blood groups of patients hospitalized between January and September 1979 for diarrheal disease due to a variety of bacterial and viral agents . A significant association was identified only for cholera, in which cholera patients were twice as likely to have blood group O and one-ninth as likely to have blood group AB as community controls . A follow-up study of family contacts of cholera patients, carried out between September 1980 and July 1982, indicated that blood group did not affect an individual's risk of having a culture-proven infection with V . cholerae 01 but was directly related to the severity of disease . Individuals with the most severe diarrhea compared with those with asymptomatic infection were more often of blood group O (68% versus 36%, p less than 0.01) and less often of AB (0% versus 7%, p less than 0.01) . It was not possible to identify the molecular basis for this genetically related protection using biologic models of cholera that are currently available . The constant selective pressure of cholera against people of O blood group may account in part for the extremely low prevalence of O group genes and the high prevalence of B group genes found among the people living in the Gangetic Delta. Arch Biochem Biophys, 1985 Jun, 239(2), 587 - 94 Adenylate cyclase from rabbit small intestine: activation by cholera toxin and interaction with calcium; Lazo PS et al.; The stimulation of adenylate cyclase in various fractions of plasma membranes from rabbit small intestinal epithelium has been studied . In crude plasma membranes cholera toxin activated 5-fold at 10 micrograms/ml; vasoactive intestinal peptide (VIP) activated at concentration from 10(-8) to 10(-7) M, the maximal stimulation being 6-fold . Fluoride activated 10-fold at 10 mM . VIP-stimulated enzyme was inhibited by Ca2+ concentrations in the micromolar range . In the presence of calmodulin a biphasic response was obtained . At low Ca2+ concentration (4 x 10(-9)-6 x 10(-8) M) the enzyme was activated . As the Ca2+ concentration was increased the enzyme was concomitantly inhibited . We have investigated the mechanism by which cholera toxin activates intestinal adenylate cyclase . We have found that cholera toxin catalyzed incorporation of 32P into proteins located in the brush-border membrane whose molecular weights are in the range of 40-45kDa . These membranes bind {3H}GTP with a Kd of 1.8 x 10(-7) M . In contrast, basal lateral membranes do not contain any protein which becomes labeled in a toxin-dependent manner when incubated with cholera toxin and {32P}NAD . The modification of brush-border membrane protein occurred in spite of the absence of adenylate cyclase in these membranes . Adenylate cyclase in basal lateral membranes was poorly activated by cholera toxin as compared to crude plasma membranes . On the other hand, the ability of VIP and fluoride to activate the enzyme was enhanced in basal lateral membranes with respect to crude membranes . The results are discussed in relation to the mechanism by which cholera toxin activates adenylate cyclase in intact intestinal cells. Blood, 1985 Jun, 65(6), 1544 - 8 Inhibitors of cholera toxin-induced adenosine diphosphate ribosylation of membrane-associated proteins block stem cell differentiation; Dexter TM et al.; Two potent inhibitors of mono-adenosine diphosphate (ADP) ribosylation have recently been described and characterized, named p-methoxylbenzylaminodecamethylene guanidine sulfate (MBAMG) and benzylaminododecylguanine hydrochloride (BADGH) . We have used these agents to investigate the role of ADP ribosylation in hematopoiesis using long-term marrow cultures . The addition of MBAMG (10(-6) mol/L) or BADGH (5 X 10(-4) mol/L) led to both an inhibition of mature cell production and the development of colony-stimulating factor (CSF-1)-responsive GM-CFC, but had no effect upon spleen colony-forming units (CFU-S) or on progenitor cells which respond to the multilineage stimulating factor present in WEHI-3B cell-conditioned medium . These data indicate that these inhibitors of mono-ADP ribosylation can block the commitment and/or differentiation of stem cells and infers that ADP ribosylation may be of some importance in the hematopoietic process. Arch Biochem Biophys, 1985 Jun, 239(2), 549 - 55 Cholera toxin A subunit: functional sites correlated with regions of secondary structure; Duffy LK et al.; The A subunit of cholera toxin contains the ADP-ribosyltransferase activity in its major constituent polypeptide A1 (Mr 23,000) which is responsible for the elevation of cAMP typically observed with most mammalian cell types after exposure to the toxin . The primary structure of the A subunit, recently established by sequence analyses, is presented and used as the basis for the secondary structure prediction according to the method of Chou and Fasman . The results indicated the presence of 27% alpha-helix, 25% beta-structure, 12% beta-turn, and 36% random coil . The majority of the beta-structure consisted of six strands located in the NH2-terminal portion of the molecule (residues 33-106) covering one-half of the region corresponding to the A1 polypeptide portion . The beta-sheet domain led immediately into the active site region characterized by the alternating structures of beta-pleated sheet and alpha-helix (residues 95-140) similar to that reported for other NAD+ binding proteins . The presence of this structural feature in the region was confirmed by the use of another predictive method (J . Garnier et al., J . Mol . Biol . 1978, 120, 97-120) . In addition, two regions (residues 14-18 and 200-214), previously identified to contain binding sites for the B subunit as evidenced by chemical modification and monoclonal antibody studies, were found to be in alpha-helix configuration. Nippon Naibunpi Gakkai Zasshi, 1985 May 20, 61(5), 541 - 53 {The effects of TSH, cholera toxin and Graves' IgG on cAMP production in cultured human thyroid adenoma cells in monolayer}; Tsuboi K; Monolayer cultures of human thyroid cells derived from thyroid adenoma were utilized for the assay of thyroid stimulating substances such as thyrotropin (TSH), cholera toxin and thyroid stimulating immunoglobulin (TSI) in patients with Graves' disease . Adenoma cells were treated with 0.1% collagenase or 2000 unit/ml dispase to thyrocytes . The cells were cultured in MEM containing 10% fetal calf serum under an atmosphere of 5% CO2 in air . Within 24 hours, the cells attached themselves to the plastic surface and formed a monolayer . Cyclic AMP responses to TSH, cholera toxin or Graves' IgG were tested in a medium (PBS) containing 0.5 mM IBMX . The cyclic AMP responses to TSH were generally maximal on the 3rd day of culture and declined thereafter . The response was dose-dependent, and 10 microU/ml of TSH produced a significant increase of cellular cyclic AMP . The response by 1 microU/ml of TSH was 28 approximately 57 fold above the basal . The response was also a function of the incubation period . The maximal response was attained after 1 h incubation . When the cultures were washed after exposure to TSH, the cellular cyclic AMP levels rapidly declined, suggesting that removal of receptor-bound TSH results in a prompt cessation of cyclic AMP production . The thyroid cells in monolayer also responded to cholera toxin . The response was dose-dependent, and cholera toxin as low as 1 ng/ml was able to increase cyclic AMP production . In contrast to the observations in TSH, the cyclic AMP responses induced by cholera were hardly affected by washing the cultures after exposure to cholera toxin . Treatment of the cells with cholera toxin for only 3 min resulted in a continuous stimulation of cyclic AMP production for more than 4 hours . Confirming recent observations by others, most of Graves' IgG stimulated cyclic AMP production in a dose-dependent manner, but some of them inhibited the response at high concentrations . IgG derived from normal subjects did not increase cellular cyclic AMP . The time course in the cyclic AMP responses induced by Graves' IgG was variable among the IgG preparations from different patients . In some patients, the maximal responses were attained after 4 hours of incubation . A significant difference was noted between TSH and Graves' IgG in the stimulation of cyclic AMP production after washing the cultures . When the cultures were treated with Graves' IgG for 30 min, washed and then incubated without Graves' IgG, cellular cyclic AMP levels remained at the levels which were almost equivalent to those observed in the continuous presence of the IgGs.(ABSTRACT TRUNCATED AT 400 WORDS) Exp Hematol, 1985 May, 13(4), 261 - 6 Cholera toxin enhances factor-dependent colony growth of murine mast cell progenitors; Saito H et al.; Mast cell colonies were observed when mouse spleen or bone marrow cells were cultured in the presence of medium conditioned by concanavalin-A-stimulated spleen cells, indicating that the medium contains the factor(s) necessary for the formation of these colonies . This factor-dependent colony growth of mast cell progenitors was enhanced by cholera toxin and prostaglandin E, which act on cellular growth mainly by elevating the intracellular cyclic-AMP level . The effect of the toxin was neutralized by preincubation of the toxin with GM1 ganglioside, the receptor substance for cholera toxin, suggesting that cholera toxin exerts its action through GM1 gangliosides present on mast cell progenitors . The toxin B subunit, which binds to GM1 ganglioside but does not elevate intracellular cyclic AMP level, did not affect the colony growth of mast cell progenitors . From these results, it is suggested that intracellular cyclic AMP levels may be involved in colony growth of mast cell progenitors. Jpn J Cancer Res, 1985 May, 76(5), 345 - 51 Induction of functional differentiation of a human monocytic leukemia cell line (THP-1) by retinoic acid and cholera toxin; Hemmi H et al.; The human monocytic leukemia cell line, THP-1, is induced to differentiate into more functionally mature monocyte (macrophage)-like cells by incubation with retinoic acid at concentrations of 10nM or higher . There is no apparent morphological change accompanying this functional maturation . These induced cells show increases in nitroblue tetrazolium reduction, immunoerythrophagocytosis, hexose monophosphate shunt activity, and 5'-nucleotidase and NAD+-glycohydrolase activities . Prostaglandin E2, dibutyryl cyclic adenosine 3':5'-monophosphate, or T-lymphocyte-derived differentiation-inducing activity, all inactive or less active alone, increase the extent of differentiation of THP-1 in combination with 10nM retinoic acid . THP-1 is also induced to differentiate by 0.1nM or higher concentrations of cholera toxin . Furthermore, 24,24-difluoro-1 alpha,25-dihydroxyvitamin D3 induces less differentiation of THP-1 compared to retinoic acid . Dimethyl sulfoxide and 12-O-tetradecanoylphorbol-13-acetate show no induction of functional differentiation . THP-1 thus joins the list of leukemic myelomonocytic cell lines (e.g., the promyelocytic HL-60 and the monoblast-like U-937) that are blocked at a relatively late stage of maturation and which differentiate in response to retinoic acid. J Cell Physiol, 1985 May, 123(2), 197 - 200 Estrogen receptor expression in serially cultivated rat endometrial cells: stimulation by forskolin and cholera toxin; Heimann R et al.; Serially propagated with 3T3 feeder layer support, epithelial cells derived from normal rat endometrium expressed estrogen receptor activity . Specific binding of 17-beta-estradiol was in the range of 30-60 fmol/mg of protein and was of high affinity (Kd = 0.3 nM) . A survey of cell lines derived from several other normal epithelia showed that rat vaginal and human cervical cultures also had high-affinity estrogen receptors (6-13 fmol/mg of protein), while rat epidermal and esophageal cells had no detectable activity . In the endometrial cultures, receptor levels were elevated nearly two- to fourfold by cholera toxin or forskolin in the medium . This effect was detectable after 4 hr but not 1 hr of treatment and did not occur in the presence of cycloheximide . We conclude that serially cultivated rat endometrial cells retain hormonal properties expressed in vivo while exhibiting some keratinocyte character . These cells may provide a useful model for study of receptor modulation. Int J Pept Protein Res, 1985 Apr, 25(4), 421 - 4 Solid phase synthesis of two cholera toxin B subunit antigens; Delmas A et al.; The 30-50 and 50-75 sequences of the cholera toxin beta chain including the amino-acids that are thought to be involved in toxin-receptor binding have been synthesized using the solid phase method . They were then purified by gel permeation and ion exchange chromatography . Both these free peptides induced serum antibodies recognising the native toxin after oral or intraperitoneal administration . Only the antibodies raised against the 50-75 peptide, however, were able to neutralize toxin activity. Lancet, 1985 Mar 30, 1(8431), 725 - 7 Successful treatment of therapy-resistant pancreatic cholera with human leucocyte interferon; Oberg K et al.; The pancreatic cholera syndrome is a serious and potentially fatal disease found in patients with endocrine pancreatic tumours and ganglioneuromas . Two patients with therapy-resistant pancreatic cholera syndrome were successfully treated with human leucocyte interferon given intramuscularly in a dose of 3 X 10(6)-6 X 10(6) IU per day . This produced a reduction in stool volume and plasma vasoactive intestinal polypeptide (VIP) within 3-5 days of the start of treatment . Tumour mass decreased in one of the patients after 3 months of treatment but some tumour tissue remained after 15 months' observation, although circulating concentrations of VIP are normal . The mechanisms of action of interferon are not known but a direct inhibition of tumour-cell hormone production and perhaps of tumour-cell proliferation might account for the rapid clinical response. Biochemistry, 1985 Mar 26, 24(7), 1791 - 7 Lipid phase separations induced by the association of cholera toxin to phospholipid membranes containing ganglioside GM1; Goins B et al.; The interactions of cholera toxin and their isolated binding and active subunits with phospholipid bilayers containing the toxin receptor ganglioside GM1 have been studied by using high-sensitivity differential scanning calorimetry and steady-state and time-resolved fluorescence and phosphorescence spectroscopy . The results of this investigation indicate that cholera toxin associates with phospholipid bilayers containing ganglioside GM1, independent of the physical state of the membrane . In the absence of Ca2+, calorimetric scans of intact cholera toxin bound to dipalmitoylphosphatidylcholine (DPPC) large unilamellar vesicles containing ganglioside GM1 result in a broadening of the lipid phase transition peak and a slight decrease (less than 5%) in the transition enthalpy . In the presence of Ca2+ concentrations sufficient to cause ganglioside phase separation, the association of the intact toxin to the membrane results in a significant decrease of enthalpy change for the lipid transition, indicating that under these conditions the toxin molecule perturbs the hydrophobic core of the bilayer . Calorimetric scans using isolated binding subunits lacking the hydrophobic toxic subunit did not exhibit a decrease in the phospholipid transition enthalpy even in the presence of Ca2+, indicating that the binding subunits per se do not perturb the hydrophobic core of the bilayer . On the other hand, the hydrophobic A1 subunit by itself was able to reduce the phospholipid transition enthalpy when reconstituted into DPPC vesicles . These calorimetric observations were confirmed by fluorescence experiments using pyrene phospholipids.(ABSTRACT TRUNCATED AT 250 WORDS) J Biochem (Tokyo), 1985 Mar, 97(3), 729 - 35 Specific binding of cholera toxin to rat erythrocytes revealed by analysis with a fluorescence-activated cell sorter; Iwamori M et al.; The direct binding of cholera toxin to the receptor on the native cell surface was analyzed with a fluorescence-activated cell sorter (FACS) by the direct membrane immunofluorescence technique using FITC-conjugated cholera toxin B subunit as a ligand and erythrocytes, but the binding was significantly affected by a change in pH, showing optimum pH of 7.2 . The optimum conditions for analysis of the cholera toxin-binding with a FACS were reaction of the target cells with 0.2 M phosphate-buffer (pH 7.2) containing 0.025% of BSA and 0.175 M of NaCl at 4 degrees C for 40 min . The binding of cholera toxin B subunit to rat erythrocytes was linear in the range of 1.2 ng to 80 ng, which corresponded to 2,469 to 163,500 molecules of toxin per cell, and the latter was almost the saturated level of binding . although erythrocytes from different strains of rats possessed equal binding ability for the cholera toxin, no binding was observed with erythrocytes from mouse, guinea pig, cow, pig, man, or rabbit, indicating that the cholera-toxin binding occurs specifically on rat erythrocytes . This is in accord with our previous analytical deta on the absence of GM1 in erythrocytes of these animals except rat, of which erythrocytes contain GM1 . Also, the structural specificity of the receptor for cholera toxin was assessed by a binding inhibition experiment using glycolipid-containing liposomes as inhibitors and GM1 was found to be the most potent inhibitor, showing complete inhibition of toxin (40 ng) binding to 5 x 10(6) erythrocytes at 505.6 pmol of GM1. Biol Reprod, 1985 Mar, 32(2), 463 - 74 Cholera toxin action on rabbit corpus luteum membranes: effects on adenylyl cyclase activity and adenosine diphospho-ribosylation of the stimulatory guanine nucleotide-binding regulatory component; Abramowitz J et al.; Cholera toxin elicited 5- to 7-fold stimulation of adenylyl cyclase activity . Half-maximal activation was at 4.42 micrograms/ml cholera toxin . Cholera toxin-mediated activation was time dependent . At 0.1 mM ATP, both guanosine triphosphate (GTP) and nicotinamide adenine dinucleotide (NAD+) were required for cholera toxin activation of luteal adenylyl cyclase . The concentrations of GTP and NAD+ required for half-maximal activation were 1 and 200 microM, respectively . The GTP requirement could be eliminated by increasing the ATP concentration to 1.0 mM . Guanosine-5'-O-(2-thiodiphosphate) {GDP beta S} did not support cholera toxin activation of the luteal enzyme . Cholera toxin treatment increased GTP-stimulated activity, did not significantly alter guanyl-5'-yl imidodiphosphate {GMP-P(NH)P}-stimulated activity, and depressed NaF-stimulated activity . Furthermore, toxin treatment resulted in a 3.4-fold reduction in the Kact values for ovine luteinizing hormone (oLH) to activate adenylyl cyclase . A similar reduction in Kact values for oLH was obtained when concentration-effect curves performed in the presence of GMP-P(NH)P were compared to those performed in the presence of GTP . In addition, luteal membranes treated with cholera toxin and {32P}NAD+ were subjected to autoradiographic analysis following sodium dodecyl sulfate-polyacrylamide gel electrophoresis . This treatment resulted in the {32P} adenosine diphospho (ADP)-ribosylation of a 45,000-dalton protein doublet, corresponding to the alpha subunit of the stimulatory guanine nucleotide-binding regulatory component (Ns) . As with activation of adenylyl cyclase activity, cholera toxin-specific {32P} ADP-ribosylation was time dependent and increased with increasing concentrations of cholera toxin . GTP, GMP-P(NH)P, and NaF, but not GDP beta S, were capable of supporting {32P} ADP-ribosylation of the protein doublet . oLH did not alter the ability of cholera toxin to ADP-ribosylate the protein activation of luteal adenylyl cyclase activity is due to the ADP-ribosylation of the alpha subunit of Ns and the concomitant inhibition of a GTPase associated with adenylyl cyclase. Immunology, 1985 Mar, 54(3), 601 - 3 Neutralization of cholera toxin by rat bile secretory IgA antibodies; Vaerman JP et al.; IgA-antibody (AB) activities have been elicited in rat bile against several antigens such as bacteria, erythrocytes, tumour cells, haptens and proteins (Lemaitre-Coelho, Jackson & Vaerman, 1978; Hall et al., 1979; Montgomery, Lemaitre-Coelho & Vaerman, 1980; Peppard et al., 1982) . However, their biological significance, except for plasma clearance of immune complexes (Peppard et al., 1982) and bacterial agglutination, remains conjectural, despite their possible major contribution to rat intestinal immunity . The importance of local intestinal immunity in protection against cholera is today widely admitted (Jertborn, Svennerholm & Holmgren, 1984) . Intraintestinally given cholera toxin (CT) is a potent immunogen in rats whose intestinal mucosa then harbours numerous anti-CT IgA plasma cells (Pierce, 1978) . Since bile IgA in rats is largely, but not entirely, derived from intestinal synthesis (Vaerman, Lemaitre-Coelho & Jackson, 1978; Manning et al., 1984), rats intestinally immunized with CT could display high levels of anti-CT IgA AB in their bile, and these AB might neutralize CT in the biologically relevant intestinal loop assay (Lange & Holmgren, 1978). Mol Cell Endocrinol, 1985 Mar, 39(3), 217 - 28 The functional activity of adult mouse Leydig cells in monolayer culture: effect of lipoproteins, pregnenolone and cholera-toxin; Hunter MG et al.; Further studies were carried out on purified mouse Leydig cells to determine why they lose their hormone responsiveness after several days in monolayer culture . The effects of cholera-toxin on cyclic AMP and testosterone production were examined . It was found that cyclic AMP production could still be maximally stimulated by cholera-toxin after 4 days in culture when response to luteinizing hormone (LH) has declined . Testosterone production was, however, not maintained . Because this decline in testosterone production may have been due to the lack of a suitable substrate after several days in culture, cells were cultured initially in the presence of exogenous pregnenolone and low-density lipoproteins (LDL) . Both substances were found to enhance basal and LH-stimulated testosterone production and to extend responsiveness of the cells until at least day 4, but by day 7 response was lost . Cells were then cultured in the presence of rat and human LDL and HDL and in both cases LDL was found to enhance consistently testosterone production, but HDL was much less effective . Scanning and transmission electron micrographs showed that changes in cell shape occurred during culture, but indicated that the cells were not depleted of lipid droplets by the end of culture or after LH stimulation . It is concluded that the eventual decline in testosterone synthesis is not due to lack of substrate, although the addition of exogenous substrate does extend the period of responsiveness . Nor is it due to a decrease in adenylate cyclase activity . At least part of the lesion is caused by a decrease in the enzymes required for the conversion of pregnenolone to testosterone. FEBS Lett, 1985 Feb 25, 181(2), 390 - 6 Comparative effects of cholera and Bordetella pertussis toxins on cyclic AMP and GTP levels and on lipolysis in rat adipocytes incubated in vitro; Lambert B et al.; The respective effects of cholera and Bordetella pertussis toxins were studied in time and concentration dependent experiments, following glycerol and fatty acid release, GTP and cAMP levels . Cholera toxin, after a lag time of 30 min, stimulated linearly GTP and cAMP accumulation and lipolysis (maximal effect: 2-fold increase at 5 micrograms/ml) . Pertussis toxin presented a biphasic effect both in time and concentration dependent studies . Up to a maximum reached after 2 h with 1.4 units LPF/ml the stimulation affected GTP (3 fold) and cAMP (7 fold) levels, glycerol and fatty acid release (15 fold) . Beyond this, an inhibition occurred, yielding a decrease towards basal values of GTP and cAMP content whereas the glycerol and fatty acid release was stopped . These results, which are the first reporting the fluctuation of the GTP content of intact cells challenged with bacterial toxins, show a close relationship between GTP and cyclic AMP levels and lipolytic activity. FEBS Lett, 1985 Feb 25, 181(2), 377 - 80 Gene expression in the polycistronic operons of Escherichia coli heat-labile toxin and cholera toxin: a new model of translational control; Yamamoto T et al.; A new model is proposed based on the suggestion that stable local secondary structures of mRNA interfere with ribosome movement on mRNA and consequently reduce the translation rate . This model accounts for a different level of translation for each cistron in the polycistronic mRNA of Escherichia coli heat-labile toxin (LT) and cholera toxin . We also conclude that the mRNA secondary structures have been conserved during the evolution of the toxin genes for its functional importance. Biochem Biophys Res Commun, 1985 Feb 15, 126(3), 1174 - 81 Pertussis but not cholera toxin inhibits the stimulated increase in actin association with the cytoskeleton in rabbit neutrophils: role of the "G proteins" in stimulus-response coupling; Shefcyk J et al.; Treatment of rabbit neutrophils with pertussis toxin, but not cholera toxin, inhibits the increases produced by formylmethionyl-leucyl-phenylalanine, leukotriene B4 and the calcium ionophore A23187 in the amounts of actin associated with the cytoskeletons . The increase in the cytoskeletal actin produced by phorbol 12-myristate, 13-acetate on the other hand is not affected by pertussis toxin . Incubation of the neutrophils with cholera toxin, unlike pertussis toxin, did not inhibit the fMet-Leu-Phe induced rise in the intracellular concentration of free calcium, and caused only a shift to the right of the dose-response curve of N-acetyl-beta-glucosaminidase release . This shift was more marked in the presence of 1-methyl-3-isobutylxanthine . In addition, the stimulated breakdown of phosphatidylinositol 4,5 bis-phosphate was inhibited by pertussis toxin . These results suggest that pertussis toxin acts at an early step in the signal transduction and does not affect the sequence of reactions initiated by the activation of the protein kinase C . Furthermore, the guanine nucleotide regulatory protein Gi, but not Gs, is closely involved in signal transduction in these cells. Lancet, 1985 Feb 2, 1(8423), 261 - 2 Cholera epidemiology in developed and developing countries: new thoughts on transmission, seasonality, and control; Miller CJ et al.; PIP: It is hypothesized that geographical regions where cholera is endemic, V . cholerae 01 may be maintained in estuarine environments . Endemic cholera, unlike epedemic cholera, occurs regularly, and sometimes seasonally, in certain geographical areas without evidence of importation . Measures used to control epedemic cholera are not very effective in preventing endemic cholera . Areas of endemic cholera include southeastern US and several African and Asian countries, including Bangladesh . It has previously been suggested that V . cholerae 01 is maintained in these areas between outbreaks by 1) living in nonhuman animals; 2) residing in chronic human carriers, who may not excrete the organism; 3) by continous transmission to individuals who manifest only mild symptoms of the disease; and 4) by living in aquatic environments . The 1st 3 explanations are to supported by available evidence . The 4th explanation, previously thought to be impossible, is now engendering new interest . Recent studies reveal that V . cholerae 01 can survive in warm water with a salinity of 0.25-3.0% and a ph of 8.0 for a considerable length of time . Estuarine environments may be suitable for the organism's survival . It is hypothesized further that outbreaks of primary cases in estuarine environments may be precipatated by eating certain types of raw or inadequately cooked estuarian plants or animals, perhaps on a seasonal basis . In areas where sanitation is poor, the disease will be transmitted to secondary cases, and in areas where sanitation is good, secondary cases will be rare . Primary cases may also develop in areas some distance from the estuarian environment by the importation of estuarian products . Outbreaks of secondary cases in nonestuarian environments may be due to the seasonal movement of estuarian inhabitants through the area . This explanation fits the epidemiological evidence available for the southeastern US and Bangladesh . If this hypothesis is correct, appropriate control measures will include educating people to refrain from eating raw and improperly cooked seafood . Eur J Biochem, 1985 Feb 1, 146(3), 533 - 8 The activation of adenylate cyclase from small intestinal epithelium by cholera toxin; Dominguez P et al.; ADP-ribosylation of membrane proteins from rabbit small intestinal epithelium was investigated following incubation of membranes with {32P}NAD and cholera toxin . Cholera toxin catalyzes incorporation of 32P into three proteins of 40 kDA, 45 kDa and 47 kDa located in the brush-border membrane . In contrast, basal lateral membranes do not contain any protein which becomes labeled in a toxin-dependent manner when incubated with cholera toxin and {32P}NAD . The modification of membrane proteins from brush border occurred in spite of the virtual absence in these membranes of adenylate cyclase activatable either by cholera toxin, vasoactive intestinal peptide (VIP) or fluoride . The three agents activated adenylate cyclase when crude plasma membrane were used . Cholera toxin activated fivefold at 10 micrograms/ml . Vasoactive intestinal peptide activated at concentrations from 10-300 nM, the maximal stimulation being sixfold . Fluoride activated 10-fold at 10 mM . When basal lateral membranes were assayed for adenylate cyclase it was found that, with respect to the crude membranes, the specific activity of fluoride-activated enzyme was 3.3-fold higher, VIP stimulated enzyme was maintained while cholera-toxin-stimulated enzyme showed half specific activity . Moreover, while fluoride stimulated ninefold and VIP stimulated fivefold, cholera toxin only stimulated twofold at the highest concentration . The results suggest that the activation by cholera toxin of adenylate cyclase located at the basal lateral membrane requires ADPribosylation of proteins in the brush border membrane. J Clin Microbiol, 1985 Feb, 21(2), 174 - 9 Enzyme-linked immunosorbent assay to measure antibodies to purified heat-labile enterotoxins from human and porcine strains of Escherichia coli and to cholera toxin: application in serodiagnosis and seroepidemiology; Levine MM et al.; Serum immunoglobulin G antibodies to purified heat-labile enterotoxin (LT) from human (LTh) and porcine (LTp) Escherichia coli strains and cholera enterotoxin (CT) were measured by an enzyme-linked immunosorbent assay . Sera from patients with LTh E . coli infection showed a prominent response with LTh, an intermediate response with LTp, and a meager response with CT . Of 47 persons with clinical LTh-producing E . coli (herein shortened to LTh E . coli) infections, significant rises in antitoxin were detected against LTh in 36 (77%), against LTp in 30 (64%), and against CT in only 13 (28%) patients; seroconversions also occurred in 11 of 14 (79%) patients with subclinical LTh E . coli infections . In North Americans with experimental LTh E . coli infection, anti-Lth did not remain at high levels for more than 3 months . Persons with cholera manifested antitoxin responses that were similarly potent against all three toxin antigens; in fact, net optical density values were often slightly higher against LTh than against CT . The ratio of CT/LTh ELISA net optical density in convalescent sera proved to be a sensitive means to differentiate LT E . coli from cholera infection . All 11 cholera patients tested had CT/LTh ratios of greater than 0.70, whereas in only 1 of 47 LTh E . coli infections did the ratio exceed that value (it was 0.71) (P less than 0.0000000001) . In single serum specimens, a net optical density of greater than or equal to 0.30 against LTh was shown to be a useful cutoff in screening sera for recent LTh E . coli or past cholera infection . The CT/LTh ratio was then used to differentiate definitively . Sera from healthy 3- to 5-year -olds and 15- to 19-year-olds in Maryland, Chile, and Bangladesh were tested against LTh and CT . The serological results fit known epidemiological observations . (i) LTh infections are rare in the United States (only 2 of 60 sera had LTh net optical density values of >/= 0.30 . (ii) In contrast, evidence of recent LTh E . coli infections was very common in Chilean (69%) and Bangladeshi (57%) 3- to 5-year-olds and not uncommon in 15- to 19-year-olds (38 and 31%, respectively) in those countries . (iii) Only Bangladeshi sera showed serological evidence of cholera infections (CT/LTh ratios of > 0.70) . The immunoglobulin G enzyme-linked immunosorbent assay measuring antibodies to purified LTh and CT represents a practical and effective tool for the serological study of LTh E . coli and cholera diarrheal infections. Gut, 1985 Feb, 26(2), 188 - 93 Increased jejunal prostaglandin E2 concentrations in patients with acute cholera; Speelman P et al.; Supraphysiologic doses of prostaglandins (PGs) mimic the effect of cholera toxin and cAMP in the small intestine, but not all observations are explicable in terms of the theory that links PGs to cAMP . Because no data exist on endogenous PGs in human cholera we measured PGE2 concentrations in jejunal fluids and fasting intestinal flow rates of PGE2 during slow marker perfusion of proximal jejunum in nine patients with high purging cholera . Nine patients in the recovery phase of cholera or other watery diarrhoeas served as controls . In acute cholera PGE2 concentrations were significantly (p less than 0.001) raised (172-1435 (n = 9) vs 60-270 (n = 9) pg/ml) and negatively correlated (r = 0.71; p less than 0.05) to the time following onset of diarrhoea . Also fasting jejunal flow rates of PGE2 were significantly (p less than 0.005) increased (0.77-8.22 (n = 7) vs 0.21-0.92 (n = 6) ng/min), and positively correlated (r = 0.84; p less than 0.01) to stool output (2.9-9.5 ml/min) . By extrapolation, at normal stool output fasting jejunal flow rates of PGE2 equalled those measured during convalescence . The results support the notion that PGs, in addition to cAMP, may play a pathophysiologic role in human cholera . As the ratio between the medians of the highest values measured during the acute phase of cholera and in late convalescence was at least 15, local intestinal PGE2 formation in full blown cholera should result in mucosal PGE2 concentrations above those required for a maximal secretory response . This observation might explain why conventional doses of aspirin and indomethacin had no significant antidiarrhoeal effect in clinical trials. J Gen Virol, 1985 Feb, 66 ( Pt 2), 267 - 73 Factors involved in interferon-induced or cholera toxin-induced steroidogenesis in Y-1 mouse adrenal tumour cells; Matsuguchi M et al.; In addition to its antiviral effect, interferon, at high concentrations, stimulates steroidogenesis and provokes cell rounding in Y-1 mouse adrenal tumour cells . This stimulation was inhibited by cytochalasin B and colchicine . In contrast, dibutyryl cAMP and cholera toxin, also able to induce steroid production and cell rounding, increased steroid production even in the presence of these cytoskeleton-disrupting agents . The initial trigger for interferon or cholera toxin thus probably involves a distinct receptor organization . However, since both inducers increased cAMP synthesis in this differentiated cell line, the further metabolic steps of ketosteroid production could be the same. FEBS Lett, 1985 Jan 21, 180(1), 9 - 12 Effects of prostaglandin E2 and cholera toxin on apical sodium uptake in thyroid epithelial cells: role of cAMP; Gerard C et al.; When cultured in tissue culture polystyrene dishes, porcine thyroid cells formed polarized monolayers . Their apical pole was oriented towards the culture medium . Influx of sodium 22 (5 min) through the apical surface was partially inhibited by amiloride . Amiloride-sensitive Na uptake was increased about 3-fold by prostaglandin E2 (1 microM, 30 min) and by cholera toxin (0.01 micrograms/ml, 1 h) . This increase, which was also obtained with forskolin and 8-(4-chlorophenylthio)adenosine-3':5'-monophosphate, cyclic (8-chloro-cAMP), is probably a consequence of the increase of the intracellular cAMP level by prostaglandin and cholera toxin. Med Biol, 1985, 63(4), 182 - 6 Alkaline phosphatases and adenylate cyclase in the normal and desensitized rat small intestine after acute cholera toxin challenge: a histochemical study; Jennische E et al.; The effect of cholera toxin (CT)-challenge on histochemically demonstrable activities of adenylatecyclase and alkaline phosphatase was investigated in rat small intestine, using an intestinal loop model . CT-challenge increased the activities of adenylatecyclase and alkaline phosphatases within 15 minutes, and the changes were confined to enterocytes in the upper third parts of the villi . There was no change in the staining of the crypt cells . There was an increased basal activity of both adenylatecyclase and alkaline phosphatases in animals desensitized to cholera toxin by multiple peroral exposures . CT-challenge in the desensitized rats did not further increase the enzyme activity . It is concluded that desensitization to secretagogues induces profound alterations in the cell systems responsible for fluid secretion. Arkh Patol, 1985, 47(3), 78 - 82 {International scientific relations in the study of the clinical aspects and pathology of cholera (1817-1850)}; Stepanov SA et al.; This review deals with scientific-medical international connections mainly in the investigation of the pathology of cholera in the first half of the XIX century . The attention is drawn to the struggle between "contagionist" (materialistic) and "myasmatic" (idealistic) trends in the epidemiology of that time . The works of Russian and foreign physicians are listed which served to the approach and reciprocal enrichment of the medical culture in European countries . The investigations in the field of pathology of the Asiatic cholera during the first half of the XIX century were summarized in a N . I . Pirogov's comprehensive publication which was highly appreciated by the German pathologists R . Virchov and V . Grizinger . It should be emphasized that N . I . Pirogov thoroughly studied and summarized clinical and pathological experience of his numerous predecessors--Russian and foreign scientists of that time who contributed to the study of cholera. Bull World Health Organ, 1985, 63(3), 569 - 83 Interventions for the control of diarrhoeal diseases among young children: rotavirus and cholera immunization; de Zoysa I et al.; PIP: The potential effects of rotavirus and cholera immunization (with an improved vaccine) on diarrhea morbidity and mortality among young children are reviewed using data from field studies and theoretical calculations . In developing countries, rotavirus may be responsible for about 6% of all diarrhea episodes and 20% of all diarrhea deaths in children under age 5 . In industrial countries, these proportions may be higher . Rotavirus immunization may reduce overall diarrhea morbidity rates by 2-3% and diarrhea mortality rates by 6-10% among children under 5 in developing countries, depending on vaccine efficacy and program coverage . The impact of improved cholera vaccines depends on the prominence of cholera as a cause of diarrhea, and this varies greatly from country to country . Taking the extreme example of Bangladesh, where cholera is endemic and may account for about 0.4% of all diarrhea episodes and 8% of all diarrhea deaths in children under 5 years of age, cholera immunization might reduce overall diarrhea morbidity rates by 0.06-0.13% and diarrhea mortality rates by 1-2% among these children . The similar incidence rates in industrial and developing countries suggest that rotavirus diarrhea may not be controlled by improvements in water supply, sanitation, or hygiene . Control may depend on the widespread use of an effective vaccine . (author's) Cell Biochem Funct, 1985 Jan, 3(1), 25 - 32 Stimulation by cholera toxin of ADP-ribosylation of membrane proteins, adenylate cyclase and insulin release in pancreatic islets; Svoboda M et al.; In rat pancreatic islet membranes exposed to {alpha-32P}NAD, cholera toxin stimulated the labelling of three peptides with Mr close to 22 000, 42 000 and 48 000, respectively . In the islets, the toxin-stimulated ADP-ribosylation of the heavy form of the Ns alpha-subunit predominated over that of the light form, in mirror image of the situation found in the exocrine pancreas . When intact islets were preincubated with cholera toxin, the adenylate cyclase activity of a subcellular particulate fraction was increased . The responsiveness of adenylate cyclase to GTP was also augmented, but that to NaF was decreased . In intact islets, the production of cyclic AMP and the glucose-stimulated release of insulin were also enhanced after pretreatment with cholera toxin . These findings reveal the presence in pancreatic islets of the guanyl nucleotide regulatory protein of adenylate cyclase, with an unusual predominance of the heavy form of the Ns alpha-subunit. J Cyclic Nucleotide Protein Phosphor Res, 1985, 10(2), 157 - 65 Cholera toxin, cyclic AMP, and the firefly flash; Nathanson JA; Cholera toxin, injected into living fireflies, caused a delayed, non-hormone-dependent activation of adenylate cyclase, an elevation of cyclic AMP levels, and a brilliant persistent glow of the firefly light organ 8-20 hours following injection . During periods of spontaneous flashing, onset of toxin-induced glowing was abrupt and step-like in character, due to a markedly prolonged off-time of individual flashes . These observations provide a striking demonstration of the mechanism of cholera toxin action, and, together with other data, suggest that the initiation of the normal adult firefly flash is mediated through elevation of cyclic AMP levels secondary to the activation of an octopamine-sensitive adenylate cyclase. Virchows Arch B Cell Pathol Incl Mol Pathol, 1985, 49(4), 325 - 40 Changes in basal cell subpopulations and tissue differentiation in human epidermal cultures treated with epidermal growth factor and cholera toxin; Jensen PK et al.; Cell kinetic studies on cultured human epidermal cells have indicated that cycling basal cells may be divided into at least two subpopulations that seem to differ with respect to the rate of DNA replication . The present study was undertaken in order to elucidate the biological significance of these subpopulations . The proliferation characteristics of cultured basal cells were changed by the addition of epidermal growth factor (EGF) and cholera toxin to the culture medium . It was shown that EGF and cholera toxin stimulated the growth of human epidermal cells in culture . Simultaneously, the terminal differentiation of the cells was inhibited resulting in a reduced multilayering and a reduced formation of the cornified envelope . However, only minor differences in the protein synthesis pattern were observed between cultures maintained in the presence or absence of the growth stimulators . The effect of EGF and cholera toxin on the basal cell subpopulations was investigated after 3H-thymidine labelling followed by cell sorting and autoradiography . In the presence of EGF and cholera toxin dramatic changes were induced in the labelling pattern of sorted S-phase cells indicating significant alterations in the balance between the subpopulations of cycling basal cells . Our results with these substances are in accord with the hypothesis that the observed cell kinetic subpopulations may be related to regeneration or early events in the differentiation process of the keratinocyte. Dev Biol Stand, 1985, 59, 51 - 62 Application of monoclonal antibodies and genetically-engineered hybrid B-subunit proteins to the analysis of the cholera/coli enterotoxin family; Finkelstein RA et al.; Single amino acid substitutions, introduced by genetic engineering, significantly modify the behavior of the B-subunits of the cholera/coli enterotoxin family in SDS-PAGE and also markedly affect the reactivity of the proteins with mouse hybridoma-derived monoclonal antibodies raised against H-LT . The results indicate that single amino acids play an important role in defining epitopes in these proteins. Life Sci, 1984 Dec 10, 35(24), 2397 - 406 Cholera toxin-induced changes in force of contraction and cyclic AMP levels in canine ventricular myocardium: inhibition by carbachol; Endoh M et al.; Cholera toxin (1-10 micrograms/ml) had a biphasic inotropic action on the isolated canine ventricular muscle: it produced a transient negative and a long lasting positive inotropic effect . The negative effect reached a maximum 43 +/- 2 min (n = 12) after administration of the toxin, while it took 3-5 hrs for the positive effect to reach a steady level . The positive inotropic effect of cholera toxin was accompanied by a prominent abbreviation of the time to peak tension and the relaxation time of individual contractions . The level of adenosine 3',5'-cyclic monophosphate (cyclic AMP) of the tissue was elevated by cholera toxin in a time- and concentration-dependent manner . Carbachol (1 mumol/l) administered 3 or 5 hrs after the administration of cholera toxin (10 micrograms/ml) reversed the increase in force of contraction and the elevation of cyclic AMP levels induced by cholera toxin . These results indicate that cholera toxin exerts a cyclic AMP-dependent positive inotropic effect and a negative inotropic effect which is not related to cyclic AMP levels in canine ventricular myocardium. Proc Natl Acad Sci U S A, 1984 Dec, 81(24), 7893 - 6 Both cholera toxin-induced adenylate cyclase activation and cholera toxin biological activity are inhibited by antibodies against related synthetic peptides; Jacob CO et al.; The immune response against six synthetic peptides corresponding to various segments of the B subunit of cholera toxin was evaluated . Conjugates in which the peptides were covalently linked to tetanus toxoid served for immunization of rabbits . As previously reported, four of these conjugates elicited antibodies cross-reactive with intact cholera toxin . We report here that antisera against two of these synthetic peptides inhibit the entire spectrum of activities of the intact cholera toxin . This is manifested both on the biochemical level (adenylate cyclase induction) and on the biological effect (intestinal fluid secretion) . These results indicate that these peptides may serve as suitable candidates for preparation of a synthetic anticholera vaccine. Bangladesh Med Res Counc Bull, 1984 Dec, 10(2), 45 - 52 Study of makeshift hospital during cholera outbreak; Faruque AS et al.; This is a report on the study of utilization pattern of a makeshift hospital during a major cholera outbreak, by analyzing data on dehydration status, distance covered, number of deaths averted, and operation-wise comparison with other permanent facilities . To avoid unnecessary deaths due to dehydration and to ensure prompt and adequate care to suddenly accumulated debilitated patients, the makeshift hospital intervened . Subsequent to the intervention, a gradual reduction in patient admissions and cholera case accumulations was noted . Nearly half the cholera cases attending the makeshift hospital came from relatively far (13 + miles) . The reporting of the majority (72%) of cholera patients with none-to-mild dehydration indicates an increased awareness of the need for early treatment during a cholera outbreak . Early attendance of diarrhoeal patients probably saved more patients by preventing shock and complications . Para-professionals given a short training accomplished similar efficacy as in a permanent facility . Nearer the affected areas, a simple but effective temporary facility is more effective than a sophisticated facility which is further away and takes several hours to reach, with risk to patients. Vaccine, 1984 Dec, 2(4), 284 - 6 Effect of storage temperatures on opacity and total nitrogen content of cholera vaccine; Gupta RK et al.; Fluid cholera vaccine was examined for its stability at 4-8 degrees C, 20-25 degrees C and 37 degrees C with regard to the number of organisms present and the total nitrogen content . Ten batches of fluid cholera vaccine manufactured at the Central Research Institute, Kasauli, India were taken for the study . The number of organisms was determined at weekly intervals for the first month and at monthly intervals for the next 11 months . The percentage loss in the number of organisms after one year averaged 6.5 at 4-8 degrees C, 20.6 at 20-25 degrees C and 21.5 at 37 degrees C . The maximum loss in number of organisms took place during the first six months at these temperatures, in the following six months the number of organisms remained almost constant even at 37 degrees C . The total nitrogen content of the vaccine remained virtually unaltered during the same period . From this study it was concluded that the number of organisms and the total nitrogen content does not reflect the antigenicity nor potency of the vaccine. Zh Mikrobiol Epidemiol Immunobiol, 1984 Dec, (12), 54 - 9 {Potentials and conditions for the reversion of pathogenic properties of the causative agent of cholera in an experiment}; Uraleva VS et al.; The passage of V . cholerae noncholerigenic strains and their mutants, both in vitro and in vivo, has demonstrated that strains in which one of such properties as mobility, viability, adhesive, lecithinase and neuraminidase activities, is sharply decreased or lost, are still capable of reversion to cholerigenic forms . V . cholerae strains which have lost two or more of these properties, as well as strains having stable hemolytic activity determined by Greig's test, seem to be incapable of such reversion. J Histochem Cytochem, 1984 Dec, 32(12), 1275 - 9 Entrance of cholera enterotoxin subunits into thymus cells; Tsuru S et al.; Analysis of the staining of cholera enterotoxin on the surface of cells with specific antibodies against each subunit of cholera enterotoxin, using a fluorescence-activated cell sorter and electron microscopy, showed that not only subunit A but also subunit B penetrates the cell membrane . The detection of subunits inside the cell was facilitated by the use of saponin, an agent that increases membrane permeability. J Immunol, 1984 Dec, 133(6), 2892 - 7 Cholera toxin feeding did not induce oral tolerance in mice and abrogated oral tolerance to an unrelated protein antigen; Elson CO et al.; The feeding of protein antigens to mice results in a state of tolerance when feeding is followed by parenteral immunization . Cholera toxin (CT) is a protein that has been used extensively as a potent oral immunogen for mucosal IgA responses, but CT feeding also stimulates a substantial plasma IgG antibody response . This latter finding prompted us to study whether or not CT induces oral tolerance . Mice were fed 5 mg keyhole limpet hemocyanin (KLH) or 10 micrograms CT at least twice before parenteral immunization with 1 microgram KLH or CT in alum i.p . Plasma and intestinal secretions were collected at intervals . The specific IgG or IgA antibody in the samples was measured by ELISA . Although KLH feeding did induce oral tolerance, CT feeding did not induce oral tolerance in any of three mouse strains tested or at any dose of CT given orally . The feeding of the B subunit of CT did not result in oral tolerance either . When both CT and KLH were fed together, CT was able to abrogate oral tolerance to KLH, an antigenically unrelated protein . Moreover, feeding CT along with KLH stimulated secretory IgA anti-KLH responses, whereas no such IgA responses were found when KLH was given alone . Thus, in these experiments with protein antigens, IgA immunization and oral tolerance were reciprocally linked and did not occur simultaneously . CT appears to abrogate oral tolerance and to stimulate secretory IgA responses by altering the regulatory environment in gut-associated lymphoid tissue, shifting it toward responsiveness. Mol Cell Biol, 1984 Dec, 4(12), 2639 - 42 Cholera toxin treatment stimulates tumorigenicity of Rous sarcoma virus-transformed cells; Gottesman MM et al.; Chinese hamster ovary cells transformed by Rous sarcoma virus form tumors poorly in nude mice . Tumorigenicity was markedly stimulated by pretreatment of the cells with cholera toxin, which raises cyclic AMP levels and activates cyclic AMP-dependent protein kinase . Increased tumorigenicity was manifested by a severalfold increase in the rate of tumor formation, as well as earlier appearance and more rapid growth of tumors . In contrast, spontaneously transformed Chinese hamster ovary cells showed decreased tumorigenicity after cholera toxin treatment . The activation of tumorigenic potential in Rous sarcoma virus-transformed Chinese hamster ovary cells by cholera toxin correlated with increased phosphorylation of the viral oncogene product pp60src and stimulation of its tyrosine kinase activity. EMBO J, 1984 Dec 1, 3(12), 2889 - 93 Neutralization of heat-labile toxin of E . coli by antibodies to synthetic peptides derived from the B subunit of cholera toxin; Jacob CO et al.; Antibodies elicited by six synthetic peptides corresponding to various fragments of B subunit of cholera toxin (CT) were evaluated for their cross-reactivity with heat-labile toxin (LT) of Escherichia coli . The antiserum directed towards the peptide CTP3 (residues 50-64) was found highly cross-reactive with the LT, in radioimmunoassay and immunoblotting . This peptide was also the most cross-reactive with intact CT . The antiserum against CTP1 (residues 8-20) was also cross-reactive with the two toxins, although to a much lower extent . Antisera to both CTP1 and CTP3, which are inhibitory towards CT, were found equally effective in neutralizing the biological activity of the E . coli LT . This was manifested by inhibition of both adenylate cyclase activity and fluid secretion into ligated ileal loops of rats . These results might indicate the potential of such synthetic peptides as the basis for a general vaccine against several types of infectious diarrhea. Am J Physiol, 1984 Dec, 247(6 Pt 1), G623 - 31 Interaction of cholera toxin and Escherichia coli enterotoxin with isolated intestinal epithelial cells; Hyun CS et al.; The interaction of biologically active 125I-labeled cholera toxin with isolated chick intestinal epithelial cells involves a large number (approx 1.7 10(6)/cell) of high-affinity (Kd = 8-9 X 10(-9) M) binding sites that belong to a single class . Binding of iodotoxin to the cells occurs rapidly, is half-maximal within 1 min, and is complete in 3-7 min (at 37 degrees C) depending on the toxin concentration . Toxin binding is saturable and includes only a small contribution from nonspecific sites . Ligand competition studies suggest that the isolated B subunit of choleragen (CT-B) behaves in an almost identical fashion to the holotoxin (CT), whereas the A subunit shows no detectable activity in competitive binding . Assays for cAMP indicate that neither that A nor the B subunits of CT contain any activity for increasing the level of intracellular cAMP . B subunit, when incubated with CT, inhibits CT-induced elevation of cAMP in a dose-dependent manner . Preincubation of 125I-CT with various concentrations of ganglioside GM1 also shows a dose-dependent inhibitory effect on the binding activity of the toxin . Pretreatment of CT with increasing concentrations of GM1 results in a progressive decrease in toxin-induced formation of cAMP . Escherichia coli heat-labile enterotoxin, which is known to alter intestinal function via a mechanism similar to that of CT, has binding and biological effects very similar to those of CT. In Vitro, 1984 Dec, 20(12), 981 - 6 Serial propagation of adult human prostatic epithelial cells with cholera toxin; Peehl DM et al.; Reproducible subculture of adult human prostatic epithelial cells from normal, benign hyperplastic and malignant tissue has been achieved . Cholera toxin is the key component in the culture system, but use of an optimal basal medium (PFMR-4) supplemented with a high level of serum in collagen-coated dishes also improves growth and serial propagation. FEBS Lett, 1984 Nov 5, 177(1), 104 - 8 Purification and characterization of a hormone-like factor which inhibits cholera secretion; Lonnroth I et al.; A factor which inhibits intestinal hypersecretion induced by cholera toxin was studied . The factor was extracted from intestinal mucosa or pituitary gland of pig . It has an isoelectric point of pH 4.7 +/- 0.1 at 10 degrees C and showed a weak affinity to dextran gel and a strong affinity to agarose gel . From agarose gel the factor was eluted with high concentrations of D-galactose or alpha-methyl-D-glucose, while D-glucose and lactose were less effective . After purification more than 2000 times by isoelectric focusing and gel chromatography, the factor was shown by SDS-polyacrylamide gel electrophoresis to be a protein consisting of subunits of molecular mass 30, 17 and possibly 15 kDa. Arch Biochem Biophys, 1984 Nov 1, 234(2), 363 - 70 The primary structure of the COOH-terminal half of cholera toxin subunit A1 containing the ADP-ribosylation site; Xia QC et al.; The sequence of 96 amino acid residues from the COOH-terminus of the active subunit of cholera toxin, A1, has been determined as (sequence; see text) This is the largest fragment obtained by BrCN cleavage of the subunit A1 (Mr 23,000), and has previously been indicated to contain the active site for the adenylate cyclase-stimulating activity . Unequivocal identification of the COOH-terminal structure was achieved by separation and analysis of the terminal peptide after the specific chemical cleavage at the only cysteine residue in A1 polypeptide . The site of self ADP-ribosylation in the A1 subunit {C . Y . Lai, Q.-C . Xia, and P.T . Salotra (1983) Biochem . Biophys . Res . Commun . 116, 341-348} has now been identified as Arg-50 of this peptide, 46 residues removed from the COOH-terminus . The cysteine that forms disulfide bridge to A2 subunit in the holotoxin is at position 91. Infect Immun, 1984 Nov, 46(2), 612 - 4 Does enteropathogenic Escherichia coli produce heat-labile enterotoxin, heat-stable enterotoxins a or b, or cholera toxin A subunits? Long-Krug SA, Weikel CS, Tiemens KT, Hewlett EL, Levine MM, Guerrant RL. Although most enteropathogenic Escherichia coli strains do not produce recognized enterotoxins, we wished to examine whether they produce any factors like heat-stable enterotoxin b or cholera toxin active subunits that might be missed by conventional assay methods . E . coli strains E851 (O142) and E2348 (O127) that had caused diarrhea in volunteers were negative for heat-labile enterotoxin and heat-stable enterotoxin a in Chinese hamster ovary cell and suckling mouse assays, failed to cause secretion in ligated small bowel loops from 6- to 8-week-old pigs after 4 to 5 h (used to show heat-stable enterotoxin b), and did not activate adenylate cyclase in pigeon erythrocyte lysates (used to demonstrate cholera toxin A subunit) . We conclude that crude, unconcentrated culture filtrates and sonicates do not mimic heat-labile or heat-stable enterotoxins or cholera toxin or its A subunit and that enteropathogenic strains of E . coli probably have yet another mechanism or group of mechanisms by which they cause diarrhea. Am J Physiol, 1984 Nov, 247(5 Pt 2), F784 - 92 PGE2, forskolin, and cholera toxin interactions in modulating NaCl transport in mouse mTALH; Culpepper RM et al.; Prostaglandin E2 (PGE2) inhibits the ADH-stimulated components of the lumen-positive transepithelial voltage (Ve) and of net chloride absorption (JnetCl) in the isolated microperfused mouse medullary thick ascending limb of Henle (mTALH), presumably by interfering with the ADH-dependent intracellular accumulation of cAMP . These experiments examined the interactions of PGE2 with two nonhormonal stimulators of adenylate cyclase--cholera toxin and forskolin--in an attempt to evaluate the means by which PGE2 inhibits ADH-stimulated transport in these mTALH segments . Forskolin (FSK) stimulated Ve in the mTALH with half-maximal stimulation at 1.4 X 10(-7) M FSK . PGE2 had no effect on FSK stimulation of Ve; 10(-6) M FSK reversed completely the PGE2 inhibition of ADH-stimulated Ve . A low concentration of cholera toxin, 5 X 10(-13) M, stimulated Ve and JnetCl in the mTALH; 10(-6) M PGE2 inhibited the stimulation by cholera toxin; and 10(-6) M FSK reversed the PGE2 inhibition of both Ve and JnetCl in cholera toxin-stimulated mTALH . A higher concentration of cholera toxin, 10(-10) M, stimulated Ve and JnetCl to values identical to those seen with maximal concentrations of ADH, but PGE2 did not inhibit the increments in either Ve or JnetCl produced by 10(-10) M cholera toxin . PGE2 appears to inhibit ADH stimulation of NaCl transport in mTALH by an action distal to hormone-receptor interactions yet proximal to the catalytic subunit of adenylate cyclase. Biochim Biophys Acta, 1984 Oct 16, 801(3), 325 - 33 Partial purification of a water-soluble liver protein that regulates adenylate cyclase activity (basal, hormone- and cholera-toxin-activated) and cholera-toxin-catalyzed ADP-ribosylation of the membrane G protein; Gordon JC et al.; We have found in water-soluble extracts of rat liver (and RL-PR-C cloned rat hepatocytes), prepared in the absence of detergent, a factor that markedly enhances basal, isoproterenol and cholera toxin activation of adenylate cyclase of rigorously washed hepatocyte membranes, in the absence of added GTP . The factor, which has characteristics of a protein with an Mr of approx . 35000, has been fractionated from crude cytosol by gel filtration, and then further purified over 50-fold by sequential ion-exchange chromatography . The site of action of the protein appears to be at the level of the guanine nucleotide regulatory (G) protein of the plasma membrane adenylate cyclase complex, as the factor, cooperatively with GTP, also permitted cholera toxin to ADP-ribosylate (from 32P-labeled NAD) two integral membrane proteins that migrated on SDS-polyacrylamide gel electrophoresis gels with the mobilities (Mr approx . 46 000 and 48 000) generally observed for the guanine nucleotide regulator protein subunits . In this system, isoproterenol did not stimulate ADP-ribosylation, in either the presence or absence of the liver protein factor. Brain Res, 1984 Oct 8, 311(2), 366 - 9 Interneuronal transfer of axonally transported proteins: studies with HRP and HRP conjugates of wheat germ agglutinin, cholera toxin and the B subunit of cholera toxin; Trojanowski JQ et al.; Experiments in the visual system and the tongue-hypoglossal nucleus pathway of the rat with HRP and HRP conjugates of wheat germ agglutinin (WGHRP), cholera toxin (CTHRP) and the B subunit of cholera toxin (BHRP) were conducted to study the interneuronal transfer of proteins . Native HRP did not undergo such transfer . Intravitreal injections of WGHRP, CTHRP or BHRP all resulted in interneuronal transfer in the visual system, but there was no evidence for this in the tongue-hypoglossal nucleus pathway . Interneuronal transfer may: (1) require a ligand-cell surface interaction; and (2) occur only after the endocytosis and/or exocytosis of these conjugates at specific sites along the neuronal plasma membrane. Pediatr Res, 1984 Oct, 18(10), 984 - 7 Microvillus membrane differentiation: quantitative difference in cholera toxin binding to the intestinal surface of newborn and adult rabbits; Bresson JL et al.; Microvillus membranes (MVM) were isolated from newborn and adult New Zealand rabbit small intestine . The isolation procedure provided a mean enrichment of 25 +/- 4 for sucrase activity in adult preparations and of 27 +/- 3 for lactase activity in newborn preparations . These purified MVM were incubated with increasing concentrations of 125I-labeled cholera toxin (CT) . 125I-CT binding to adult MVM reached saturation at 6.4 X 10(-9) M; in contrast 125I-CT binding to newborn MVM did not reach saturation but instead continued to increase with increasing 125I-CT concentrations . Scatchard plot analysis of adult data supported the existence of a single binding site (Kd = 1.2 +/- 0.2 X 10(-9) M); analysis of newborn data, however, suggested the existence of additional binding sites, as 125I-CT binding to newborn MVM was inhibited by preincubation with unlabeled CT . These results show that CT binding to both preparations is quantitatively different and is higher in newborn preparations . This difference may be accounted for by the existence of additional binding sites in newborn MVM preparations in contrast to the presence of only the unique receptor previously reported in adult MVM preparations. J Immunol, 1984 Oct, 133(4), 1818 - 24 Cholera toxin B subunit as a carrier protein to stimulate a mucosal immune response; McKenzie SJ et al.; Horseradish peroxidase (HRP) was covalently coupled to the binding subunit of cholera toxin (CTB) via a two-step glutaraldehyde procedure . The HRP-CTB conjugate was characterized by physiochemical as well as immunochemical methods . Mice were immunized intraduodenally with the HRP-CTB conjugate, with HRP alone, or with a mixture of uncoupled CTB and HRP . The functionally active dose of CTB was 50 micrograms and the HRP dose was in the 30- to 90-micrograms range . Both IgA and IgG antibody responses were measured in serum, intestinal washes, and bile by using a solid phase immunoradiometric assay . Mice immunized with the HRP-CTB conjugate showed a significantly higher level of IgA anti-HRP in intestinal washes and bile, as well as increased levels of serum IgG anti-HRP . Animals that received only HRP or the mixture of CTB and HRP had reduced levels of HRP-specific antibody of either class in both gut washes and bile . The IgA anti-HRP responses in the gut washes were 33- to 120-fold higher when the conjugate was used as the immunogen in comparison with immunization with the CTB + HRP or the HRP alone . Vaccines to stimulate mucosal immunity to any antigenic determinant might thus be prepared by covalent conjugation to effective mucosal immunogens such as CTB. Br J Exp Pathol, 1984 Oct, 65(5), 549 - 56 Development of mucous cells in mouse intrapulmonary airways induced by cholera toxin, dibutyryl cyclic AMP and prostaglandin E1; Nygren H et al.; The effect of cholera toxin (CT), dibutyryl cyclic adenosinemonophosphate (DE-cAMP) and prostaglandin E1 (PGE1) on the morphological appearance of mouse intrapulmonary airway epithelium has been studied . All three secretagogues, known to mediate their activity via cAMP, induced increased numbers of mucous cells, but with different kinetics . After exposure to DB-cAMP, mucous cells appeared after 7 h with a peak value at 48 h . The corresponding values for PGE1 were 24 h for the appearance and 48 h for peak value whereas exposure to CT gave a lower response with a peak at 72 h . Autoradiography of lungs from mice injected with 3H-thymidine showed that the mucous cells appeared without any foregoing DNA synthesis, however, increased numbers of labelled cells were seen as a late response to the secretagogues. Biochim Biophys Acta, 1984 Sep 12, 795(2), 363 - 71 Modulation of lipoprotein lipase in the intact rat by cholera toxin--an irreversible agonist of cyclic AMP; Knobler H et al.; Rats were injected intravenously with cholera toxin, a potent stimulator of adenylate cyclase, and lipoprotein lipase was determined in various organs and plasma . 16 h after cholera toxin injection, lipoprotein lipase activity increased 2-6-fold in heart, diaphragm and lung and decreased to one-third in adipose tissue . An increase in lipoprotein lipase activity was seen in the plasma and in the liver, as determined by antiserum to lipoprotein lipase . The increase in heart lipoprotein lipase was preceded by a rise in cyclic AMP and continued for 24 h when cyclic AMP returned to base-line levels . Both heparin-releasable and residual lipoprotein lipase increased in the heart, but to an unequal extent . The more pronounced rise in residual activity (up to 10-fold) could have contributed to an increase in the t1/2 of heart lipoprotein lipase from 1.5 to 2.6 h . The relatively lower increase in heparin-releasable lipoprotein lipase could have been due to a loss of the enzyme from this compartment into the circulation . The effect of cholera toxin on heart and adipose tissue lipoprotien lipase was observed in fasted, fed and super-fed animals and thus appears to be independent of the nutritional state of the animal . Since cholera toxin not only mimics hormonal stimulation, but causes an exaggerated response to hormones, it made studies on some aspects of regulation of both the functional and storage forms of lipoprotein lipase in the intact organism possible. Zh Mikrobiol Epidemiol Immunobiol, 1984 Sep, (9), 90 - 3 {Effect of gamma radiation on the immunobiological and immunochemical properties of cholera exotoxin . IV . The biological and immunochemical properties of purified irradiated choleragen}; Nedugova GI et al.; The results of investigations carried out to study the effect of gamma radiation on the properties of the purified preparations of cholera exotoxin are presented . Irradiation has been shown to decrease the anterotoxicity of purified choleragen and the activity of its permeability factor, depending on the radiation dose . The investigations have revealed that in purified toxin enterotoxicity is completely inactivated with a lover radiation dose than in crude toxin filtrate (25 kGy) . In immunochemical reactions the increase of the electrophoretic mobility of the choleragen components, correlated with the increase of the radiation dose, and the reduced number of protein zones have been observed . The irradiated preparations of purified choleragen have been found to retain their immunogenic properties and serological activity. J Clin Microbiol, 1984 Sep, 20(3), 506 - 8 Characterization of uterine growth response to cholera toxin in hamsters and test of heat-labile enterotoxin from Escherichia coli; Alleva JJ et al.; Cholera toxin (CT) and the heat-labile enterotoxin from Escherichia coli, when injected intraperitoneally into cycling hamsters but not rats or mice, induced a massive uterine growth similar to that normally induced by the implanting blastocyst during pregnancy . CT and heat-labile enterotoxin are the only known agents that have this action in any species . Uterine weight reached a maximal sixfold increase 48 h after injection of CT . Concurrent injection of estrogen, progesterone, and CT increased the maximal response to eightfold and eliminated differences in the response to CT injected on different days of the 4-day hamster estrous cycle . The dose response for CT, heat-labile enterotoxin, and CT plus estrogen plus progesterone was most linear (r greater than 0.93) when the logarithm of uterine weight was plotted against the dose of toxin . The hamster uterine weight response can serve as a simple, highly precise, and highly specific bioassay for CT and heat-labile enterotoxin. Nippon Naibunpi Gakkai Zasshi, 1984 Aug 20, 60(8), 995 - 1004 {The effects of cholera toxin in the release of beta-endorphin from the dispersed cells of the rat neurointermediate lobe}; Furuki Y et al.; The intermediate lobe cells of pituitary gland synthesize and secrete bioactive peptides derived from proopiomelanocortin . In the present study, we investigated the effects of cholera toxin on the release of beta-endorphin (beta-Ep) from dispersed-intermediate lobe cells of rats . Cholera toxin added into culture medium, enhanced the intracellular accumulation of adenosine 3', 5'-monophosphate (cAMP) and the release of beta-endorphin like immunoreactive substance (beta-END-LIS) . A positive dose-response relationship existed between the concentration of cholera toxin and the release of beta-END-LIS or the accumulation of cAMP . Maximal response was obtained with approximately 3 X 10(-10) M (in beta-END-LIS release) and 1 X 10(-9) M (in cAMP accumulation) concentration of cholera toxin . Incubation with cholera toxin (3 X 10(-8) M) resulted in a significant rise of cAMP accumulation after 20-30 min, and a 2-2.5 fold increase of beta-END-LIS release occurred after 60 min in comparison with nontreated cells . cAMP analog and phosphodiesterase inhibitor also increased the beta-END-LIS release) . These results suggested the close relationship between cAMP accumulation and its biological effect (i.e . beta-END-LIS release). Eur J Biochem, 1984 Aug 15, 143(1), 213 - 9 Artificial low-molecular-mass substrates of cholera toxin; Tait RM et al.; A model system, measuring the rate of cholera-toxin-catalysed release of nicotinamide from NAD+, has been used to identify novel compounds which may serve as substrates for toxin-directed ADP-ribosylation . In a series of guanidine-containing compounds, those in which the guanidine group was connected to a large hydrophobic domain greatly stimulated the rate of toxin-catalysed nicotinamide release . The introduction of a charge centre near the guanidine group destroyed all activity . The compounds thus identified were found to inhibit the action of cholera toxin on rat liver adenylate cyclase, and this was associated with a reduction in the amount of {32P}ADP-ribosylation of a 42-kDa protein in the membranes . Guanidine-containing compounds which did not enhance toxin-catalysed release of nicotinamide from NAD+ had no effect on toxin action on adenylate cyclase, and there was a good correlation between the two activities . The results are discussed in relation to the known properties of the guanine nucleotide regulatory protein associated with adenylate cyclase systems, which is the toxin's natural substrate. Mol Cell Biochem, 1984 Aug, 63(1), 83 - 91 Effects of ganglioside GM1 on the thermotropic behavior of cholera toxin B subunit; Dalziel AW et al.; The B, or binding, subunit of cholera enterotoxin forms a pentameric ring structure in the intact toxin, and also when the subunit is isolated from the A subunit . The thermal denaturation of the B subunit ring was examined by differential scanning calorimetry in the presence and absence of ganglioside GM1, its natural 'receptor' . In the absence of ganglioside an irreversible endotherm was observed with maximal excess apparent heat capacity, Cmax, at 74.6 degrees C . When the ganglioside was added in increasing amounts, multiple transitions were observed at higher temperatures, the most prominent having a Cmax at 90.8 degrees C . At high ganglioside concentrations, the 74.6 degrees C transition was not observed . In addition to the thermodynamic results a model is proposed for the interaction of GM1 and B subunit pentamer . This model is derived independently of the calorimetric results (but is consistent with such data) and is based upon considerations of the geometry of the GM1 micelle B subunit pentamer. Zh Mikrobiol Epidemiol Immunobiol, 1984 Aug, (8), 55 - 8 {Standardization of cholera vaccine and preparation of a national reference standard}; Nenkov P et al.; The antigenic potency of the proposed national reference preparations in comparison with that of the corresponding international reference preparations was studied by means of the active protection test in mice . The antigenic potency of the proposed national reference preparations for Inaba and Ogawa was found to be the same or even greater than the antigenic potency of the international reference preparations for cholera vaccine . A high level of antigenic activity was observed during comparison of a production lot of cholera divaccine with the international reference preparation and the national reference preparation in parallel tests . The proposed national reference preparations for Inaba and Ogawa may be used for evaluating the antigenic potency of the lot of cholera vaccine produced in Bulgaria as the standard preparation. Biull Eksp Biol Med, 1984 Aug, 98(8), 190 - 2 {Antitoxic system of the small intestine and liver in rats exposed to cholera enterotoxin}; Iurkiv VA et al.; The effect of cholera enterotoxin on glutathione-S-transferase (GT), glutathione peroxidase (GP) and superoxide dismutase (SOD) in cytosols of the rat small intestinal mucosa and liver was studied in an experimental ligated jejunal loop . It was found that changes in the detoxication enzymatic activity were phasic in nature in both the mucous membrane of all parts of the small intestine and in the liver: the decrease within the first 30 min to 1 h was replaced by activation after 2 h followed by repeated fall 4 h after toxin administration . The time course of the activity of GT and GP in all the parts of the small intestinal mucosa and liver was marked by the same line of changes . SOD activity underwent dissimilar changes in different parts of the mucous membrane . The mechanisms and pathogenetic significance of the impairment of the small intestinal and liver detoxication system by cholera toxin are discussed. Am J Physiol, 1984 Aug, 247(2 Pt 1), G140 - 8 Cholera-induced mucin secretion from rat intestine: lack of effect of cAMP, cycloheximide, VIP, and colchicine; Roomi N et al.; Purified cholera enterotoxin (20-50 micrograms) and dialyzed cholera filtrate (50-125 mg) increased net glycoprotein synthetic and secretory rates in rat intestinal epithelium . Specific goblet cell mucin secretion was increased 5- to 10-fold . However, other agents that increase intestinal cAMP and accelerate glycoprotein synthesis did not enhance mucin secretion . This was true for dibutyryl cAMP (10(-3) and 10(-2) M) with or without theophylline (10(-3) M) and isoproterenol (10(-4) M) with or without dibutyryl cAMP (10(-3) M) . Hyperosmotic mannitol (450 mosmol/l), which increases fluid secretion but does not affect cAMP, and vasoactive intestinal peptide (2 X 10(-7) M), which increases both fluid secretion and cAMP, both failed to increase mucin secretion, implying that fluid "washout" of mucin adherent to the mucosal surface is not responsible for cholera-induced mucin secretion . Cycloheximide, an inhibitor of cholera diarrhea in vivo (20 mg/kg) or in vitro (1 mM), effectively abolished {3H}leucine incorporation into protein but did not affect cholera-induced mucin secretion . Colchicine (10-50 mg/kg) given to block microtubule assembly was similarly without effect on mucin secretion . These findings suggest that there is a dissociation of electrolyte/fluid and mucin secretory processes and cast doubt on the widely accepted notion that all cholera effects are mediated via the well-known adenylate cyclase-cAMP mechanism. Bull Soc Pathol Exot Filiales, 1984 Jul-Aug, 77(4), 415 - 22 {7th epidemic of cholera in Maghreb . Epidemiologic data compared}; Jeddi M et al.; Epidemiological characteristics are very similar into the concerned countries . From official data of Algeria and Tunisia, authors described the main variables: distribution in age, seasonal variations and secular trends . Case fatality rate is similar in the two countries (between 5 and 8%). Avian Dis, 1984 Jul-Sep, 28(3), 770 - 3 Possible genetic variation in resistance of turkeys to erysipelas and fowl cholera; Saif YM et al.; Natural disease outbreaks of erysipelas and fowl cholera occurred in several lines of turkeys maintained for genetic studies . There were line differences in mortality during both outbreaks, suggesting that there is genetic variation in resistance to these diseases . A line developed by selection for increased egg production had a higher mortality rate from fowl cholera than did the randombred control line from which it was developed . Both the egg line and its control line had a lower mortality rate in the erysipelas outbreak than did a line selected for increased growth rate . Both diseases induced high mortality in a line selected for increased growth. Rev Infect Dis, 1984 Jul-Aug, 6(4), 563 - 6 Enhancement by lipid A of mucosal immunogenicity of liposome-associated cholera toxin; Pierce NF et al.; Two methods that might enhance the mucosal immunogenicity of a protein antigen, cholera toxin (CT), were studied in rats: association of CT with liposomes, and coadministration of CT with lipid A . Enteric priming by CT was not enhanced when the antigen was trapped within liposomes or bound to their surface via GM1 ganglioside, nor was it improved when CT was mixed with lipid A or with liposomes containing lipid A . However, lipid A did enhance priming by liposome-associated CT when the lipid A was incorporated into CT-bearing liposomes . It is concluded that lipid A can act as an adjuvant for a local IgA response to a mucosally applied antigen, at least when lipid A and the antigen are associated on a liposome carrier. J Biol Chem, 1984 Jun 25, 259(12), 7983 - 9 The role of gangliosides in the interaction of human chorionic gonadotropin and cholera toxin with murine Leydig tumor cells; Fishman PH et al.; A clonal line of murine Leydig tumor cells (MLTC-1) bound both human chorionic gonadotropin (hCG) and cholera toxin (CT) with high affinity and accumulated cyclic AMP in response to either effector . The major cellular ganglioside was GM3 with small amounts of GM2, GM1, and GD1a . The gangliosides became labeled when the cells were grown in medium containing {3H} galactose or were exposed to galactose oxidase or NaIO4 followed by NaB3H4 . CT specifically protected GM1 from surface labeling whereas hCG did not protect any gangliosides from being labeled . When the cells were exposed to sialidase, surface GD1a was eliminated, and GM1 increased with a corresponding increase in CT binding . When sialidase-treated cells were first incubated with the B component of CT, binding and action of CT was blocked . The cells, however, retained their ability to bind and respond to hCG . Addition of purified gangliosides to the medium effectively inhibited the binding and action of CT but not hCG . The cells incorporated the exogenous gangliosides and exhibited increased binding of and responsiveness to CT but not hCG . Both hCG- and CT-receptor complexes were extracted from the cells with nonionic detergent and analyzed by sucrose gradient centrifugation . The hCG-receptor complex had an apparent molecular weight of 190,000 whereas the CT-receptor complex sedimented only slightly faster than CT itself . MLTC-1 gangliosides were separated on thin layer chromatograms which were overlayed with either iodinated CT or hCG . The toxin bound to a ganglioside corresponding to GM1 whereas the hormone did not bind to any of the gangliosides . When the cells were incubated overnight with hCG, they lost their hCG receptors but exhibited an increase in CT binding and gangliosides . Our results indicate that GM1 is the specific receptor for CT whereas gangliosides are not involved in the binding and action of hCG. Nippon Naibunpi Gakkai Zasshi, 1984 Jun 20, 60(6), 788 - 99 {The measurement of N-protein activity in plasma membranes: the comparison of assay methods by reconstitution of cyc- membranes and cholera toxin-catalyzed ADP ribosylation}; Akita Y et al.; The relationship between the assay values of the activities of the stimulatory guanine nucleotide-binding regulatory component (N-protein) of adenylate cyclase by two different methods: reconstitution of plasma membranes of cyc- S49 cells and ADP ribosylation catalyzed by cholera toxin, have not been fully elucidated yet . In the present study, the reconstitution and ADP ribosylation assay methods were utilized, and the relationship between the assay values of the N-protein activities of the erythrocyte membranes from normal subjects and patients with pseudohypoparathyroidism type I (PHP-I) measured by the two methods was investigated . When Lubrol extracts of human erythrocyte membranes were reconstituted with cyc- membranes, the rate of cyclic AMP synthesis reached a constant rate after incubation of 80 minutes at 30 degrees C . This reaction depended on the concentrations of cyc- membranes and Lubrol extracts of the erythrocyte membranes . N-protein activity in the erythrocyte membranes of normal subjects and PHP-I patients assayed by reconstitution of cyc- membranes (mean +/- SD, expressed by % of pooled standard preparation) was 100.1 +/- 13.5 (n = 29) and 82.3 +/- 28.0 (n = 19) respectively . The cholera toxin-catalyzed transfer of {32P}ADP ribose from {32P}NAD to the 42,000-dalton peptide subunit of human erythrocyte N-protein reached a plateau after incubation of 10 minutes at 30 degrees C . This reaction depended on the concentrations of cholera toxin, NAD, and the erythrocyte membranes . N-protein activity in the erythrocyte membranes from normal subjects and PHP-I patients assayed by cholera toxin-catalyzed ADP ribosylation (pmol/mg of protein) was 1.33 +/- 0.20 (n = 7) and 1.15 +/- 0.43 (n = 5) respectively . The correlation between assay values of erythrocyte N-protein activity in PHP-I patients by reconstitution of cyc- membranes (X, % of pooled standard) and cholera toxin-catalyzed ADP ribosylation (Y, % of pooled standard) was recognized as a linear regression: Y = 0.77X + 16.8 (r = 0.981, P less than 0.001) . It is thus concluded that the activity of N-protein in human erythrocyte membrane can be assayed by the ADP ribosylation method more simply than by the method of reconstitution of cyc- membranes, with highly close correlation between the values determined by these two methods. Science, 1984 Jun 15, 224(4654), 1245 - 7 Reinitiation of growth in senescent mouse mammary epithelium in response to cholera toxin; Daniel CW et al.; Several lines of mouse mammary tissue that had been serially transplanted until mitotic senescence was reached were exposed in vivo to plastic implants that slowly released cholera toxin . Gland tissue surrounding the implants displayed new end buds, indicating reinitiation of growth and morphogenesis . The ability of cholera toxin, which elevates intracellular adenosine 3',5'-monophosphate, to temporarily reverse the senescent phenotype suggests that this mitotic dysfunction results not from generalized cellular deterioration but from specific changes in cell regulation. Zh Mikrobiol Epidemiol Immunobiol, 1984 Jun, (6), 76 - 9 {Effect of gamma radiation on the immunobiological and immunochemical properties of cholera exotoxin . III . The serological activity and immunochemical properties of irradiated unpurified toxin}; Nedugova GI et al.; The immunochemical properties and serological activity of irradiated preparations of crude cholera exotoxin have been studied . This study has revealed that with the increase of the dose of ionizing radiation changes occur in the physico-chemical properties of the preparations of the toxin, which leads to an increase in the electrophoretic motility of the protein components of the toxin, to the aggregation and polymerization of individual fragments . The preparations of antigen exotoxins have been shown to retain their serological activity within the range of radiation doses under study (10-350 kGy). J Infect Dis, 1984 Jun, 149(6), 1014 - 7 From the National Institute of Allergy and Infectious Diseases . Summary of the 19th United States-Japan Joint Cholera Conference; Edelman R et al.; Classical cholera has reappeared in Asia after a 20-year hiatus, reminding us that we still have much to learn about the epidemiology of this disease . The unexpected recovery of V . cholerae from nonendemic estuarine waters suggests that the continued occurrence of clinical cholera may not be entirely dependent on repeated contamination of environmental waters by man . Of critical importance has been the discovery and partial characterization of new enterotoxins produced by V . cholerae and ETEC, a finding that further complicates the already complex problem of fully elucidating the virulence mechanism of these organisms . The recent purification of Shiga toxin is beginning to provide clues as to its structure, function, and possible pathogenic role in EPEC-related hemorrhagic colitis and diarrhea . The conversion of virulent V . cholerae into less virulent strains by genetic engineering provides hope for the ultimate development of safe and effective live oral cholera vaccines . Intestinal Peyer's patches process living and killed enteropathogens differently, and this discovery may afford insights into ways to improve antigen potency . Enterotoxins differ fundamentally in their biochemical effects, and not all of them evoke active electrolyte secretion by altering cyclic-nucleotide levels in mucosal cells . Finally, the mucosal response to a protein toxin may be under some genetic control . The complete proceedings of this conference will be published by KTK Publishers (Tokyo) . The next Joint Conference on Cholera has been scheduled for early November 1984 in Nara, Japan. Arch Biochem Biophys, 1984 Jun, 231(2), 271 - 9 Induction of synthesis of bovine adrenocortical cytochromes P-450scc, P-45011 beta, P-450C21, and adrenodoxin by prostaglandins E2 and F2 alpha and cholera toxin; Boggaram V et al.; To further elucidate the mechanisms by which ACTH (adrenocorticotropin) exerts its long-term action to maintain normal levels of adrenocortical cytochromes P-450 and related enzymes, the abilities of cholera toxin and prostaglandins E2 and F2 alpha to induce the synthesis of cytochromes P-450scc, P-45011 beta, and P-450C21 and adrenodoxin have been examined . These effectors stimulate the production of cyclic AMP and thus steroidogenesis in the adrenal cortex . Using bovine adrenocortical cells in primary monolayer culture, we have shown that treatment with cholera toxin results in increased synthesis of cytochromes P-450scc and P-45011 beta and adrenodoxin, similar to the effect observed upon ACTH treatment . Prostaglandins E2 and F2 alpha are less effective at inducing the synthesis of the mitochondrial cytochromes P-450, and do not seem to induce the synthesis of adrenodoxin . Furthermore, cholera toxin was found to be less effective at inducing the synthesis of microsomal cytochrome P-450C21 than ACTH, and no more effective than the prostaglandins . Thus, while it appears that elevation of cyclic AMP levels is a necessary step leading to increased synthesis of adrenocortical forms of cytochrome P-450, the detailed mechanism of this induction will be found to be different for each of the different enzymes. J Immunol, 1984 Jun, 132(6), 2736 - 41 Generalized systemic and mucosal immunity in mice after mucosal stimulation with cholera toxin; Elson CO et al.; Cholera toxin (CT) has been found to be an extremely potent immunogen for mucosal IgA responses when administered via the intestine . This study has examined both mucosal and systemic immune responses after feeding CT and compared these responses with those obtained after feeding keyhole limpet hemocyanin (KLH), another protein that is strongly immunogenic in mice . Feeding CT to mice resulted not only in IgA antibody in intestinal secretions but also resulted in substantial plasma IgG and IgA antibody levels . Feeding KLH in much larger quantity resulted in little or no antibody response in intestinal secretions or plasma . Lymphoid cells from various tissues of mice fed CT were cultured in vitro for 10 days and the supernatant was tested for antibody to CT . Spontaneous antibody synthesis (no antigen added to cultures) was present in cultures of each cell type, but IgG anti-CT was found mainly in cultures of spleen and mesenteric lymph node cells and IgA anti-CT mainly in cultures of Peyer's patch and lamina propria cells . Peyer's patch cells cultured with CT as antigen synthesized both IgG and IgA anti-CT, suggesting that the antibody response to both isotypes originated in this site . Helper T cell activity for both IgA and IgG anti-CT was detected in spleens, mesenteric lymph nodes, and Peyer's patches . Lastly, when KLH and CT were fed to mice at the same time, an intestinal IgA anti-KLH and plasma IgG anti-KLH response was stimulated, a response pattern similar to that occurring to CT after CT was fed alone . We conclude that mucosal stimulation by CT generates both a systemic IgG and mucosal IgA response to this antigen, and that CT can cause a similar pattern of response to an unrelated protein antigen when both are administered into the intestine at the same time . The data favor the idea that both the IgG and IgA responses originate in GALT and then disseminate to other tissues . We propose that CT accomplishes these effects by altering the regulatory environment within GALT. J Biol Chem, 1984 May 25, 259(10), 6686 - 93 Characterization of transducin from bovine retinal rod outer segments . Mechanism and effects of cholera toxin-catalyzed ADP-ribosylation; Navon SE et al.; Transducin, a guanine nucleotide-binding protein consisting of two subunits (T alpha and T beta gamma), mediates the signal coupling between rhodopsin and a membrane-bound cyclic GMP phosphodiesterase in retinal rod outer segments . The T alpha subunit is an activator of the phosphodiesterase, and the function of the T beta gamma subunit is to physically link T alpha with photolyzed rhodopsin . In this study, the mechanism of cholera toxin-catalyzed ADP-ribosylation of T alpha has been examined in a reconstituted system consisting of purified transducin and stripped rod outer segment membranes . Limited proteolysis of the labeled T alpha with trypsin indicated that the inserted ADP-ribose is located exclusively on a single proteolytic fragment with an apparent molecular weight of 23,000 . Maximal incorporation of ADP-ribose was achieved when guanosine 5'-(beta, gamma-imido)triphosphate (Gpp(NH)p) and T beta gamma were present at concentrations equal to that of T alpha and when rhodopsin was continuously irradiated with visible light in the 400-500 nm region . The stimulating effect of illumination was related to the direct interaction of the retinal chromophore with opsin . These findings strongly suggest that a transient protein complex consisting of T alpha X Gpp(NH)p, T beta gamma, and a photointermediate of rhodopsin is the required substrate for cholera toxin . Single turnover kinetic measurements demonstrated that the ADP-ribosylation of T alpha coincided with the appearance of a population of transducin molecules having a very slow rate of GTP hydrolysis . The hydrolysis rate of the bound GTP for this population was 1.1 X 10(-3)/s, which was 22-fold slower than the rate for the unmodified transducin. J Biol Chem, 1984 May 25, 259(10), 6228 - 34 Purification of a protein cofactor required for ADP-ribosylation of the stimulatory regulatory component of adenylate cyclase by cholera toxin; Kahn RA et al.; A factor (ARF) that is required for the cholera toxin-dependent ADP-ribosylation of the stimulatory, GTP-binding regulatory component (Gs) of adenylate cyclase has been purified about 2000-fold from cholate extracts of rabbit liver membranes . ARF is an intrinsic membrane protein with Mr = 21,000 . The final product can be resolved into two polypeptides with very similar molecular weights; each of these has ARF activity . The ADP-ribosylation of Gs can now be studied with defined components . GTP and ARF are both necessary cofactors . The data imply that the substrates for the activated toxin are NAD and a GTP X Gs X ARF complex, and the reaction proceeds in a lipid environment . The apparent ability of ARF to bind to the alpha subunit of Gs suggests that it may play another, unknown role in the regulation of adenylate cyclase activity. J Biol Chem, 1984 May 25, 259(10), 6110 - 6 Modulation of thyroid hormone nuclear receptors by cholera toxin in cultured GH1 cells; Aranda A et al.; The cellular actions of the thyroid hormones L-thyroxine and L-triiodothyronine are mediated by the association of hormone with a chromatin-associated receptor . In cultured GH1 cells, a hormone-responsive rat pituitary cell line, thyroid hormone decreases the concentration of its receptor at early incubation times by reducing the accumulation of newly synthesized receptor . In this study, we demonstrate that cholera toxin also reduces the amount of nuclear receptor in GH1 cells in a time- and dose-dependent fashion, without altering the affinity of the receptor for hormone . The reduction of receptor mediated by cholera toxin is not secondary to a generalized inhibition of cell protein synthesis or cell replication rates and this effect can be abolished by pretreatment of the cholera toxin with soluble ganglioside II3-alpha-N- acetylneuraminosylgangliotetraosylceramide . This effect requires an intact cholera toxin molecule and does not occur at similar concentrations of the membrane-binding B subunit of cholera toxin . In order to study the influence of cholera toxin on thyroid hormone receptor turnover, we have used a dense amino acid-labeling technique . The results indicate that cholera toxin does not change the half-life of receptor, but decreases the rate of appearance of newly synthesized receptor . This decreased rate completely accounts for the lowered steady state receptor levels . The extent of cAMP stimulation by cholera toxin does not correlate with the extent of receptor reduction and forskolin, which stimulates cAMP 25- to 500-fold, does not decrease thyroid hormone receptor abundance . These studies suggest that cholera toxin modulates receptor levels by a mechanism(s) that is not mediated by cAMP in GH1 cells. Biochemistry, 1984 May 22, 23(11), 2520 - 6 Interaction of cholera toxin with gangliosides: differential effects of the oligosaccharide of ganglioside GM1 and of micellar gangliosides; Tomasi M et al.; Ultraviolet difference absorption spectra of cholera toxin and its B protomer produced by the oligosaccharide moiety of the monosialoganglioside GM1 were measured as a function of the oligosaccharide concentration . In the presence of oligosaccharide, the spectrum is characterized by three peaks at 282, 288, and 292 nm . A linear increase in difference absorption was observed at these wavelengths vs . oligosaccharide concentration; a saturation effect occurred when the molar ratio of oligosaccharide to cholera toxin was higher than 5 . The features of the spectra indicated that the binding with the oligosaccharide affected the environment of tryptophan and tyrosine residues of protomer B . In good agreement with the above results, circular dichroic spectra indicated also a local effect of the binding, mostly restricted to protomer B, while the residues of protomer A remained largely unperturbed . Difference absorption spectra were also measured for cholera toxin in the presence of ganglioside and detergent micelles . The employed gangliosides GD1a and GT1b, unable to bind cholera toxin, interact with the protein by way of contaminating traces of GM1 . The preparations of GD1a and GT1b contained 0.8-1.0% (w/w) and 0.4-0.5% (w/w) of GM1, respectively . The results obtained with ganglioside GD1a and GT1b in contrast with the observations made with the oligosaccharide of GM1 indicated a major conformational change of the toxin structure . Upon comparison of the conformational change induced by ganglioside micelles with that induced by sodium dodecyl sulfate it may be suggested that the ganglioside micelle, behaving as a detergent, alters the structure of the toxin such as to induce the penetration of protomer A into the lipid milieu.(ABSTRACT TRUNCATED AT 250 WORDS) Biochemistry, 1984 May 22, 23(11), 2339 - 47 Light-regulated biochemical events in invertebrate photoreceptors . 1 . Light-activated guanosinetriphosphatase, guanine nucleotide binding, and cholera toxin catalyzed labeling of squid photoreceptor membranes; Vandenberg CA et al.; The occurrence of a guanine nucleotide binding protein activated by squid rhodopsin was established by examination of GTPase activity, guanine nucleotide binding, and cholera toxin catalyzed labeling of squid photoreceptor membranes . Purified squid (Loligo opalescens) photoreceptors exhibited GTPase activity that increased 3-4-fold by illumination . Half-maximal GTPase activity was observed when 2% of the rhodopsin was photoconverted to metarhodopsin . The Km of the light-regulated activity was 1 microM GTP . Binding of the hydrolysis-resistant GTP analogue guanosine 5'-(beta, gamma-imidotriphosphate) {Gpp(NH)p} was enhanced greater than 10 times by illumination . A protein, Mr 44 000, was identified as a component of the light-activated guanine nucleotide binding protein/GTPase through its specific labeling with {32P}NAD catalyzed by cholera toxin: light increased the extent of 32P incorporation 7-fold . The addition of ATP to the membrane suspension enhanced labeling, while guanine nucleotides inhibited labeling with the relative potency GTP gamma S much greater than GDP greater than GTP greater than Gpp(NH)p . The 44 000-dalton protein was membrane bound irrespective of variations in ionic strength and divalent ion concentration over a wide range . These results suggest that a G protein, which incorporates both GTP binding and hydrolysis functions, is intimately involved in the visual process of invertebrate photoreceptors. Infect Immun, 1984 May, 44(2), 474 - 8 Resistance of bovine colostral anti-cholera toxin antibody to in vitro and in vivo proteolysis; McClead RE et al.; Pregnant cows immunized with cholera enterotoxin produce an immunoglobulin G class 1 antibody that enters the colostrum in high titer . After exposure to intestinal enzymes, this antibody remains immunologically reactive and inhibits intestinal fluid secretion in infant and adult rabbits exposed to cholera enterotoxin . Specific bovine colostral antibodies may be a source of passive immune protection for human infants and adults at risk for cholera and other enteric diseases. Infect Immun, 1984 May, 44(2), 469 - 73 Enhanced mucosal priming by cholera toxin and procholeragenoid with a lipoidal amine adjuvant (avridine) delivered in liposomes; Pierce NF et al.; The mucosal adjuvant activity of avridine, a synthetic lipoidal amine {N,N-dioctadecyl-N',N'-(2-hydroxymethyl) propanediamine, previously designated CP-20,961), was studied in rats immunized intraintestinally with cholera toxin or procholeragenoid . Avridine was most efficient as an adjuvant when incorporated into liposomes; liposomes that lacked avridine had no adjuvant effect . Coadministration of avridine-containing liposomes with enteric priming doses of cholera toxin or procholeragenoid enhanced the efficiency of priming for secondary mucosal anti-cholera toxin responses, i.e., the establishment of memory, five- to sevenfold . Avridine-containing liposomes had no significant effect, however, on either the primary mucosal anti-cholera toxin response, when given with the primary dose of antigen, or on the secondary response, when given with the booster dose to previously primed animals . Little or no adjuvant effect occurred when avridine-containing liposomes were given concurrently with antigen, but at a separate mucosal site or parenterally, or at the site of enteric immunization, but 1 day earlier or later . These results support the notion that adjuvants may be developed which enhance the mucosal immunogenicity of locally applied antigens and suggest that liposomes may be effective vehicles for delivery of such adjuvants. Cancer Res, 1984 May, 44(5), 1998 - 2010 Direct mitogenic effects of insulin, epidermal growth factor, glucocorticoid, cholera toxin, unknown pituitary factors and possibly prolactin, but not androgen, on normal rat prostate epithelial cells in serum-free, primary cell culture; McKeehan WL et al.; Selective nutritive conditions were used to isolate normal epithelial cells from fibroblasts in primary cell cultures prepared from adult rat prostate . The pure population of normal epithelial cells proliferated at an exponential rate on a simple polystyrene substratum with doubling times of 35 to 50 hr for 10 to 12 days in the absence of high epithelial cell density, other cell types, or added extracellular matrix elements . Optimization of the nutritive environment allowed direct analysis of the hormone:growth factor requirements for sustained proliferation of the isolated epithelial cells in serum-free medium . An in situ videometric method was used to assay the effect of over 30 known hormones and growth factors on proliferation of the prostate epithelial cell population . The results revealed direct mitogenic effects of insulin, epidermal growth factor, glucocorticoid, cholera toxin, one or more unidentified factors from bovine pituitary, and possibly prolactin . No direct mitogenic effect of androgen on isolated prostate epithelial cells could be demonstrated . Radioimmunoassay of androgen in the primary cultures showed that endogenous androgen was about 34 pM on Day 1 of culture and thus probably too low to mask a response to exogenous androgen . Deletion of any single active growth factor did not reveal an androgen response . The results demonstrate a multihormonal control of normal prostate epithelial cell maintenance and proliferation without the direct participation of androgen. Biol Reprod, 1984 May, 30(4), 787 - 94 Calcium-dependent regulation of progesterone production by isolated rat granulosa cells: effects of the calcium ionophore A23187, prostaglandin E2, dl-isopro