Cholera

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-isoproterenol and cholera toxin; Tsang BK et al.; The role of calcium in the regulation of ovarian steroidogenesis was investigated in granulosa cells from estradiol-treated immature rats . Incubation of granulosa cells with various calcium channel blockers (verapamil, cobalt or manganese) and a calcium chelator (EGTA) resulted in marked decreases in progesterone production in response to follicle-stimulating hormone (FSH), cholera toxin, prostaglandin E2, dl-isoproterenol and dibutyryl cyclic AMP (Bt2cAMP) . Cyclic AMP production, however, was unaffected by treatment with EGTA and verapamil at concentrations which attenuated steroidogenesis (0.1-1.0 mM and 125 microM, respectively) . Two inhibitors of the calcium-dependent regulatory protein, calmodulin {trifluoperazine, 40 microM and 1{bis-(p-chlorophenyl)methyl} 3-{2,4-dichloro-beta-(2,4- dichlorobenzyloxy )-phenethyl}imidazolium chloride, ( R24571 ) 20 microM} significantly inhibited both cyclic AMP and progesterone production elicited by these stimulatory agents . Over the concentration range of 62.5 ng/ml-1.0 micrograms/ml, the calcium ionophore A23187 increased basal progesterone production in a dose-dependent manner, with half-maximal stimulation at approximately 0.14 microgram/ml . Maximal steroidogenic response to the calcium ionophore (1 microgram/ml) however, was only 50% of that evoked by FSH (0.33 microgram/ml) . A23187 (0.5 microgram/ml) significantly enhanced progesterone production stimulated by a low concentration of FSH (0.025 microgram/ml) but failed to potentiate the maximally stimulatory action of the gonadotropin (0.33 microgram/ml) . These findings support our earlier suggestion that the calcium-calmodulin system plays a central role in the gonadotropic regulation of ovarian steroidogenesis and suggest that a transmembrane flux of extracellular calcium may be an important and common step in the mechanism of stimulation of granulosa cell progesterone production.

Biochem Biophys Res Commun, 1984 Apr 30, 120(2), 700 - 6
Inhibition of ADP-ribosyltransferase activity of cholera toxin by MDL 12330A and chlorpromazine; Bitonti AJ; ADP-ribosylation by cholera toxin of the guanine nucleotide binding regulatory protein (Gs) of rat liver membrane adenylate cyclase was inhibited by 0.1-1 mM MDL 12330A or 0.1-1 mM chlorpromazine . Basal as well as cholera toxin activated adenylate cyclase activity in liver membranes was also inhibited by the two drugs . NAD glycohydrolase activity and self-ADP-ribosylation of cholera toxin were also inhibited by MDL 12330A and chlorpromazine . These effects of MDL 12330A and chlorpromazine may be related to their effects on cholera toxin-induced fluid secretion in vivo.

Biochem Biophys Res Commun, 1984 Apr 30, 120(2), 540 - 7
Effects of forskolin and cholera toxin on cyclic AMP release in a neurotensin-secreting rat C-cell line; Zeytin F et al.; The effects of forskolin and cholera toxin on the regulation of cAMP release were studied in a neurotensin-secreting rat C-cell line . The interaction of these agents with norepinephrine, a potent neurotensin secretagogue, was also investigated . Forskolin stimulated cAMP release 10(2)-10(3) fold while it increased neurotensin release 2-3 fold . Cholera toxin caused a 10(2)-10(3) fold increase in cAMP release and had no effect on neurotensin release . We conclude that the 44-2 C-cells provide a new model for studying the regulation of the concomitant (via forskolin) or independent (via cholera toxin) secretion of cyclic AMP and/or neurotensin.

FEBS Lett, 1984 Apr 24, 169(2), 241 - 6
Evolution and structure of two ADP-ribosylation enterotoxins, Escherichia coli heat-labile toxin and cholera toxin; Yamamoto T et al.; Nucleotide sequence comparisons of the heat-labile enterotoxin (LTh) genes of E . coli pathogenic for humans with cholera toxin (CT) genes suggest that the two toxin genes have evolved from a common ancestry by a series of single base changes, while conserving the catalytic fragment A1 (ADP-ribose transferase) . Based on the local hydrophilicity profiles of LTh and CT peptides, a transmembrane segment appears to be present in A1 in both toxins.

Am J Physiol, 1984 Mar, 246(3 Pt 1), E288 - 91
Estrogen-like stimulation of uterine ornithine decarboxylase by cholera toxin; Webster RA et al.; Cholera toxin administered by intrauterine injection to ovariectomized rats increased uterine ornithine decarboxylase activity as much as systemic estradiol at 4 h after treatment . At 45-60 min after treatment, however, cholera toxin did not increase nuclear estrogen receptor or stimulate synthesis of the uterine "induced protein," which is closely correlated with nuclear receptor, whereas estradiol caused substantial increases in both nuclear receptor and induced protein synthesis . Intrauterine injection of cholera toxin also produced an estrogen-like elevation of the uterine protein/DNA ratio at 24 h . Because both cholera toxin and estradiol are known to increase vascular permeability, our results support the hypothesis that some uterine effects of estradiol are not mediated by receptor-genome interaction but involve another mechanism that is associated with increased vascular permeability.

Infect Immun, 1984 Mar, 43(3), 811 - 6
Differences in cross-protection in rats immunized with the B subunits of cholera toxin and Escherichia coli heat-labile toxin; Klipstein FA et al.; Although cholera toxin (CT), Escherichia coli heat-labile toxin (LT), and their B subunits are known to be immunologically related, the ability of each to raise an antitoxin response that provides equally strong cross-protection against active challenge with pure heterologous toxin has not been examined previously . We immunized rats with pure preparations of the B subunits of human LT, porcine LT, and CT . Immunization with either of the LT B subunits raised greater than or equal to fourfold increases in specific mucosal immunoglobulin A antitoxin titers to homologous and heterologous LT and CT B subunits, thereby providing strong protection against active challenge in ligated ileal loops with all three respective holotoxins and with a viable LT-producing E . coli strain . In contrast, immunization with the CT B subunit raised a greater than or equal to fourfold increase in antitoxin titers only to itself and provided strong protection only against challenge with the CT holotoxin . Conjugation of the CT B subunit with the E . coli heat-stable toxin by the carbodiimide reaction yielded a cross-linked immunogen with equal antigenicity for both components; immunization with this conjugate raised greater than or equal to fourfold increases in antitoxin titers to both components, but it provided significant protection only against challenge with a viable heat-stable toxin-producing E . coli strain and not to an LT-producing E . coli strain . These observations indicate that immunization with the LT B subunits raises a heterologous antitoxin response that extends to the CT B subunit, thereby providing equally strong protection against LT and CT; however, immunization with the CT B subunit raises principally a homologous antitoxin response, so that this immunogen provides strong protection only against CT.

Endocrinology, 1984 Mar, 114(3), 904 - 13
Human pancreatic tumor growth hormone (GH) - releasing factor and cyclic adenosine 3',5'- monophosphate evoke GH release from anterior pituitary cells: the effects of pertussis toxin, cholera toxin, forskolin, and cycloheximide; Cronin MJ et al.; Both synthetic human pancreatic tumor GH-releasing factor (hpGRF) and prostaglandin E2 (PGE2) rapidly stimulate cellular cAMP accumulation in and GH release from primary cultures of rat anterior pituitary cells . SRIF inhibits both of these actins . A 1-h treatment with the protein synthesis inhibitor cycloheximide potentiates hpGRF-induced cAMP accumulation for hours and GH release for the first hour . This indicates that a rapidly turning over protein tonically mutes the degree of hpGRF-stimulated cAMP accumulation . Pretreatment of the cells with pertussis toxin amplifies hpGRF- and PGE2-stimulated cAMP levels and GH release; pertussis toxin also attenuates the ability of SRIF to affect these variables . This suggests that an inhibitory coupling protein contributes to these events . Finally, cholera toxin and forskolin are also potent stimulators of cAMP accumulation and GH release . We conclude that hpGRF-evoked GH release and the inhibitory action of SRIF are closely correlated with the cAMP-generating system.

Zh Mikrobiol Epidemiol Immunobiol, 1984 Feb, (2), 47 - 51
{Effect of gamma radiation on the immunobiological and immunochemical properties of cholera exotoxin . I . Change in the biological activity of nonpurified cholera exotoxin as affected by ionizing radiation}; Nedugova GI et al.; Crude cholera exotoxin (filtrate toxin) was irradiated with increasing doses of gamma radiation . A significant drop in enterotoxicity, in the activity of the permeation factor and a decrease in toxicity were shown to occur as radiation doses increased . Radiation doses of 50-70 kGy were found to completely inactivate enterotoxicity in liquid toxic preparations . A higher radioresistance of dried preparations in comparison with liquid ones was registered: inactivation occurred at 150-200 kGy . Different batches of the initial filtrate toxin had varying radiosensitivity . The sterilizing effect of gamma radiation was achieved at doses of 20 kGy for liquid preparations and 30 kGy for dried preparations . During the prolonged storage of the irradiated preparations of crude toxin (the term of observation being 1.5 years) at different temperatures no reversion of toxicity was found to occur, while their immunogenic properties remained unchanged.

J Biol Chem, 1984 Jan 25, 259(2), 696 - 8
Amino acid sequence of retinal transducin at the site ADP-ribosylated by cholera toxin; Van Dop C et al.; Transducin was {32P}ADP-ribosylated by cholera toxin in bovine retinal rod outer segments and then partially purified on omega-amino octyl agarose to remove other ADP-ribosylated proteins . Trypsin digestion of the ADP-ribosylated transducin and further purification using boronate-polyacrylamide beads and high performance liquid chromatography yielded a single radiolabeled tetrapeptide, Ser-Arg-Val-Lys . The ADP-ribose is linked to the guanidinium group of arginine.

Soc Sci Med, 1984, 18(5), 429 - 40
Identification of the cholera diffusion process in Ibadan, 1971; Adesina HO; The paper tries to examine and identify which spatial diffusion process was responsible for generating the pattern of cholera diffusion (an epidemic spread which was apparently wave-like) within Ibadan City in 1971 . In this paper one of Moran's statistics, the BW join-count measure of spatial autocorrelation is employed . Five different plannar graphs are used in the study . The results show that contagion was apparent on the various models of spatial processes employed and on their different combinations . But it is the radial contact diffusion which was discovered to be most important in the spread of the epidemic . But even, though such a radial contact diffusion process was discovered to be the most important, during the advance and peak phases of the epidemic wave; as the epidemic intensity rose to a spread phase, a mixture of the various models became a best contributor to the contagion.

Soc Sci Med, 1984, 18(5), 421 - 8
The diffusion of cholera outside Ibadan City, Nigeria, 1971; Adesina HO; This paper examined the spread of cholera epidemic to the towns of the former Western State of Nigeria and to the neighbouring villages of Ibadan after the initial introduction of the disease to Ibadan city by early January, 1971) . In the diffusion process, an hierarchical diffusion was discovered at the town-village dichotomy while a distance decay function was justified at the purely urban level analyses . In the spread of cholera to all the surrounding villages of Ibadan, the epidemic speed was discovered to be too rapid that neither population size nor distance from Ibadan was relevant to the pattern of spread . The rate of cholera infection was observed to decline with distance from the city of Ibadan, while the duration of the epidemic obeyed the rank-size principles.

Int Arch Allergy Appl Immunol, 1984, 74(3), 221 - 5
Intestinal resistance to cholera toxin in mouse . Antitoxic antibodies and desensitization of adenylate cyclase; Lange S et al.; Mice were immunized perorally with cholera toxin (CT), cholera B-subunit (CB), or buffer as control . The response of anti-CT antibodies of the IgG, IgA and IgM class in bile, IgA being predominating, were similar in both immunized groups . The same number of anti-CT containing plasma cells (ACC) were determined in the intestinal lamina propria of CT - as well as of CB-immunized mice 20 days after the last immunization, while ACC at day 4 in the CB group were 50% higher than in the CT group . In contrast to the vigorous antibody response to CT in both groups of immunized mice, only animals immunized with CT displayed resistance to CT-induced intestinal hypersecretion and to CT stimulation of adenylate cyclase . The CB-treated group responded to CT with fluid accumulation and enzyme activation similar to controls . The results suggest that intestinal resistance to CT in mouse is due to desensitization of adenylate cyclase rather than to CT-neutralizing antibodies.

Indian J Med Res, 1984 Jan, 79, 96 - 102
Safety of oral rehydration solutions in non-cholera diarrhoea; Deorari AK et al.; PIP: The safety of 2 oral rehydration solutions (ORS) containing 60 and 90 mEq/1 of sodium respectively was evaluated in 50 children with mild to moderate dehydration secondary to noncholera diarrhea . Hypernatremia developed in 1 patient (3.7%) on high sodium formula . The risk of hypernatremia and hyponatremia in the 2 groups did not differ significantly . 3 patients (6.1%) showed hypokalemia 24 hours after oral rehydration was initiated . While these data confirm that ORS containing 90 mEq/1 of sodium is safe, an increase in potassium content should be considered . author's modified

Zh Mikrobiol Epidemiol Immunobiol, 1984 Jan, (1), 85 - 9
{Experimental justification for combined subcutaneous immunization against cholera and typhoid fever}; Gapochko KG et al.; The article presents materials on experiments substantiating the method for the subcutaneous administration of chemical adsorbed typhoid vaccine by means of a jet-injector and the possibility of using this preparation in combination with cholerogen toxoid.

Forensic Sci Int, 1984 Jan, 24(1), 95 - 8
Sudden, unexpected death following typhoid-cholera vaccination; Pounder DJ; A previously healthy 33-year-old Australian male died suddenly and unexpectedly 8 h after a typhoid-cholera vaccination . Such fatalities are extreme rarities and the present case is the first in which postmortem measurement of serum immunoglobulins has been undertaken . The clinical course and necropsy findings suggest that death was the result of a slowly evolving systemic anaphylactic reaction which terminated in hypotension and right heart failure . The deceased was probably atopic . The current recommendations for the vaccination of international travellers against typhoid and cholera are discussed.

J Neurosci Res, 1984, 12(2-3), 335 - 41
Tryptophan fluorescence properties of cholera toxin upon interacting with ganglioside GD1b; Mestrallet MG et al.; A blue shift of the tryptophan fluorescence emission spectra of cholera toxin or the B protomer is induced by disialoganglioside GD1b with a capacity similar to that of monosialoganglioside GM1 . Both gangliosides were also capable of decreasing or reversing the fluorescence quenching by iodide ion of the toxin . The quenching constants (Ksv) for the toxin fluorescence in absence of gangliosides was 2.8 M-1; in presence of GM1 or GD1b, Ksv was 0.8 M-1 and 0.7 M-1, respectively . Gangliosides GD1a and GT1b were unable to decrease the quenching effect . The results suggest that GD1b induces a perturbation in the tryptophan environment of the toxin molecule similar to that induced by GM1.

Int Arch Allergy Appl Immunol, 1984, 75(2), 143 - 8
Protection against experimental cholera in the rat . A study on the formation of antibodies against cholera toxin and desensitization of adenylate cyclase after immunization with cholera toxin; Lange S et al.; The importance of antibodies to cholera toxin (CT) versus desensitization of intestinal adenylate cyclase for protection against experimental cholera in the rat was investigated . Animals were immunized five times with CT either perorally or intravenously; antitoxic antibodies were measured in both serum and bile, and intestinal anti-CT-containing plasma cells (ACC) as well as eosinophilic leucocytes were counted . Both peroral and intravenous immunizations induced high levels of serum antibodies, while antibodies in bile appeared only after peroral immunization . The number of eosinophilic leucocytes in the intestinal mucosa increased in response to peroral immunization . CT-induced stimulation of intestinal adenylate cyclase was significantly suppressed, i.e., desensitized, after peroral immunization, but was equally stimulated in intravenous immunized and in control animals . A constant and long-lasting protection against CT-induced secretion was obtained after peroral immunization . This protection correlated neither to the concentrations of antitoxic antibodies in serum, nor to those in bile, nor to the number of ACC, but did correlate to the desensitization of adenylate cyclase.

Int Arch Allergy Appl Immunol, 1984, 74(3), 226 - 31
Studies on cholera-toxin-induced desensitization of adenylate cyclase in the mouse intestinal mucosa; Lonnroth I et al.; Repeated peroral pretreatment ('immunization') with cholera toxin (CT) in mice induces protection against CT as well as against prostaglandin E1 (PGE1), as evaluated by fluid accumulation in intestinal loops . The fluid response to CT is depressed for more than 1 month, while the response to PGE1 is inhibited for 4-7 days after the pretreatment . Immunofluorescence microscopy revealed that neither the binding nor the penetration of CT into the intestinal epithelial cells is affected by the toxin pretreatment . Furthermore, the CT-induced release of mucus in goblet cells is not influenced by the toxin pretreatment . In contrast, the enzyme adenylate cyclase (AC), which mediates the actions of CT and PGE1, shows a long-lasting desensitization to CT, as estimated in mucosal membrane preparations . Chlorpromazine and cycloheximide revert the desensitization to CT as well as to PGE1 . The present data suggest that intestinal resistance to CT in the mouse is due to desensitization of the reaction between CT and AC and requires stimulation of AC, as well as an active protein synthesis.

Bull Soc Pathol Exot Filiales, 1984 Jan-Feb, 77(1), 13 - 6
{Initial controlled clinical trials of an oral anticholera vaccine during the cholera epidemic in the district of Malemba-Nkulu (Shaba-Zaire)}; Bwanga M; The authors have utilized the Institut Pasteur anticholeric vaccine by oral passage amid the Malemba - Nkulu area population . The choleric epidemic beginning six months after the vaccine oral passage . The results had been 67 cholera case on 18,377 subjects non inoculated 25 case on 6,249 subjects inoculated by inject vaccine, 3 case on 12,014 subjects inoculated by vaccine oral passage.

Acta Pathol Microbiol Immunol Scand {A}, 1984 Jan, 92(1), 15 - 21
Internalization in vivo of cholera toxin in the small intestinal epithelium of the rat; Hansson HA et al.; The binding and internalization of cholera toxin (CT) into the intestinal epithelium were studied in vivo in rats . The distribution of CT was ascertained using immunofluorescence and by immunoenzyme-electron microscopy, with horse-radish peroxidase anti-CT antibodies as the conjugate . The toxin was rapidly bound and internalized into both epithelial and goblet cells; CT was evenly distributed on the microvilli at the bases of which it appeared in invaginations (coated pits) . Though not found in nuclei, CT appeared intracellularly in coated vesicles, and dissolved in the cytoplasm where it was enriched at the terminal web . The basolateral membrane, except for the tight junctions, was outlined with CT; some staining also appeared in the basement membrane, in fibroblasts, macrophages and in the blood-vessel walls in the submucosa . The lysosomatotrophic agent chloroquine simultaneously inhibited CT-induced fluid secretion and intracellular distribution of CT in the cytoplasmic matrix, but not in the vesicles . The inhibitor of CT-action on adenylate cyclase, chlorpromazine, did not affect the cellular distribution of CT . Our results suggest that CT mainly is internalized by endocytosis into the intestinal epithelium . The toxin is probably released from vesicles into the cytoplasm via secondary lysosomes.

Int Arch Allergy Appl Immunol, 1984, 73(1), 86 - 8
Cholera toxin-binding T cells in the human peripheral blood at different ages; Tsuru S; The capacity of human T cells to bind cholera toxin was shown to decrease with age . In aged humans, the number of cells capable of binding high concentrations of cholera toxin was lower than that in young humans . The method presented in this paper may be useful as one of the indicators of aging of the immune system.

Arch Oral Biol, 1984, 29(4), 303 - 9
Cholera toxin and dibutyryl cyclic-AMP stimulate the growth of epithelial cells derived from epithelial rests from porcine periodontal ligament; Brunette DM; The epithelial cells (E-cells) grew best at high (greater than 5 per cent) concentrations of fetal bovine serum (FBS) . Growth could be obtained at low concentrations of dialysed FBS (DFBS) if the medium (alpha-MEM) was modified so that the levels of Ca2+ and K+ were reduced to 0.1 and 1.0 mM, respectively (beta-MEM) . The addition of 0.5 per cent DFBS to the beta-MEM did not initiate good growth but was sufficiently supportive to enable the effects of various growth promoters to be tested . Cholera toxin and dibutyryl cyclic-3'5'-adenosine monophosphate (Bt2cAMP), which are known to increase intracellular cAMP levels, at concentrations of 1 ng/ml and 0.5 mM, respectively increased cell number . Cholera toxin caused the E-cells to be more flattened when viewed by phase-contrast; this appeared to be due to spread of the cells . No difference in cell-size distributions obtained between the trypsinized E-cells grown in the presence or absence of cholera toxin was observed . Epithelial proliferation that occurs in dental cysts could result from a rise in intracellular cAMP levels in epithelial cell rests.

Int J Biochem, 1984, 16(3), 275 - 80
Effect of cholera enterotoxin on calcium uptake and cyclic AMP accumulation in rat basophilic leukemia cells; Knoop FC et al.; Cholera enterotoxin (CT), at an optimal concentration of 2.38 X 10(-10) M, stimulated calcium uptake (P less than 0.01) and cyclic AMP accumulation (P less than 0.02) in cultured rat basophilic leukemia cells . No significant effect of CT on calcium release or cyclic GMP accumulation was detected . Pharmacologic and chemical agents which block calcium uptake or prostaglandin synthesis antagonized the effect of CT.

Trans R Soc Trop Med Hyg, 1984, 78(1), 106 - 7
Episode resembling immune complex disease after cholera vaccination; Mall T et al.; The case of a 25-year-old patient is reported who suffered from a syndrome similar to immune complex disease following cholera revaccination . The clinical picture included fever, muscle, joint and abdominal pain, vomiting, serositis, hepatitis, suspected myocarditis, anaemia and thrombocytopenia . Clinical symptoms subsided spontaneously within two weeks . This case illustrates a hazard of cholera vaccination so far not reported in the literature.

Mol Cell Endocrinol, 1984 Jan, 34(1), 67 - 80
Desensitization of the cAMP system in mouse Leydig cells by hCG, cholera toxin, dibutyryl cAMP and cAMP: localization of the 'lesion' to the guanine nucleotide regulatory protein-adenylate cyclase complex; Schumacher M et al.; The mechanism of hCG-induced desensitization of the cAMP system was studied in Percoll-purified mouse Leydig cells . Pretreatment of Leydig cells with hCG resulted in a time- and dose-dependent decrease in the capacity of hCG-induced cAMP formation . Maximal desensitization (approximately 90%) was induced by only partial prior stimulation . Desensitization, however, was not observed without a prior increase in cAMP or testosterone production . Pretreatment of the cells with N6,O2'-dibutyryl cAMP (DBcAMP) also induced a dose- and time-dependent densensitization . cAMP was only effective in the presence of the phosphodiesterase inhibitor 1-methyl-3-isobutylxanthine (MIX) . Cholera toxin desensitized the hormone-induced cAMP response as drastically as hCG . Cholera toxin was unable to reverse the refractory state induced by one of the agonists . hCG-induced desensitization was not associated with a loss in {125I}hCG binding or an increase in maximal phosphodiesterase activity, and appeared not to be dependent on protein synthesis . Membranes from hCG, cholera toxin of DBcAMP-desensitized cells showed an impaired adenylate cyclase activity in response to hCG, hCG plus beta-gamma-imidoguanosine 5'-triphosphate (GPPNP) and NaF . In conclusion, hCG-induced desensitization of the adenylate cyclase system in mouse Leydig cells can be mimicked by cholera toxin, DBcAMP and cAMP, indicating a cAMP-mediated process . The site of the 'lesion' has to be localized to the guanine nucleotide regulatory protein-adenylate cyclase complex rather than to its uncoupling from the hormone receptor.

Comp Biochem Physiol C, 1984, 79(2), 243 - 8
Effects of concanavalin A and cholera toxin on epidermal cAMP and migration rate during wound closure in adult newts; Donaldson DJ et al.; Following removal of a skin patch from each hind limb of a series of adult newts, the limbs were explanted into small dishes of Holtfreter solution containing various combinations of test drugs . Later, the amount of wound epithelium that formed on each limb was determined using a planimeter on wound tracings obtained with the aid of a drawing tube-equipped microscope . Exposure of migrating cells to the plant lectin, concanavalin A (con A), lowered cyclic AMP (cAMP) levels and depressed migration . Exposure to cholera toxin and theophylline (CTX) significantly elevated cAMP levels and significantly depressed migration rate . Exposure of CTX-treated cells to con A tended to lower CTX-elevated cAMP levels while depressing the migration rate well beyond the depression caused by CTX alone . These results provide further evidence that cAMP can regulate the rate of newt epidermal cell migration . They also show that the inhibitory effect of con A on motility in these cells is independent of its effects on cAMP.

Comp Biochem Physiol C, 1984, 78(1), 89 - 98
Sensitivity to injected cholera toxin of the sodium efflux in single barnacle muscle fibers; Bittar EE et al.; A study has been made of the effect of microinjected cholera toxin (CT) on the efflux in single barnacle muscle fibers . Characteristically, injected CT causes sustained stimulation of the ouabain-insensitive Na efflux but only after a lag phase . An effect is seen with as little as a 10(-7) M-solution of CT . Sustained stimulation after a lag phase is also seen following injection of subunit A fragment . Enrichment of fibers with NAD+ fails to enhance the response to CT . Prior injection of GTP or its non-hydrolyzeable analogue, Gpp(NH)p, markedly reduces the response to CT, whilst prior injection of CT reduces the response to guanine nucleotides . Evidence is also brought forward that omission of external Ca2+ reversibly reduces the response to CT and that pre- or postinjection of EGTA markedly reduces the response to CT . In addition, fibers preinjected with CT show increased aequorin light emission . Whereas verapamil and Cd2+ are ineffective, both Mg2+ and trace metals, e.g . Fe and Zn, reverse the response to CT following injection . Prior injection of protein kinase inhibitor reduces the response to CT . As for calmodulin inhibitors, e.g . chlorpromazine, imipramine and mepacrine, they are effective in reducing the response to CT but not calmodulin antibody (IgG) . Collectively, the above results are compatible with the view that sustained stimulation of the ouabain-insensitive Na efflux by injected CT is due to persistent activation of adenylate cyclase by the toxin and that a fall in myoplasmic pCa facilitates or augments this activation mechanism.

Med Biol, 1984, 62(5), 290 - 4
Inhibition of cyclic AMP-mediated intestinal hypersecretion by pituitary extracts from rats pretreated with cholera toxin; Lonnroth I et al.; Peroral pretreatment with cholera toxin (CT) in rats induced protection against intestinal hypersecretion by CT or prostaglandin E1 (PGE1) . Pituitary glands from these CT-pretreated rats were homogenized and injected intravenously or intraluminally into untreated rats . These recipients became resistant to CT- as well as to PGE1-induced hypersecretion; recipients given pituitary extracts from control animals responded normally . Extracts of intestinal mucosa from CT-pretreated, but not from control rats, also inhibited secretion by CT . Ultrafiltration experiments with the pituitary or intestinal extracts indicated that the antisecretory factors had a molecular weight between 10,000 and 50,000.

J Neurosci Res, 1984, 12(2-3), 325 - 34
Biological activity of preformed cholera toxin-ganglioside GM1 complex; Fiani ML et al.; Synthetic and natural amphiphiles, octyl glucoside, Nonidet P40, sodium dodecyl sulfate (SDS), gangliosides GM1 and GD1a, interact with cholera toxin (CLT) and with its active region (promoter A) . The formation of CLT-amphiphile complex leads to inhibition of ADP-ribosyltransferase activity, a characteristic of promoter A elicited after thiol-reagents treatment . In all cases the interaction produces the maximum inhibitory effect above the critical micellar concentration of amphiphiles, although monomers of SDS show inhibition activity as well . The gangliosides appear to be capable of altering bilayer organization of membrane, similar to synthetic detergents . When CLT-ganglioside complexes were incubated with cell culture medium containing 10% fetal calf serum (FCS) and ADP-ribosyltransferase activity was completely restored both in cholera toxin and in promoter A . Some protein of FCS, which is avid of gangliosides, seems to be responsible for reversibility of inhibition . The results indicate that the active site of promoter A may be located in a hydrophobic pocket of the toxin structure . Furthermore, CLT was bound to reconstituted Sendai virus envelopes (RSVEs), containing a small amount of GM1 . The RSVEs are made of membranous vesicles, capable of binding and fusing with host cell membrane . The incubation for 1 1hr of RSVE bearing CLT with Friend's erythroleukemic cells produced the stimulation of adenylate cyclase . This stimulation appears to be due to the translocation of the active subunit of CLT in the inner half of plasma membrane.

Lancet, 1983 Dec 24-31, 2(8365-66), 1439 - 42
Reduction of fluid-loss in cholera by nicotinic acid: a randomised controlled trial; Rabbani GH et al.; A randomised controlled clinical trial was conducted to investigate the ability of nicotinic acid to reduce intestinal secretion in patients with severe cholera . Of the 62 adults investigated, 29 received either 1 or 2 g of nicotinic acid given orally in divided doses and 33 served as controls . Patients who received the 2 g dose had less fluid loss than did their controls during the first (p less than 0.01) and second (p less than 0.05) 8 h post-treatment periods . During the third and fourth 8 h periods, the rates were lower in the treatment groups, but not significantly so . The drug-specific stool reduction was 31%-47% during the first 16 h . Patients receiving 1 g consistently had lower rates of purging than had their controls during each 8 h observation period, but the differences were not significant . The effect of the 2 g dose was significantly better than that with the 1 g dose . The peak inhibition occurred 8-16 h after start of therapy . The drug was well tolerated, the only side-effect being transient flushing of the body in 1 patient.

Biochemistry, 1983 Dec 20, 22(26), 6291 - 6
Effects of guanine nucleotides on cholera toxin catalyzed ADP-ribosylation in rat adipocyte plasma membranes; Graves CB et al.; ADP-ribosylation of rat adipocyte plasma membrane proteins was investigated following incubation of membranes with {alpha-32P}NAD and cholera toxin in the presence and absence of various guanine nucleotides . In membranes incubated without guanine nucleotides, cholera toxin induced incorporation of 32P into three discrete proteins of 48, 45, and 41 kDa . In membranes containing 100 microM GTP or GDP, toxin-catalyzed incorporation of 32P into the 41-kDa protein was inhibited . GMP and Gpp(NH)p (100 microM) allowed moderate incorporation of 32P into the 41-kDa protein . Toxin-catalyzed labeling of all proteins was rapid, reaching maximal levels between 5 and 10 min . Toxin-catalyzed ADP-ribosylation of the 48- and 45-kDa proteins was stimulated by GTP, reaching maximal levels at 10(-5) M GTP . Inhibition of toxin-dependent labeling of the 41-kDa protein required GTP concentrations above 10(-7) M with complete inhibition occurring between 10(-5) and 10(-4) M GTP . Cholera toxin catalyzed ADP-ribosylation was increased up to 2-fold in membranes supplemented with adipocyte cytosol . These results indicate that cholera toxin catalyzes ADP-ribosylation of three distinct adipocyte plasma membrane proteins, each of which is regulated by the amount and type of added guanine nucleotides.

Proc Natl Acad Sci U S A, 1983 Dec, 80(24), 7611 - 5
Antibodies against synthetic peptides of the B subunit of cholera toxin: crossreaction and neutralization of the toxin; Jacob CO et al.; Six peptides corresponding to various segments of the B subunit of cholera toxin have been synthesized and covalently linked to tetanus toxoid . Of the antibodies raised in rabbits against these conjugates, four crossreacted to different extents with the intact B subunit and whole native cholera toxin . Antisera to the peptide of sequence 75-85 were not crossreactive, whereas elongation by six amino acid residues resulted in a peptide (69-85) leading to antibodies crossreactive with the cholera toxin . Of most interest was peptide CTP3 (50-64), which was the only one that reacted with antisera to cholera toxin and which led to antibodies reacting with the cholera toxin to a similar level as its homologous peptide-antipeptide reaction . Indeed, antisera to CTP3 neutralized significantly the biological activity of cholera toxin, as followed by skin vascular permeability and by fluid accumulation in ligated small intestinal loops of rabbits.

Exp Cell Res, 1983 Dec, 149(2), 577 - 81
Lectin-resistant B16 melanoma cells exhibit an altered response to MSH and cholera toxin; Sheppard JR et al.; Mouse B16 melanoma cells respond to melanocyte-stimulating hormone (MSH) or cholera toxin (CT) with an accumulation of cAMP . The kinetics and dose-response of MSH were examined in the B16 parent line and two cell clones derived from it that exhibited wheat germ agglutinin (WGA) resistance {1} . These WGA lectin-resistant cells, designated W4 and W5 showed a greater response to MSH and CT than the parent B16 cells . Exposure of the W4 and W5 cells to lotus lectin or ricin respectively, led to the previously described {2} selection of cell clones that were resistant to lotus lectin (W4L) and ricin (W5R) . The W4L and W5R cells which were shown {2} to be as sensitive as the B16 parent to WGA (i.e., were phenotypically reverted to WGA sensitivity), were also found to respond to MSH in a manner similar to the B16 parent . Since lectin sensitivity has been directly correlated in these cell clones with the membrane's oligosaccharides and glycopeptide pattern, these data suggest that the cellular binding and/or biological response to hormones is influenced by the carbohydrate composition of the plasma membrane.

Infect Immun, 1983 Dec, 42(3), 914 - 23
Characterization of monoclonal antibodies that react with unique and cross-reacting determinants of cholera enterotoxin and its subunits; Holmes RK et al.; Seventeen selected hybridoma cell lines that produced monoclonal antibodies against cholera enterotoxin (CT) were isolated and characterized . All of the monoclonal antibodies contained the kappa light chain; 14 were of the immunoglobulin G1 (IgG1) isotype and 3 were IgG2a . The 17 monoclonal antibodies were divided into a minimum of seven different specificity groups based on their abilities to bind to the following purified test antigens in solid-phase radioimmunoassays: CT, the A and B polypeptides of CT (CT-A and CT-B, respectively), and the heat-labile enterotoxins designated LTh and LTp from Escherichia coli . The binding of these antibodies to the following subunits and fragments of CT was also determined in Western blots: pentameric CT-B, monomeric CT-B, intact CT-A, and the A1 fragment of CT-A . Each of the monoclonal antibodies was tested for neutralization of CT and for precipitation with CT in immunodiffusion tests . Antigenic determinants were identified on CT that were not present either on CT-A or CT-B . One class was unique for CT and another was shared with LTh and LTp . Antibodies directed against these holotoxin-specific determinants had no neutralizing activity . Most of the monoclonal antibodies that reacted strongly with CT-A or CT-B also reacted strongly with CT holotoxin; however, one class of antibody reacted strongly with CT-A but weakly with CT . Among the monoclonal antibodies against CT-A or CT-B, some were specific for CT and others cross-reacted with LTh and LTp or with LTh only . The most potent neutralizing antibodies were against CT-B, and all of our monoclonal antibodies against CT-B had some neutralizing activity . In contrast, only some of the monoclonal antibodies against CT-A had neutralizing activity, and their specific activities were low . We found no direct correlation between the ability of monoclonal antibodies to neutralize CT and to cross-react with LTh or LTp . None of the epitopes recognized by our monoclonal anti-CT antibodies was present on CT-A and CT-B.

FEBS Lett, 1983 Nov 28, 164(1), 132 - 4
A conjugate of the A1 peptide of cholera toxin and the lectin of Wisteria floribunda that activates the adenylate cyclase of intact cells; van Heyningen S; The active A1 peptide of cholera toxin was linked by a disulphide bond to the lectin of Wisteria floribunda . The resulting conjugate activated the adenylate cyclase of intact U937 or K562 cells at the same concentrations as native toxin did, but to a greater extent . Activation was inhibited by N-acetyl-D-galactosamine or by antisera to the lectin or peptide . The characteristic lag phase between addition of toxin to cells and activation of cyclase was not found with the conjugate or with free A1 peptide.

Exp Lung Res, 1983 Nov, 5(3), 173 - 82
Cholera toxin stimulates secretion of saturated phosphatidylcholine and increases cellular cyclic AMP in isolated rat alveolar type II cells; Mescher EJ et al.; We compared the time course of saturated phosphatidylcholine secretion and cellular cyclic AMP concentrations in isolated rat alveolar type II cells following stimulation by cholera toxin, terbutaline, and 12-0-tetradecanoyl-13-phorbol acetate . Secretion of saturated phosphatidylcholine was stimulated by cholera toxin at concentrations from 1.2 X 10(-11) M to 5.0 X 10(-7) M . In time course experiments there was no significant stimulation with cholera toxin before 1 hr; all subsequent points between 90 and 180 min were significantly different from controls . Secretion stimulated by 10 microM terbutaline was similar in magnitude to stimulation by 1.2 X 10(-9) M cholera toxin; stimulation following either of these agonists was higher than control secretion and lower than secretion stimulated by 10 nM 12-0-tetradecanoyl-13-phorbol acetate . Terbutaline caused an early rise in cellular cyclic AMP that peaked within 5 min and then returned to basal level by 60 min . In contrast, cholera toxin did not increase cellular cAMP levels until 60 min after addition, but then produced a sustained increase in cyclic AMP levels for up to 3 hr . In conclusion, we have presented evidence that more than one mechanism exists by which secretion can be stimulated in type II cells . It is likely that both terbutaline and cholera toxin act by stimulating cellular cyclic AMP and that 12-0-tetradecanoyl-13-phorbol acetate acts by a mechanism different from terbutaline or cholera toxin.

Infect Immun, 1983 Nov, 42(2), 683 - 91
Immunological relationships between cholera toxin and Escherichia coli heat-labile enterotoxin; Gilligan PH et al.; The antigenic relationships between heat-labile enterotoxin (LT) produced by a human strain of enterotoxigenic Escherichia coli (strain 286C2) and cholera toxin (CT) were examined by using antisera raised against LT and CT and specific antisera prepared against each subunit of both enterotoxins . Double immunodiffusion analysis revealed reactions of partial identity between the A subunits of LT and CT, as well as between the B subunits . Rabbit antisera raised against LT subunit A reacted with only subunit A, whereas rabbits immunized with LT subunit B produced antibodies which reacted with only subunit B . A high degree of CT neutralization was observed with antisera raised against LT . Data from neutralization assays with specific antisera to each enterotoxin showed that LT was more effectively neutralized by homologous anti-LT than CT (3.7-fold); however, anti-CT was only slightly more effective in neutralization of homologous CT compared with LT (1.9-fold) . In contrast, antisera raised against the B subunit of CT (choleragenoid) exhibited significantly higher neutralization activity against CT than LT (5.8-fold); however, the amount of CT neutralized by anticholeragenoid was less (4.1-fold) than anti-CT . These results suggested that anti-CT serum contained neutralizing antibodies reactive with a shared determinant formed by interaction of the A and B subunits, whereas anti-LT and anti-choleragenoid sera did not . Sensitive solid-phase radioimmunoassays were developed to examine the affinity and degree of specificity involved in homologous and heterologous antigen-antibody interactions between LT, CT, their subunits, and specific antibodies . Only unlabeled LT competed with radiolabeled LT in polystyrene tubes coated with anti-LT, and only unlabeled CT competed with radiolabeled CT in tubes coated with anti-CT . However, when radiolabeled CT was incubated in tubes coated with anti-LT, competitive inhibition responses were observed with both unlabeled toxins . When radiolabeled LT was incubated with tubes coated with anti-CT, competitive inhibition responses were observed with both unlabeled toxins . Similar competitive inhibition responses were observed with the A subunits of LT and CT and with the B subunits using antisera specific for the subunits of each enterotoxin . Double immunodiffusion analysis and radioimmunoassay data supported the presence of unique and shared immunodeterminants in each subunit.

Biochem Biophys Res Commun, 1983 Oct 14, 116(1), 341 - 8
Location and amino acid sequence around the ADP-ribosylation site in the cholera toxin active subunit A1; Lai CY et al.; Renatured, S-carboxymethylated subunit A1 of cholera toxin possess the ADP-ribose transferase activity (Lai, et.al., Biochem . Biophys . Res . Commun . 1981, 102, 1021) . In the absence of acceptor self ADP-ribosylation of A1 subunit was observed . Stoicheometric incorporation of ADP-ribose moiety was achieved in 20 min at room temperature in a 0.1 - 0.2M PO4(Na) buffer, pH 6.6 . On incubation of the complex with polyarginine, 75% of the enzyme-bound ADP-ribose moiety was transferred to the acceptor in 25 min . The ADP-ribosylated A1 was stable at low pH, and on cleavage with BrCN, the ADP-ribose moiety was found associated with peptide Cn I, the COOH-terminal fragment of A1 subunit . On further fragmentation with cathepsin D, a dodecapeptide containing ADP-ribose moiety was isolated whose structure was determined as: Asp-Glu-Glu-Leu-His-Arg-Gly-Tyr-Arg*-Asp-Arg-Tyr . The Arg* in the peptide was indicated to be the site of ADP-ribosylation.

J Biol Chem, 1983 Oct 10, 258(19), 11908 - 14
A second guanyl nucleotide-binding site associated with adenylate cyclase . Distinct nucleotides activate adenylate cyclase and permit ADP-ribosylation by cholera toxin; Gill DM et al.; There are two functionally and physically distinct types of guanyl nucleotide site associated with the adenylate cyclase system of pigeon erythrocytes . One is on the well known regulatory protein, N, that mediates the adenylate cyclase response to hormones, guanyl nucleotides and fluoride, and is the substrate for ADP-ribosylation by cholera toxin . We now describe a second site that must be occupied by GTP or an analog of GTP before N can be ADP-ribosylated . We call this second site S . It differs from the site on N in many respects . GTP appears to be rapidly hydrolyzed when it is bound to N but not when bound at S . GTP analogs such as guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) bind stably to both sites but the binding of GTP gamma S to N is more sensitive to EDTA and is more easily prevented by guanosine 5'-O-(2-thiodiphosphate) . The nucleotide binding only to S is promoted by the cytosolic protein required by cholera toxin . Isoproterenol decreases GTP gamma S binding to S while indirectly increasing GTP gamma S binding to N . By adjusting the binding conditions, the nucleotides bound functionally to N and S can be varied independently and then the effect of ADP-ribosylation upon the adenylate cyclase activity can be seen to depend on the type of nucleotide bound to N . This activity rises, falls slightly, or remains at zero, if N is occupied by GTP, GTP gamma S, or guanosine 5'-O-(2-thiodiphosphate, respectively.

Biol Reprod, 1983 Oct, 29(3), 671 - 9
Intrauterine injection of cholera toxin induces estrogen-like uterine growth; Stewart PJ et al.; Uterine growth in response to estrogen involves nuclear interaction of cytoplasmic estrogen receptors . The magnitude of the uterine growth response, however, and other observations have led to the suggestion that an additional mechanism or mechanisms of estrogen action may be at work in the uterus . This was investigated indirectly by examining estrogen-like growth responses of the uterus to the intrauterine (i.u.) injection of cholera toxin and water . Ovariectomized rats received either a subcutaneous injection of estradiol or vehicle and an i.u . injection of cholera toxin, water, or nothing . At 4 h after treatment of uterine wet weight was determined, and at 24 h the uterine protein/DNA ratio, {3H} leucine incorporation into protein, and {3H} thymidine incorporation into DNA were measured . Estradiol caused a 1.5-fold increase in the protein/DNA ratio and cholera toxin caused a 2.0-fold increase . Similarly, estradiol increased uterine wet weight 2.1-fold and cholera toxin increased it 1.9-fold . Estradiol and cholera toxin caused similar increases in the incorporation of {3H}leucine into protein: 3.3-fold and 2.3-fold, respectively . The effects of the treatments on the incorporation of {3H} thymidine into DNA were even greater, with estradiol producing a 10.3-fold increase, cholera toxin a 12.0-fold increase, and water a 3.6-fold increase . Since vascular permeability is known to be increased in the uterus by estrogen and in other systems by cholera toxin, these results support the idea of a relationship between uterine growth and increased vascular permeability.

J Lab Clin Med, 1983 Oct, 102(4), 509 - 21
Effects of amphotericin B and cholera toxin on intestinal transport in the rat . An in vivo model for the effects of dihydroxy bile acids and fatty acids on intestinal transport; Ammon HV et al.; In vivo perfusion experiments were performed in the rat jejunum and colon to test the hypothesis that the changes in intestinal solute transport induced by dihydroxy bile acids and fatty acids are the result of the combined effects of fluid secretion and enhancement of mucosal permeability . The hypothesis predicts that absorption of organic solutes will be reduced in inverse relationship to the absorption rates under control conditions and that absorption of small, nonabsorbable solutes such as mannitol will be enhanced by these agents . Fluid secretion was induced either by administering cholera toxin or by increasing the osmolality of the perfusion solution to 365 mOsm/L . Permeability was enhanced by adding amphotericin B, 50 micrograms/ml, to the perfusion solutions . The isotonic perfusion solutions contained 11.2 mM glucose and 4 mM triethylene, tetraethylene, pentaethylene, and hexaethylene glycol or mannitol as probes of passive permeability . In the jejunum cholera toxin induced fluid and electrolyte secretion and reduced organic solute absorption to a small but significant degree (p less than 0.05) . Amphotericin B alone enhanced absorption of organic solutes, water, and electrolytes (p less than 0.01) . In the presence of fluid secretion induced by an osmotic load, only absorption of triethylene and pentaethylene glycol was reduced . Addition of amphotericin B after exposure to cholera toxin or to the hypertonic solutions resulted in a further significant reduction of absorption of glucose and ethylene glycols (p less than 0.05) . The combination of amphotericin B and cholera toxin resulted in enhanced absorption of mannitol (p less than 0.02) . Similarly, 5 mM deoxycholate enhanced jejunal absorption of mannitol (p less than 0.01) and reduced the absorption of glucose and the low-molecular-weight ethylene glycols (p less than 0.01) . In the colon the administration of amphotericin B after the exposure to cholera toxin resulted in enhanced absorption of glucose (p less than 0.05) in spite of continuing fluid secretion . The combination of fluid secretion and enhancement of mucosal permeability, therefore, reproduced all in vivo effects of bile acids and fatty acids on intestinal transport of organic solutes.

Biosci Rep, 1983 Sep, 3(9), 879 - 86
Presence of a dinucleotide fold in cholera toxin: possible approach to chemoprophylaxis?
Dorai DT.
The ADP-ribosyltransferase activity of the A1 subunit of cholera toxin is specifically inhibited by the dye cibacron blue 3GA . The presence of a 'dinucleotide fold' in the A1 subunit is thus established for the first time . This specific inhibition observed in vitro is successfully exploited in vivo for the inhibition of te diarrheal response brought out by the pure toxin in the rabbit ileal-loop test.

Biochim Biophys Acta, 1983 Aug 23, 759(1-2), 117 - 24
Cyclic AMP in the regulation of exocytosis in the rat parotid gland . Evidence obtained with cholera toxin; Spearman TN et al.; The effects of cholera toxin on rat parotid gland function were determined in order to further characterize the relationship between cyclic AMP and exocytosis in this tissue . Cholera toxin induced the release of alpha-amylase from rat parotid minces in vitro . This release was accompanied by an activation of adenylate cyclase, elevated cyclic AMP levels, an elevated protein kinase activity ratio, and changes in the degree of phosphorylation of three endogenous phosphoproteins . Two of the phosphoproteins became more phosphorylated upon cholera toxin stimulation while the phosphorylation of the other decreased . The effects of cholera toxin on endogenous phosphoprotein labelling appeared to mimic those of the beta-adrenergic agonist isoproterenol but were of a smaller magnitude . These results are consistent with cyclic AMP functioning as a major mediator of exocytosis in this gland exerting its effects, at least in part, via activation of cyclic AMP dependent protein kinase . The mechanism by which an increased cyclic AMP level results in the decreased phosphorylation of an endogenous phosphoprotein is not known.

FEBS Lett, 1983 Aug 8, 159(1-2), 75 - 8
GDP does not support activation of adenylate cyclase nor ADP-ribosylation of a guanine nucleotide binding protein by cholera toxin; Shimada N et al.; The actions of cholera toxin (i.e., activation of adenylate cyclase and ADP-ribosylation of a guanine nucleotide binding protein in purified membranes from rat liver) were GTP dependent . Neither of these actions of cholera toxin was reproduced with GDP . Simultaneous addition of ATP and MgCl2 along with GDP allowed cholera toxin to exert these actions . The role of GDP in adenylate cyclase regulation was discussed.

J Cell Biol, 1983 Aug, 97(2), 447 - 54
Capping of cholera toxin-ganglioside GM1 complexes on mouse lymphocytes is accompanied by co-capping of alpha-actinin; Kellie S et al.; We used cholera toxin, which binds exclusively and with a high affinity to the ganglioside GM1, as a probe to investigate the distribution of this glycolipid on the surface of mouse lymphocytes . When lymphocytes are incubated with cholera toxin (or its B subunit) and then sequentially with horse anti-toxin and FITC-swine anti-horse Ig at 37 degrees C, the cholera toxin-ganglioside GM1 complex is redistributed to a cap at one pole of the cell . The capping of cholera toxin-GM1 complexes is slower than the capping of surface-Ig complexes, requires two antibodies, and is inhibited at high toxin concentrations . Cholera toxin-GM1, like surface-Ig capping, is an energy-dependent process and is inhibited by sodium azide, low temperatures, or cytochalasin B, but is unaffected by demecolcine . An affinity-purified antibody against alpha-actinin was used to examine the distribution of this cytoskeletal component during the capping process . 88% of the cells that had a surface Ig cap displayed a co-cap of alpha-actinin, and 57% of the cells that had a cholera toxin-GM1 cap displayed a co-cap of alpha-actinin . Time course studies revealed similar kinetics of external ligand cap formation and the formation of alpha-actinin co-caps . We conclude that capping of a cell-surface glycolipid is associated with a reorganization of the underlying cytoskeleton . The implications of such an association are discussed in the context of current models of the mechanism of capping.

Cancer Lett, 1983 Aug, 20(1), 61 - 8
Influence of cholera toxin on the growth and development of N-methyl-N-nitrosourea-induced rat mammary carcinomas; Welsch CW et al.; Daily injections of cholera toxin (2.0 micrograms/rat/day) for 4 weeks to female Sprague-Dawley rats did not significantly affect the growth of palpable N-methyl-N-nitrosourea (MNU)-induced rat mammary carcinomas . Percent increase in tumor volume was + 78.8% for control animals and +72.8% for cholera toxin treated animals . Daily treatment for 16 weeks of female Sprague-Dawley rats with cholera toxin (1.0 micrograms/rat/day), commencing 3 days after MNU treatment, resulted in a significant (P less than 0.05) increase in mean mammary carcinoma weight per rat at the termination of the study; mammary carcinoma incidence was not significantly affected by cholera toxin treatment . Retinyl acetate feeding (1.0 mM/kg diet) for 16 weeks significantly (P less than 0.05) reduced mammary carcinoma incidence and weight of mammary carcinoma per rat at the termination of study; feeding of retinyl acetate to cholera toxin treated rats blocked the stimulatory effect of cholera toxin on mammary carcinoma development . Thus, the reported striking inhibitory effect of cholera toxin on the growth of dimethylbenzanthracene (DMBA)-induced rat mammary carcinomas was not duplicated in our study, using the MNU-induced rat mammary carcinoma; indeed the toxin appeared to enhance the early developmental stage of this neoplastic process.

Acta Pathol Microbiol Immunol Scand {B}, 1983 Aug, 91(4), 215 - 20
Influence of bile acids on cholera toxin-induced secretion in mouse jejunum; Lange S et al.; The influence of bile acids on intestinal secretion induced by cholera toxin (CT) was studied in mice . The secretion was examined in ligated loops after the bile flow had been stopped by ligation of the common bile duct (CD) . Bile depletion was found to inhibit both the secretion induced by CT, the degradation of CT, and the binding of CT to epithelial cells--all of which could be restored to normal by the application, before CT challenge, of bile acids in the loops of CD-ligated mice . Out of the nine bile acids tested, cholic acid, deoxycholic acid and chenodeoxycholic acid were the most potent, their ED50 values being 0.2, 0.2, and 0.6 mM respectively.

Biull Eksp Biol Med, 1983 Aug, 96(8), 37 - 9
{Biosynthesis and metabolism of prostaglandins in the small intestine of rats exposed to cholera enterotoxin}; Zolotukhin SV et al.; The effect of cholera enterotoxin on biosynthesis and metabolism of prostaglandins in the rat small intestine was studied . It was shown that in the course of action of cholera enterotoxin maximal synthesis and metabolism of prostaglandins (PG) was observed within the first 30 minutes after enterotoxin administration into the isolated intestinal loop . It was found that cholera enterotoxin induced, on the one hand, the shift in the correlation of different types of prostaglandins synthetized in vitro and, on the other, differentially activated PG synthesis and metabolism after pretreatment with the PG-synthetase inhibitor indomethacin.

Neuroendocrinology, 1983 Aug, 37(2), 161 - 3
Luteinizing hormone secretion is enhanced by pertussis toxin, cholera toxin, and forskolin . Evidence for the involvement of the cyclic AMP-generating system; Cronin MJ et al.; Pertussis toxin, cholera toxin and forskolin, all of which can increase adenylate cyclase activity, stimulated luteinizing hormone LH release from cultured rat anterior pituitary cells . Although cellular cyclic AMP and growth hormone were increased rapidly by cholera toxin and forskolin, enhanced LH release occurred significantly later with no change in total radioimmunoassayable LH (i.e., released plus stored) . These data suggest that changes in cyclic AMP levels may regulate the tonic availability of releasable LH in the gonadotroph.

J Invest Dermatol, 1983 Aug, 81(2), 131 - 6
Adenylate cyclase activation by cholera toxin in pig epidermis: an obligatory role of the GTP-regulatory protein; Takeda J et al.; Cholera toxin (CT) stimulates the epidermal adenylate cyclase system in vitro . This stimulation was demonstrated in the skin (slice) floating system and the homogenate (membrane) assay system . With the floating system, the addition of CT to the incubation medium caused a marked accumulation of cAMP intracellularly, which was both dose- and time-dependent . A 1-h lag time was present before activation started . Pretreatment of the skin with CT changed the nature of the stimulatory effect caused by epinephrine and histamine, i.e., the transient accumulation of cAMP (a peak at 5 min and subsequent decrease) was no longer observed but the stimulation became persistent . With the membrane assay system in which the receptor components had been uncoupled, adenylate cyclase activities were markedly stimulated by CT (with guanosine-5'-triphosphate, GTP), guanylyl-beta,gamma-imidodiphosphate (GTP-analog, Gpp{NH}p), or sodium fluoride . The stimulation was both dose- and time-dependent without an initial time lag . Either CT or Gpp{NH}p could fully activate adenylate cyclase, and the simultaneous addition of both did not cause further additive stimulation . These data are consistent with the view that the GTP-regulatory protein plays a key role in the activation of adenylate cyclase, and that CT both activates the catalytic unit and modifies the response to receptor hormones through its action on this protein.

Avian Dis, 1983 Jul-Sep, 27(3), 836 - 8
Chronic cholera-like lesions caused by Moraxella osloensis; Emerson FG et al.; Cholera-like lesions appeared in four house-confined flocks of tom turkeys on one farm from October 30, 1980, to December 2, 1980; Moraxella osloensis was isolated from the tissues . All flocks were treated with 0.04% sulfaquinoxaline in the water for 3 days . The flocks returned to normal and had normal condemnation rates at slaughter . An experiment was conducted in which six hen turkeys were inoculated with a M . osloensis isolate . The same gross lesions were produced as seen in the field cases.

Infect Immun, 1983 Jul, 41(1), 50 - 3
Analysis of antigenic determinants in cholera enterotoxin and heat-labile enterotoxins from human and porcine enterotoxigenic Escherichia coli; Takeda Y et al.; Antigenic determinants of cholera enterotoxin (CT) and heat-labile enterotoxin from a human strain (LTh) and a porcine strain (LTp) were analyzed by Ouchterlony double-gel diffusion test against anti-CT, anti-LTh, and anti-LTp, which were treated by immunoaffinity column chromatography . The results showed the existence of the following antigenic determinants: (i) antigenic determinants unique to CT, LTh, and LTp, respectively; (ii) an antigenic determinant common to CT, LTh, and LTp; (iii) an antigenic determinant common to CT and LTh, but not LTp; and (iv) an antigenic determinant common to LTh and LTp, but not CT . On the basis of these results, an antigenic scheme for CT, LTh, and LTp is proposed.

Nucleic Acids Res, 1983 Jun 25, 11(12), 3855 - 61
Nucleotide sequences within the cholera toxin operon; Gennaro ML et al.; Nucleotide sequences coding for the N- and C-terminus of the A subunit and the N-terminus of the B subunit of cholera toxin were determined . These results show that the genes for the A and B subunits overlap out of phase by one nucleotide and that each subunit is synthesised as a precursor molecule which is subsequently processes after translation . It is proposed that the synthesis of each subunit is regulated at the translational level . Considerable homology with the heat labile toxin genes of enteropathogenic E.coli was noted.

Biochim Biophys Acta, 1983 Jun 16, 752(1), 106 - 17
Modulation of lipoprotein lipase activity in cultured rat mesenchymal heart cells and preadipocytes by dibutyryl cyclic AMP, cholera toxin and 3-isobutyl-1-methylxanthine; Friedman G et al.; We have compared the effects of cellular cyclic AMP modulation on the regulation of lipoprotein lipase in cultures of rat epididymal pad preadipocytes and mesenchymal heart cells . Addition of dibutyryl cyclic AMP (dibutyryl cAMP) or 3-isobutyl-1-methylxanthine (IBMX) to preadipocytes grown in serum-containing culture medium resulted in a progressive decrease in lipoprotein lipase activity released into the culture medium so that at 6-8 h enzyme activity ranged between 20 and 30% of that recovered in the control dishes . Similar short-term (6-8 h) studies of the heart cell cultures showed a variable and much less pronounced depression of lipoprotein lipase activity . Thus, following dibutyryl cAMP and IBMX treatment, lipoprotein lipase activity ranged between 70 and 95% of control values . Incubation for 6 h with cholera toxin was followed by a 4-fold rise in the concentration of cellular cyclic AMP in both types of culture, but while in heart cell cultures enzyme activity was unchanged, lipoprotein lipase activity in preadipocytes decreased to 30% of control value . After 24 h incubation with all three effectors, an increase in lipoprotein lipase activity was seen . In the preadipocytes the increase ranged between 50 and 150% above control value, in the heart cell cultures it was 100-250% . 24-h incubation of heart cell cultures with dibutyryl cAMP resulted in a 6-fold increase of heparin-releasable lipoprotein lipase activity while residual activity was doubled . The rise in surface-bound lipoprotein lipase was evidenced also by an increase in the lipolysis of chylomicron triacylglycerol . In the presence of cycloheximide, the dibutyryl cAMP-induced heparin-releasable and residual lipoprotein lipase activity declined at the same rate as the basal activity . The reason for the difference in response of cultured preadipocytes and heart cells to the effectors during the first 8 h of incubation has not been elucidated, but could be related to a possible absence of hormone-sensitive lipase in the heart cells, and hence in a difference in intracellular metabolism of triacylglycerol . On the other hand, a common mechanism can be postulated for the long-term effect of cyclic AMP on the induction of lipoprotein lipase activity in both types of cultures . It probably involves mRNA and protein synthesis, which culminates in an increase in enzyme activity.

Biochem J, 1983 Jun 15, 212(3), 669 - 78
Human platelets are defective in processing of cholera toxin; Hughes RJ et al.; Cholera toxin is unable to elevate cyclic AMP levels in intact human platelets despite being very efficacious in this respect in other mammalian cells; in the presence of 0.5 mM-isobutylmethylxanthine, we found that 3-6nM-cholera toxin over 3h at 37 degrees C elevated platelet cyclic AMP from 33 +/- 13 to 39 +/- 12pmol/mg of protein (means +/- S.D.; n = 12) . We have investigated the basis for this lack of response . 125I-labelled cholera toxin bound to platelets both saturably and with high affinity (Kd congruent to 60pM; Bmax . congruent to 50fmol/mg of protein) . Incubation of platelets with the putative cholera toxin receptor monosialoganglioside GM1 enhanced 125I-labelled cholera toxin binding at least 40-fold but facilitated only a minimal (less than or equal to 3-fold) elevation of platelet cyclic AMP levels . In contrast, dithiothreitol-activated cholera toxin markedly stimulated adenylate cyclase activity in platelet membranes . Platelet cytosol both enhanced stimulation of adenylate cyclase activity by activated cholera toxin (A1 subunit) and supported stimulation by the A1-A2 subunit of cholera toxin . Neither GTP nor NAD+, both necessary for response to cholera toxin, was lacking in intact platelets . However, we found that platelets were unable to cleave cholera toxin to the active A1 subunit (as assessed by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis) . By contrast, murine S49 lymphoma cells were able to generate the A1 subunit with a time course that closely resembled the kinetics of toxin-mediated cyclic AMP accumulation in these cells . Thus we conclude that human platelets are defective in their ability to process surface-bound cholera toxin . These results indicate that binding of cholera toxin to surface receptors is necessary, but not sufficient, for expression of the toxin effect and the generation of the A1 subunit of the toxin may be rate-limiting for expression of cholera toxin response.

Gut, 1983 Jun, 24(6), 542 - 8
5-Hydroxytryptamine and cholera secretion: a histochemical and physiological study in cats; Nilsson O et al.; The effect of cholera toxin on the content of 5-hydroxytryptamine (5-HT) in the enterochromaffin cells of the cat small intestine was estimated by cytofluorimetry of individual enterochromaffin cells at varying times after exposing the intestinal mucosa to the toxin . The observed changes in 5-HT levels in the enterochromaffin cells were correlated with the simultaneously measured rate of net fluid transport across the intestinal epithelium . Intestinal segments exposed to cholera toxin showed a statistically significant decrease in 5-HT levels of enterochromaffin cells compared with segments exposed to heat-inactivated cholera toxin . A good correlation (r = 0.73) was found between relative 5-HT fluorescence in enterochromaffin cells and net fluid transport across the intestinal epithelium . Thus, a diminished 5-HT content was associated with a decreased rate of fluid absorption or an increased rate of secretion . A hypothesis is presented for explaining the possible role of the enterochromaffin cells in the pathophysiology of cholera secretion.

Endocrinology, 1983 Jun, 112(6), 2095 - 106
Induction of estrogen and progesterone receptors and decidualization in the hamster uterus by cholera toxin; Alleva JJ et al.; Cholera toxin (CT) injected ip on day 1 (day of ovulation) of the 4-day hamster estrous cycle, when circulatory progesterone is high and estrogen low, induced a massive uterine decidual reaction, a progesterone-dependent growth normally triggered by the implanting blastocyst . However, CT injected ip on day 3, when circulatory estrogen is high and progesterone low, did not induce a decidual reaction but, instead, intensified the effects of estrogen (stromal edema and stimulation of the mucosa) . These cycle day effects were reproduced in one uterine horn injected intraluminally with CT, but not in the other horn of the same animal given solvent alone as a control . The intrauterine injection of CT had no effect on the concentration of serum estrogen or progesterone . The decidual reaction resulting from intrauterine injection of CT on day 1 was accompanied by increases in estrogen receptor (femtomoles per mg DNA) in both cytoplasm and nucleus . In long term ovariectomized hamsters, an ip or intrauterine injection of CT induced only histological effects of estrogen (stromal edema and mucosal mitosis) without affecting circulatory estrogen . These estrogenic effects were accompanied by increases in receptors for estrogen and progesterone in both cytoplasm and nucleus . CT injected ip into ovariectomized hamsters primed with estrogen intensified the stromal edema and mucosal mitosis and resulted in progesterone and estrogen receptor levels equal to or greater than those after the administration of CT or estrogen alone . When progesterone was included in the priming (estrogen + progesterone + CT), all receptor levels were decreased, and a massive decidual reaction resulted . Thus, the induction of estrogen receptor by CT may have been the primary event that triggered the decidual reaction . Whether CT-induced estrogen receptor is mediated by cAMP, a known mediator of CT, remains to be determined.

Biochim Biophys Acta, 1983 May 5, 730(2), 321 - 6
The effect of cyclic nucleotides and cholera toxin on in vivo and in vitro phosphorylation of small intestinal brush border membranes; Scalera V et al.; The effect of cyclic nucleotides and cholera toxin on the phosphorylation of the brush border membrane proteins of the rat jejunum was studied . Phosphorylation was analyzed by autoradiography of brush border membrane proteins separated by SDS-polyacrylamide gel electrophoresis . Phosphorylation was performed either in vivo by perfusion of the jejunum with {32P}orthophosphate followed by an analysis of the isolated membranes or in vitro by phosphorylation of isolated brush border membranes by {gamma-32P}ATP in the presence of saponin . The addition of cholera toxin (10 micrograms/ml) or dibutyryl-cAMP (5 mmol/l) to the perfusate was unable to produce significant changes in the phosphoprotein pattern . On the other hand, cAMP (at 5 mumol/l) induced an increase of the phosphorylation of a 86 kDa protein when freshly isolated brush border membranes were phosphorylated by {gamma-32P}ATP . However, the same effect could also be induced by low concentrations of cGMP (0.1 mumol/l) . It is concluded that brush border membranes from rat jejunum do not contain cAMP-dependent protein kinase activity and that cAMP-dependent protein phosphorylation of this membrane does probably not represent the final event of cholera toxin-induced secretion.

J Clin Lab Immunol, 1983 May, 11(1), 37 - 42
Augmented delayed footpad reaction in thymus cell-depleted mice induced by cholera toxoid; Tsuru S et al.; One hundred microgram of cholera toxoid was injected intravenously into DDD and AKR mice and its effects on lymphoid tissues and immune responses against sheep erythrocytes (SRBC) were examined at various times after the injection . (1) A remarkable reduction of thymus cells was revealed from day 1 to 7 and from day 1 to 4 in DDD and AKR mice, respectively . (2) Cholera toxoid exhibited only slight effects on the numbers of spleen cells and peripheral blood leukocytes in both strains . (3) Delayed footpad reactions to SRBC were augmented by a pretreatment with cholera toxoid 4 or 7 days before immunization in both strains . The delayed reactions were not suppressed in the presence of a prominent antibody production and were accompanied by positive macrophage migration inhibition . (4) Antibody production against SRBC, especially of IgG class, was facilitated, when cholera toxoid was given 7 days before the immunization through the footpad in DDD mice . On the other hand, antibody production was suppressed irrespective of immunizing routes and mouse strains, when cholera toxoid was given 1 day before immunization.

Infect Immun, 1983 May, 40(2), 570 - 6
Monoclonal antibodies to cholera toxin with special reference to cross-reactions with Escherichia coli heat-labile enterotoxin; Lindholm L et al.; Seventy monoclonal antibodies to cholera toxin were prepared and characterized . All were of immunoglobulin G (IgG) isotypes (39 IgG1, 29 IgG2, and 2 IgG3) . A total of 61 clones produced antibody directed against the B subunit, and 9 clones produced antibodies with specificity for the cholera toxin A subunit . Among both the anti-B and anti-A antibodies, there were representatives which showed full cross-reactivity with the heat-labile enterotoxin of Escherichia coli (14 clones), others which gave partial cross-reactions (12), and still others (44) which did not cross-react . Although 24 of 25 tested anti-B monoclonal antibodies could neutralize cholera toxin, none of the 9 anti-A clones had any detectable neutralizing ability . Among the anti-B antibodies, those which cross-reacted completely with E . coli heat-labile enterotoxin all had strong cholera toxin-neutralizing capacity, whereas those with lesser or no degree of cross-reactivity varied more in their neutralizing potency . The isolation of monoclonal antibodies that distinguish between enterotoxins of different bacterial origin suggests the possibility of developing immunodiagnostic methods allowing species-specific enterotoxin detection in stools of patients with diarrheal disease.

J Cell Physiol, 1983 May, 115(2), 151 - 8
Insulin-cholera toxin binding unit conjugate: a hybrid molecule with insulin biological activity and cholera toxin binding specificity; Roth RA et al.; The polypeptide hormone insulin and the binding unit of cholera toxin (CTB) were coupled via a disulfide bond . This hybrid molecule had 1/30 the ability of native insulin to bind to the insulin receptor and 1/30 the biological activity of native insulin in H35 rat hepatoma cells and rat adipocytes . Thus, in these two cell types that are very sensitive to insulin, the biological activity of the hybrid molecule was as predicted on the basis of the ability of the molecule to interact with the insulin receptor . In contrast, in HTC rat hepatoma cells and rat thymocytes, two poorly responsive cell types, the insulin-CTB conjugate had 1/3 the biological activity of native insulin, a value 10 times greater than its insulin receptor binding potency . This increased activity of the conjugate did not appear to be due to cholera toxin in the preparation, since a control of uncoupled CTB had no biological activity . Furthermore, native cholera toxin increased intracellular levels of cAMP by 20-fold, whereas the conjugate had no effect on cAMP levels . The CTB moiety did, however, contribute to the biological activity of the conjugate, since the activity of the hybrid molecule, like cholera toxin, was inhibited by gangliosides, whereas the activity of native insulin was not . Finally, the binding to thymocytes of insulin-CTB conjugate, but not insulin, was inhibited by gangliosides . Thus, a hybrid hormone molecule has been constructed which has insulin-like biological activity with the receptor specificity of cholera toxin in poorly responsive cells.

Arkh Anat Gistol Embriol, 1983 Apr, 84(4), 53 - 61
{Ultrastructural changes in rat colon epitheliocytes after treatment with cholera toxin}; Shakhlamov VA et al.; Ultrastructural changes in adenylate cyclase activity in the columnar and goblet cells of the rat colon were studied 10, 30 min and 1, 4, 6, 12 and 24 h following injection of cholera toxin (choleragen) into the small intestine of 60 animals . The toxin rather quickly (within 30 min) reached the colon and stimulated membrane-bound adenylate cyclase activity in the colonic columnar and goblet cells . Ultrastructural alterations in the columnar cells, which were comparable to those previously described in choleragen-treated small intestine but were considerably less pronounced, could be detected 1-12 h following the toxin injection . In the colon, stimulation of mucus production by the goblet cells was much more pronounced than in the small intestine . The data obtained suggest that colon is succeptible to the action of choleragen but they cannot be interpreted as an indication of its disturbed re-absorptive capacity.

J Clin Lab Immunol, 1983 Apr, 10(4), 209 - 14
Effects of cholera toxin on the lymphoid system . III . In vivo generation of cytotoxic lymphocytes; Tsuru S et al.; Cytotoxic T lymphocytes (CTL) were generated in the spleen by subcutaneous inoculation of EL-4 leukaemia cells (H-2b) into C3H/He mice (H-2k) . (1) Cytotoxicity of spleen cells was profoundly suppressed by an intravenous injection of 1 microgram cholera toxin on the day of tumor inoculation . Tumors continued to grow progressively . (2) Differentiation of memory cells to mature CTL was suppressed by cholera toxin injection on the day of booster inoculation of EL-4 cells . The tumors grew progressively in immune mice given cholera toxin on the day of the booster . (3) In vitro treatment of CTL with cholera toxin suppressed the expression of their cytotoxic activity to a considerable extent . However, tumors regressed very rapidly after rechallenge to immune mice carrying CTL, even when cholera toxin was injected on the day of rechallenge . Cholera toxin suppressed not only the functional expression of CTL but also the induction phase of CTL generation.

Cancer Res, 1983 Apr, 43(4), 1473 - 6
Arrest of hormone-dependent mammary cancer growth in vivo and in vitro by cholera toxin; Cho-Chung YS et al.; Growth of 7,12-dimethylbenz(alpha)anthracene-induced mammary carcinoma in rat was arrested by daily s.c . injections of cholera toxin . At a dose of 2 micrograms/200-g rat, tumors regressed to 50% of their initial size within 2 weeks, and 85% of tumors regressed completely within 4 to 5 weeks . The same response to cholera toxin was observed with another hormone-dependent mammary tumor, MTW9, but not with the hormone-independent tumors, DMBA No . 1 and MT 13762 . The latter result was consistent with the lack of response of these hormone-independent tumors to hormone removal (ovariectomy) or N6,O2'-dibutyryl cyclic adenosine 3':5'-monophosphate treatment . The growth-inhibitory effect of cholera toxin was dose dependent, and upon cessation of treatment tumors resumed growth; after complete regression, however, tumors did not reappear until 6 months after termination of the treatment . An amount of cholera toxin as high as 10 micrograms/day/200-g rat s.c . injected over a 6-week period showed no systemic toxicity in the animals . The growth of human breast cancer cells (MCF-7) in culture was also inhibited by a daily supplement of cholera toxin . At a concentration of 100 ng/ml, the cell replication ceased completely within 2 days . The growth inhibitions, both in vivo and in vitro, were accompanied by marked increases in the cellular cyclic adenosine 3':5'-monophosphate content and type II cyclic adenosine 3':5'-monophosphate-dependent protein kinase activity as well as a decrease of estrogen binding activity . Thus, extinction of mammary cancer can be achieved by cholera toxin, an agent that stimulates the intracellular cyclic adenosine 3':5'-monophosphate system.

Proc Natl Acad Sci U S A, 1983 Mar, 80(5), 1275 - 9
Mechanism of action of glycopeptide hormones and cholera toxin: what is the role of ADP-ribosylation?
Rebois RV, Beckner SK, Brady RO, Fishman PH.
The cultured murine Leydig tumor cell line MLTC-1 and the normal rat thyroid strain FRTL have adenylate cyclase activities that are stimulated by human chorionic gonadotropin (hCG) and thyrotropin, respectively . Both cell types also respond to choleragen . Activation of adenylate cyclase in membranes by choleragen required NAD whereas stimulation of the enzyme by hormones did not . With {alpha-32P}NAD as a donor, ADP-ribosylation of membrane proteins was determined under the same conditions used to assay adenylate cyclase activity . Under these conditions, choleragen, but not the hormones, caused the ADP-ribosylation of subunits of the regulatory component (G/F) of adenylate cyclase in both FRTL and MLTC-1 membranes . In the absence of any effectors, several membrane proteins became labeled but the hormones did not cause the specific labeling of these or any other membrane proteins . Pretreatment of intact MLTC-1 cells with hCG did not block the ability of choleragen to ADP-ribosylate G/F in isolated membranes; labeling was actually enhanced in a manner related to the length of exposure to hCG . In contrast, pretreatment of the cells with choleragen inhibited ADP-ribosylation of G/F by the toxin in isolated membranes . Extracts of membranes from untreated, hCG-treated, and choleragen-treated MLTC-1 cells were used to reconstitute adenylate cyclase activity in membranes from the cyc- variant of S49 lymphoma cells which lacks a functional G/F . Toxin but not hormone treatment caused an increase in the basal activity of adenylate cyclase in the reconstituted system . Our results indicate that ADP-ribosylation of the regulatory component of adenylate cyclase is required for choleragen action but not for hormone action.

Gastroenterology, 1983 Mar, 84(3), 516 - 23
Effect of dopamine and bromocriptine on rat ileal and colonic transport . Stimulation of absorption and reversal of cholera toxin-induced secretion; Donowitz M et al.; Water and electrolyte transport were determined in rat ileum and colon using the single-pass perfusion technique . Intraperitoneal dopamine caused prompt stimulation of both ileal and colonic water absorption . The dopamine effect was mediated by both specific dopamine and alpha 2-adrenergic receptors . Haloperidol, a specific dopamine antagonist, and yohimbine, an alpha 2-adrenergic antagonist, inhibited the effect of dopamine in ileal absorption; both antagonists alone had no effect on basal water transport . Bromocriptine (intravenous and intraluminal) stimulated ileal and colonic water absorption, which was inhibited by haloperidol and yohimbine, and reversed cholera toxin-induced ileal secretion . Magnitude and time-courses of the increased water absorption in ileal loops, inoculated with saline, were the same as in loops, inoculated with saline, suggesting that bromocriptine acted to reverse cholera toxin-induced secretion by stimulating absorption . Bromocriptine had no effect on the cyclic adenosine monophosphate increase caused by cholera toxin . We conclude (a) dopamine stimulates water absorption in vivo in rat ileum and colon; (b) this dopamine effect is via specific dopamine and alpha 2-receptors; (c) bromocriptine stimulates water absorption in ileum and colon and also acts by dopamine and alpha 2-receptors; and (d) bromocriptine reverses cholera toxin-induced secretion.

Life Sci, 1983 Feb 21, 32(8), 839 - 46
Separation of rabbit ileum mucus secretion from electrolyte and water secretion by cholera enterotoxin, verapamil and A23187; George MH et al.; Net water, Na+, Cl- and HCO3- fluxes were measured in in vivo rabbit ileal loops, while mucus secretion was assessed by measuring the glycoprotein or total sialic acid secreted into the lumen, or by measuring the luminal fluid viscosity . Inoculating loops with cholera enterotoxin (CT) produced a sustained secretion of electrolytes and water, but a more transient secretion of mucus . A dose of verapamil was found which, when included in the luminal fluid, inhibited or delayed the CT-induced mucus secretion while not affecting the ongoing electrolyte and water secretion . Exposure of the ileal mucosa to the ionophore, A23187, in the presence of 2mM Ca++ resulted in a brief secretion of mucus, with no change in basal water absorption . Verapamil inhibited this A23187-induced mucus secretion . The ionophore was not effective in the absence of luminal Ca++ . Thus rabbit ileum mucus secretion can be separated from electrolyte and water secretion by agents that affect Ca++ movement.

Acta Physiol Scand, 1983 Feb, 117(2), 195 - 202
The involvement of intramural nerves in cholera toxin induced intestinal secretion; Cassuto J et al.; In previous reports we have suggested that nervous reflexes are involved in the pathophysiology of cholera secretion and that these nervous reflexes involve a cholinergic synapse and a neuron with vasoactive intestinal polypeptide (VIP) as neurotransmitter . These proposals were further analyzed in this study . Tetrodotoxin (TTX) and lidocaine applied on the serosal surface inhibited cholera secretion in segments of rat small intestine . Fluid absorption in control rats was not significantly changed . Hexamethonium given i.v . decreased cholera secretion in the cat . No additional inhibition of cholera secretion was observed after giving TTX close i.a . Furthermore, the intestinal secretion evoked by VIP was not influenced by hexamethonium given i.v . or TTX given close i.a . The present observations support the hypothesis of a role for nervous reflexes in cholera secretion . The results suggest that at least a major part of the proposed nervous reflex(es) in cholera have a cholinergic synapse . Furthermore, the VIP-ergic neuron is situated "distal" to the cholinergic neuron in the reflex(es) closer to the effector cells.

Br Med J (Clin Res Ed), 1983 Jan 15, 286(6360), 184 - 6
Epidemiology of cholera in travellers, and conclusions for vaccination recommendations; Morger H et al.; All cases of cholera imported to Europe and North America between 1975 and 1981 were reviewed to assess the danger of cholera for visitors to endemic areas . Data were obtained from the health authorities of the respective countries . From a total of 129 cases notified to the World Health Organisation detailed reports were obtained on 117 patients . Of these, 66 (56%) were immigrants, refugees, from endemic areas, or foreign workers returning from leave in their native countries . Only 51 (44%) were citizens of countries in Europe or North America . The incidence per journey for foreign travellers visiting Africa or Asia was about 1 in 500 000 . Stay in hospital was always short, and fewer than 2% of patients died . In view of the minimal risk and lack of reliability of cholera vaccination, such protection is not indicated for ordinary tourists visiting developing countries.

Int Arch Allergy Appl Immunol, 1983, 72(2), 119 - 27
IgA isotype restriction in the mucosal but not in the extramucosal immune response after oral immunizations with cholera toxin or cholera B subunit; Lycke N et al.; Intestinal mucosal as well as extramucosal antibody responses were studied in mice after peroral immunizations with cholera toxin or cholera B subunit . The immunizations with cholera toxin gave rise to a marked response with antitoxin-secreting cells (PFC) in Peyer's patches (PP), mesenteric lymph nodes (MLN) and spleen showing isotype distribution of IgG greater than IgA greater than IgM and with PFC kinetics in MLN and spleen that suggested migration of cells from PP after peroral administration rather than cells stimulated in situ by adsorbed antigen . Highest numbers of PFC were obtained after 2 immunizations, and further administrations resulted in a decrease in the PFC response in MLN and spleen, while the PP responsiveness was relatively unchanged, and interestingly, protective immunity and IgA-dominated antitoxin titers in intestinal washings increased markedly by the additional boosters . Animals immunized with cholera B subunit, which lacks the adenylate cyclase-stimulating capacity of cholera toxin, showed similar PFC responses in extramucosal organs as those receiving cholera toxin but were poorly protected and had correspondingly lower IgA antitoxin titers in intestinal washings . These results suggest that the mucosal IgA antitoxin predominance is mainly due to regulatory mechanisms operating on the end-stage differentiation of the committed B cells in lamina propria and that this differentiation, as judged from the different results with cholera toxin and its B subunit, might be influenced by cyclic AMP.

Mol Cell Biol, 1983 Jan, 3(1), 91 - 101
Endocytosis of exogenous GM1 ganglioside and cholera toxin by neuroblastoma cells; Gonatas NK et al.; Cholera toxin (CT) covalently linked to horseradish peroxidase (HRP) is a specific cytochemical marker for its receptor, the monosialoganglioside GM1 . The binding and endocytosis of exogenous {3H}GM1 by cultured murine neuroblastoma cells (line 2A {CCl-131} ), which contain predominantly GM3, was examined by quantitative electron microscope autoradiography . The relationship between exogenous receptor, {3H}GM1, and CT HRP was studied in double labeling experiments consisting of autoradiographic demonstration of {3H}GM1 and cytochemical visualization of HRP . Exogenous {3H}GM1 was not degraded after its endocytosis by cells for 2 h at 37 degrees C . Quantitative studies showed similar grain density distributions in cells treated with {3H}GM1 alone and in cells treated with {3H}GM1 followed by CT-HRP . Qualitative studies conducted in double labeling experiments showed autoradiographic grains over the peroxidase-stained plasma membrane, lysosomes, and vesicles at the trans aspect of the Golgi apparatus . The findings indicate that exogenous glycolipid is associated with the plasmid membrane of deficient cells and undergoes endocytosis . The quantitative ultra-structural autoradiographic studies are consistent with the hypothesis that the spontaneous endocytosis of exogenous {3H}GM1 controls the subsequent uptake of CT-HRP.

Natl Inst Anim Health Q (Tokyo), 1983 Fall, 23(3), 101 - 2
Replication of hog cholera virus in porcine alveolar macrophage cultures; Nakamura S et al.; Eight hog cholera viral strains were tested for virulence for pigs and ability to replicate in porcine alveolar macrophage cultures . Of them, five were virulent and replicated well in the macrophage culture . The other three were avirulent and grew less remarkably in this culture than them . It was suggested that the ability of the hog cholera viral strains to replicate in porcine macrophages might be correlated with their virulence.

Digestion, 1983, 27(3), 174 - 84
Separation of cholera enterotoxin-induced mucus secretion from electrolyte secretion in rabbit ileum by acetazolamide, colchicine, cycloheximide, cytochalasin B and indomethacin; Njoku OO et al.; In vivo rabbit ileal loops were prepared and inoculated with purified cholera enterotoxin (CT) . After a lag period of about 1 h there was persistent stimulation of water and electrolyte secretion and a transient stimulation of mucus secretion into the luminal fluid . Repeated intraluminal inoculation of prostaglandin E1 (PGE1) caused a pattern of water, electrolyte and mucus secretion which was qualitatively the same as that following CT, except that no lag period was observed . Doses of the protein synthesis inhibitor, cycloheximide, the microtubule disrupter, colchicine, and the microfilament disrupter, cytochalasin B, were found that inhibited CT-induced mucus secretion but not water and electrolyte secretion . The carbonic anhydrase inhibitor, acetazolamide, inhibited CT-induced water and electrolyte secretion without inhibiting the mucus secreted over a 5-hour test period . Thus a variety of agents can be used to demonstrate a separation of intestinal water and electrolyte secretion from mucus secretion . The prostaglandin synthesis inhibitor, indomethacin, also inhibited CT-induced water, electrolyte and mucus secretion, but no dose of this agent was found that completely separated the water and electrolyte from the mucus secretion.

Arch Oral Biol, 1983, 28(8), 765 - 71
The effect of cholera toxin and epidermal growth factor on the in-vitro growth of human oral epithelial cells; Muller-Glauser W et al.; Human epithelial cells isolated from adult gingival and infant palatal biopsies were cultured using 3T3 feeder cells . The colony-forming efficiency was about 0.8 per cent with cholera toxin and epidermal growth factor (EGF) . The cell yield of cultures from infant palates depended on the concentration of cholera toxin and the presence of EGF in the culture medium; the culture lifetime and the number of cell generations were higher for oral epithelial cells originating from infants than from adults; the mean thickness of well-developed areas was 15 micron in control cultures and slightly smaller with cholera toxin and EGF . It is concluded that cultivation of epithelial cells from the human oral mucosa is easier with culture media containing cholera toxin and EGF . The same is true for cells originating from infants rather than from adults.

C R Seances Acad Sci III, 1983, 297(12), 557 - 62
{Biological properties of a synthetic sequence (10-24) of the gamma chain from sub-unit A of cholera toxin}; Dodin A et al.; The biological activity of a synthetic fragment of choleric toxin . gamma Chain A subunit, was studied in vivo . This pentadecapeptide (10-24) was synthesized by solid phase method and finally purified by HPLC . Associated with the B subunit, the peptide inhibits the tissue water loss induced by commercial choleric toxin.

Prog Food Nutr Sci, 1983, 7(3-4), 143 - 6
Mode of action of the cholera-coli family of enterotoxins; Gill DM; Described here are the biochemical changes whereby cholera toxin and similar toxins cause adenylate cyclase activity to rise.

Dev Biol Stand, 1983, 53, 93 - 5
Antigenic and structural variations in the cholera/coli family of enterotoxins; Finkelstein RA; Recent observations establish the existence of a family of ADP-ribosylating, adenylate cyclase-activating, heat-labile enterotoxins which are structurally, functionally, and immunologically related to the cholera enterotoxin . Despite their overall similarity, it is clear that there are significant structural and immunological differences within the group . These conclusions are supported by extensive experiments comparing the precipitin activity and neutralizing effects of various specific antisera, before and after purification by immunoaffinity chromatography and solid phase immunoaffinity adsorption of common and cross-reactive antibody species, on the various pure enterotoxins . It will be of great importance to determine the full range of the antigenic and structural drift among this family of enterotoxins . The results will have significance in efforts to develop effective broad spectrum antitoxic immunity and also in the development of rapid and reliable techniques for the identifications of enterotoxic enteropathogens.

Natl Inst Anim Health Q (Tokyo), 1983 Fall, 23(3), 103 - 4
Properties of hog cholera viruses recently isolated in Japan; Shimizu M et al.; Hog cholera (HC) viruses newly isolated in Japan in 1980 and 1981 were examined for pathogenicity and serological properties by the neutralization test with antisera against bovine viral diarrhea-mucosal disease (BVD . MD) and HC viruses . Five of 23 isolates examined were neutralized poorly by BVD . MD antibody, but well by HC antibody . On the contrary, 15 isolates were neutralized readily and two isolates moderately by BVD . MD antibody . The other one reacted poorly with either HC or BVD . MD antibodies . The isolate neutralized poorly by BVD . MD antibody was more highly pathogenic than those neutralized readily . It was concluded that the antigenic properties and pathogenicity of the HC viruses were not monotype , and that HC viruses varying in antigenicity and pathogenicity were present in the field.

Adv Biochem Psychopharmacol, 1983, 36, 281 - 91
Labeling of a GTP-binding regulatory protein of rat brain adenylate cyclase system by cholera toxin-catalyzed ADP-ribosylation; Enomoto K et al.; Cholera toxin ADP-ribosylated several proteins in synaptic plasma membranes of rat brains such as peptides of 66,000, 48,000, 38,000, 25,000, and 18,000 daltons . A 42,000-dalton protein reported to be the GTP-binding regulatory protein in several tissues was faintly labeled and its content varied from preparation to preparation . Adenylate cyclase activity was concomitantly enhanced by more than twice in proportion to the ADP-ribosylation . Accumulated evidence indicated that a protein of 48,000 daltons (48K protein) was a component which constituted the GTP-binding regulatory protein of adenylate cyclase system in rat brain . Among ADP-ribosylated proteins, the 48K protein was the major substrate for the ADP-ribosylation, and the content of this protein was estimated to be about 15 pmol/mg membrane protein . When synaptic membranes were solubilized and applied on a Ultrogel AcA34 gel filtration column to separate the regulatory protein from other components of adenylate cyclase, the 48K protein--but not other ADP-ribosylated proteins--co-eluted with the regulatory protein activity assayed by the reconstitution with catalytic unit of adenylate cyclase . The 48K protein was also detected in myelin fraction which lack adenylate cyclase activity . When solubilized proteins from myelin were subjected to the gel filtration in the AcA34 column, the regulatory protein activity eluted in the 48K protein fractions where the regulatory protein from synaptic membranes appeared . Myelin contained basic protein-like peptides of 20,000 and 18,000 daltons ADP-ribosylated by cholera toxin . These peptides were solubilized from the membranes with acid . Soluble proteins in brain cytosol were ADP-ribosylated by cholera toxin, which was dependent on the presence of nucleotides.

Int Arch Allergy Appl Immunol, 1983, 71(1), 88 - 92
Regulation of lymphocyte responses in cancer patients . I . Study of cell-surface gangliosides by cholera toxin and their induction of impaired activation; Tsuru S et al.; The gangliosides compose a major portion of the surface membrane structure of the lymphocytes and their expression may relate with functional properties of different lymphocyte subpopulations . In the present study, the binding of the ganglioside GM1 to the human peripheral blood mononuclear cells (PBMC) and their effects on the lymphocyte function were examined . Cholera toxin (CT) was used as an indicator for detection of GM1 on the cell surface . It was shown that the exogenous GM1 binds to normal PBMC and inhibits proliferative responses in vitro by various mitogens (Con A, PHA, PWM) . It was also revealed that more than 18 h is required to induce unresponsiveness of lymphocytes by preincubation with GM1, even though GM1 bound very rapidly (10-30 min) to lymphocytes . Some intracellular event may be needed to induce unresponsive state of the lymphocytes by GM1 binding . Moreover, the number of CT binding lymphocytes and the amount of CT bound to each cell showed to increase in the cancer patients . These results suggest that the increase of GM1 in the serum and the lymphocyte surface may be one of the mechanisms of suppressed lymphocyte responsiveness in the various pathological states especially in the cancer patients.

Circ Res, 1983 Jan, 52(1), 111 - 7
Stimulation of slow action potentials in guinea pig papillary muscle cells by intracellular injection of cAMP, Gpp(NH)p, and cholera toxin; Li T et al.; To test the hypothesis that intracellular cAMP has a regulatory role in cardiac slow channel function, intracellular pressure injections of cAMP and adenylate cyclase activators, Gpp(NH)p and cholera toxin, were carried out . Guinea pig papillary muscles were depolarized to about -45 mV by superfusion with 22 mM K+-Tyrode's solution to inactivate the fast Na+ channels . Induction of slow action potentials or enhancement of ongoing slow action potentials was observed in about 70% of all cells in which a successful intracellular injection of the testing compounds was obtained . The slow AP is highly dependent on slow inward current and is known to be enhanced by catecholamines . The effect of the injected cyclic nucleotides and related compounds occurred within 3 minutes after starting the injection, whereas superfusion with these compounds (dibutyryl cAMP was used in place of cAMP) required 10-30 minutes to show an effect . This difference is attributed to the intracellular injection of the compound . The effect on stimulating slow action potentials persisted (greater than 5 minutes) after termination of the application of either Gpp(NH)p or cholera toxin, indicating the long-lasting nature of their action . The effect of the cAMP injections decayed within 1 minute . Intracellular injection of 5'-AMP was without effect . These results support the view that a causal relationship exists between intracellular cAMP level and slow channel function . Phosphorylation of a protein constituent of the slow channel by a cAMP-dependent protein kinase may be involved.

J Cyclic Nucleotide Protein Phosphor Res, 1983, 9(3), 245 - 58
Pertussis toxin actions on the pituitary-derived 235-1 clone: effects of PGE1, cholera toxin, and forskolin on cyclic AMP metabolism and prolactin release; Cronin MJ et al.; Pertussis toxin (PT) modulation of the cyclic AMP (cAMP) accumulation induced by prostaglandin E1 (PGE1), cholera toxin (CT), and forskolin was used to study the role of cAMP in the regulation of prolactin release . The clonal cell line 235-1, derived from a rat anterior pituitary tumor, served as the major target tissue . While PT had no effect on basal cAMP levels, in the presence or absence of a phosphodiesterase inhibitor, this novel bacterial toxin potentiated the cAMP response to each stimulus . The PT enhancement of PGE1-stimulated cAMP production was maximal after 24 hr of PT exposure, whether the toxin was left in the medium or removed after as little as 30 sec . Although PGE1, CT, and forskolin are all secretagogues for prolactin, increasing release by about 50%, PT had no apparent effect on these responses . These data support the hypothesis that cAMP may facilitate prolactin release, but may not be the primary stimulus for secretion.

Acta Physiol Scand, 1982 Dec, 116(4), 443 - 6
The effect of stimulating somatic afferents on cholera secretion in the rat small intestine; Cassuto J et al.; The effect on rate of cholera secretion in the small bowel of activation of the group III and A delta afferent fibres in the sciatic nerve was studied in rats . Activation of these fibres at 3 Hz for 60 min significantly diminished choleraic secretion from 121 +/- 29 to 25 +/- 9 microliters x min-1 x 100 cm-2 serosal surface (mean +/- SE; n = 9) . The effect was apparent after the nerve stimulation . Stimulation of the sciatic nerve had no significant effect on choleraic fluid secretion after interrupting the autonomic nerves to the intestine, nor did it significantly alter net fluid transport in non-choleraic intestine with intact nervous supply . It is proposed that the observations may explain the clinical reports of an effect of acupuncture on cholera secretion.

Am J Epidemiol, 1982 Dec, 116(6), 959 - 70
Endemic cholera in rural Bangladesh, 1966-1980; Glass RI et al.; Since 1963, the International Centre for Diarrhoeal Disease Research, Bangladesh (ICDDR,B), formerly the Cholera Research Laboratory, has maintained a field station in Matlab to treat patients from a surveillance population of 240,000 who have cholera and other diarrheal diseases . Since 1966, the authors have analyzed hospital records of 7141 surveillance-area patients culture-positive for v . cholerae 01 to relate the seasonality, age and sex distribution, and geographic trends with hypotheses concerning transmission, immunity, and risk groups . From this review, they have found that: 1) children 2-9 years old and adult women are most commonly hospitalized for cholera; 2) V . cholerae 01 emerges simultaneously throughout the area of surveillance, with the early cases being of different phage types; 3) three patients were hospitalized twice for cholera compared with 29 expected on the basis of life-table analysis (p less than 0.01), suggesting that immunity to severe disease conferred by previous illness may be stable and long-lasting; 4) no constant relationship was found between the times of onset or peaks of the yearly cholera epidemic and the times of onset or peaks of the monsoon rains or river water levels; and 5) an outbreak of multiply antibiotic-resistant V . cholerae 01 infection documented in 1979 raises questions about the dissemination of resistance plasmids, antibiotic-use patterns, and the need for other drugs in addition to tetracycline . While little progress has been made in understanding the mode of transmission of v . cholerae 01, and in identifying practices for prevention, fluid therapy in this area has decreased the case fatality rate significantly and provides guidance for similar programs elsewhere.

Biochim Biophys Acta, 1982 Nov 22, 692(3), 339 - 44
Micellar gangliosides mediate the lipid insertion of cholera toxin protomer A; Tomasi M et al.; The topology of the interaction of cholera toxin with ganglioside and detergent micelles was studied with the technique of hydrophobic photolabelling . Cholera toxin alpha and gamma polypeptide chains appear to penetrate into the hydrophobic core of ganglioside micelles . Micelles of SDS cause the labelling also of the beta polypeptide chains, while Triton X-100 micelles have little ability to mediate the labelling of the toxin . The specific reduction of the alpha-gamma disulfide bond allows the penetration of the alpha polypeptide chain into Triton X-100 micelles, but does not affect the interaction of cholera toxin with either ganglioside or SDS micelles . Thus, ganglioside micelles appear to cause a conformational change of the native toxin, such as to induce the penetration of the alpha chain into the micelle hydrophobic core.

S Afr Med J, 1982 Nov 13, 62(21), 753 - 5
Determination of the mode of transmission of cholera in Lebowa . An epidemiological investigation; Sinclair GS et al.; In early November 1981 an epidemic of cholera broke out in the Mokerong district of Lebowa . A large proportion of patients originated from the village of Moletlane . A matched-pair case-control study was conducted in the village at the height of the epidemic (18-24 November), and the investigation revealed that the consumption of water from the Gumpies River was significantly more common among cholera patients than among matched controls . The drinking of water sold by water-vendors was also positively associated with an increased risk of contracting cholera.

Infect Immun, 1982 Nov, 38(2), 424 - 33
Rabbit intestinal glycoprotein receptor for Escherichia coli heat-labile enterotoxin lacking affinity for cholera toxin; Holmgren J et al.; The receptors for cholera toxin and Escherichia coli heat-labile toxin (LT) in rabbit small intestinal epithelium were characterized and compared . (i) In vivo studies in ligated intestinal loops showed that whereas LT B subunits could block the fluid secretogenic action of purified LT as well as cholera toxin, cholera toxin B subunits did not inhibit the LT response even when tested in a concentration 100-fold higher than one which gave complete blocking of cholera toxin action . (ii) In vitro studies indicated that isolated intestinal epithelial cells or brush-border membranes could bind about 10-fold more of E . coli LT than of cholera toxin . (iii) All binding sites for cholera toxin in duodenal, jejunal, or ileal mucosal cells or brush-border membranes were extracted by chloroform-methanol-water (4:8:3), which removed lipids quantitatively but did not extract glycoproteins . The extracted cholera toxin binding sites were to greater than 95% recovered in a monosialoganglioside fraction; quantitatively these sites closely corresponded to the concentration of chromatographically identified mucosal GM1 ganglioside (1 nmol of cholera toxin was bound per 1 to 2 nmol of GM1) . In contrast, a substantial fraction of mucosal binding sites for E . coli LT remained in the delipidized tissue residue, and these sites had properties consistent with a glycoprotein nature . Thus, whereas cholera toxin appeared to bind highly selectively to GM1 ganglioside receptor sites of rabbit small intestine, E . coli LT bound both to GM1 ganglioside and to a main glycoprotein receptor for which cholera toxin lacks affinity.

Gastroenterology, 1982 Nov, 83(5), 1051 - 6
Evidence for cholera secretion emanating from the crypts . A study of villus tissue osmolality and fluid and electrolyte transport in the small intestine of the cat; Hallback DA et al.; Villus tissue osmolality and fluid and electrolyte transport were measured in intestinal segments exposed to cholera toxin . The osmolality of the luminal fluid was kept at about 100, 300, or 600 mOsm X kg-1 by use of appropriate concentrations of mannitol . A net fluid secretion was seen in all experiments, the magnitude being dependent on the osmolality in the lumen . A secretion of sodium, potassium, and chloride was also seen in all experiments but the secretion rate of electrolytes was independent of the osmolality in the intestinal lumen . The hydraulic conductivity of the villus epithelium, calculated from the lumen and tissue osmolality, was the same as that estimated in the normal intestines . A villus tissue osmolality gradient was apparent in all experiments regardless of the mannitol concentration in the lumen, the tip osmolality being hypertonic while the tissue osmolality at the base was isotonic . This was the case also when the luminal fluid was hypotonic, a finding opposite to what we found in an earlier study on the normal feline intestine . A likely explanation for this observation is that the crypts of Lieberkuhn secrete fluid containing sodium chloride, which is absorbed by the villus epithelial cells . Hence, a luminal "circulation" of electrolytes between crypts and villi was suggested in the present experimental circumstances.

Biochim Biophys Acta, 1982 Oct 28, 719(1), 58 - 64
Cholera toxin does not impair hormonal inhibition of adenylate cyclase and concomitant stimulation of a GTPase in adipocyte membranes; Aktories K et al.; The influence of cholera toxin on hormonal inhibition of adenylate cyclase and concomitant stimulation of low Km GTPase was studied in adipocyte membrane preparations . In hamster adipocyte ghosts, cholera toxin caused an about 8-fold activation of the adenylate cyclase . The antilipolytic hormonal factors, prostaglandin E1 (1 micro M), N6-phenylisopropyladenosine (1 micro M) and nicotinic acid (30 micro M) reduced both basal and cholera toxin-stimulated enzyme activities . Similar data with regard to inhibition of cholera toxin-stimulated adenylate cyclase were obtained in mouse and rat adipocyte ghosts . As studied in hamster adipocyte ghosts, prostaglandin E1 (1 micro M) increased GTP hydrolysis by a low Km GTPase by about 3-4 fold . Pretreatment of the membrane preparation with cholera toxin did not impair prostaglandin E1-induced GTPase stimulation . The data suggest that cholera toxin does not directly affect the GTPase enzyme stimulated by adenylate cyclase inhibitory hormones.

J Biol Chem, 1982 Oct 25, 257(20), 12148 - 52
Mechanism of action of cholera toxin on intact cells . Generation of A1 peptide and activation of adenylate cyclase; Kassis S et al.; When intact mouse neuroblastoma NB cells were incubated with choleragen at 4 degrees C, washed, and incubated at 37 degrees C, activation of adenylate cyclase occurred rapidly after a delay of 15 min . The cells were incubated under the same conditions with 125I-labeled toxin, lysed, and solubilized with sodium dodecyl sulfate under mild conditions . Soluble proteins were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the absence of dithiol reductants to separate labeled toxin products . Initially, only 0.1 to 0.2% of the cell-associated radioactivity migrated on the gels as the A1 peptide of choleragen . After a 15-min delay, the amount of A1 peptide increased rapidly with time and paralleled the activation of adenylate cyclase . Similar results were observed with human skin fibroblasts, Friend erythroleukemic cells, and II3-alpha-N-acetylneuraminosyl-gangliotetraosylceramide-treated rat glioma C6 cells . When toxin-treated NB cells were incubated at increasing temperatures, generation of A1 peptide and activation of adenylate cyclase increased in parallel . Both processes were prevented by incubation of cells at 4 or at 37 degrees C in the presence of anticholeragen antibodies . These results indicate that there is delay both in the formation of A1 peptide and in the activation of adenylase cyclase in intact cells . As A1 is believed to be the catalytically active component of choleragen, it is suggested that the lag period may be related in part to the time required to generate A1 peptide from choleragen.

Gann, 1982 Oct, 73(5), 798 - 804
The mechanism of cholera toxin-induced suppression of natural killer cytotoxicity; Fuyama S et al.; In a previous publication, it was reported that the pretreatment of mouse spleen cells with cholera toxin (CT) greatly inhibited their natural killer (NK) cytotoxicity . In the present study, it was found that ganglioside GM1 blocked the inhibitory effect of CT, and mature T cells and nylon-wool-adherent cells were not involved in the CT-mediated inhibition of NK cytotoxicity . It was also demonstrated that subunit A or B dissociated from CT lacked the capacity to inhibit NK cytotoxicity . In addition, CT-mediated inhibition was persistent and irreversible . The implications of CT-mediated inhibition of NK cytotoxicity are discussed.

J Cell Physiol, 1982 Oct, 113(1), 35 - 9
Temperature-dependent alteration of cellular morphology by cholera toxin in rat liver epithelial cells which are ts for maintenance of transformed properties; Niles R et al.; Cholera toxin via its ability to increase intracellular cyclic AMP levels can induce drastic changes in cell morphology . This report describes a temperature sensitive mutant of chemically transformed rat liver epithelial cells which only display cell shape alterations in response to cholera toxin at the permissive temperature . Shift up-shift down experiments indicate that the change in the response occurs fairly rapidly, i.e., within 2 hours at the new temperature . The behavior of the temperature sensitive cells at the nonpermissive temperature mimics that of the untransformed rat liver epithelial cells (i.e., no morphological change in response to cholera toxin) while at the permissive temperature the positive cell shape change is identical to that exhibited by chemically transformed rat liver epithelial cells . The temperature sensitive response to cholera toxin is not a function of cyclic AMP production, since the amount of cyclic AMP found as a function of either time or concentration of cholera toxin is quite similar in cells treated at either temperature.

Infect Immun, 1982 Oct, 38(1), 267 - 72
Isolation of hybridoma cell lines and characterization of monoclonal antibodies against cholera enterotoxin and its subunits; Robb M et al.; Hybridoma cell lines which produced monoclonal antibodies against cholera toxin were isolated . These cell lines were detected with an enzyme-linked immunosorbent assay screening procedure with purified cholera toxin or subunit A of cholera toxin . Seven cell lines were characterized with respect to their reactivity with cholera toxin subunits by Western blot analysis . Five clones produced antibodies which were directed against subunit A, and two clones produced antibodies which reacted with subunit B . These antibodies were also characterized by Western blot analysis for reactivity with the heat-labile enterotoxin produced by porcine and human enterotoxinogenic strains of Escherichia coli . Monoclonal antibodies which reacted with subunit A of cholera toxin also reacted with subunit A of both porcine and human heat-labile enterotoxins . In contrast, monoclonal antibodies to subunit B of cholera toxin did not react with subunit B of the heat-labile enterotoxin . Antibodies directed against subunit B neutralized the biological activity of cholera toxin in vitro in the S49 mouse lymphosarcoma assay . In contrast to polyclonal anti-subunit A antisera, monoclonal anti-subunit A from four of five clones had small but measurable neutralizing capacities in vitro.

J Biol Chem, 1982 Sep 25, 257(18), 10540 - 3
Functional homology between signal-coupling proteins . Cholera toxin inactivates the GTPase activity of transducin; Abood ME et al.; Both the light-stimulated cGMP phosphodiesterase of retinal rod outer segments (ROS) and hormone-stimulated adenylate cyclase are regulated by guanine nucleotide-binding regulatory proteins (N) . Transducin serves as the signal-carrying regulatory protein in ROS, and the N protein (also called G or G/F) performs this role in the adenylate cyclase system . The GTP form of these regulatory proteins activates the corresponding enzyme, whereas the GDP form does not . Both transducin and the N protein possess a GTPase activity that restores the regulatory protein to the unstimulated state . Cholera enterotoxin catalyzes the transfer of ADP-ribose from NAD+ to the N protein, which inhibits its GTPase activity and activates adenylate cyclase . We report here that the toxin also catalyzes ADP-ribosylation of the alpha-subunit of transducin in ROS membranes . This modification of the guanine nucleotide-binding subunit of transducin is markedly enhanced by the bleaching of rhodopsin and by the addition of guanosine-5'-(beta, gamma-imino)triphosphate . In contrast, GDP, GTP, and guanosine-5'-(3-O)thiotriphosphate inhibit the reaction, while GMP and ATP have no effect . Under optimal conditions, toxin catalyzes labeling of 0.7 mol of the alpha-subunit of transducin/mol of bound {3H}guanosine-5'-(beta, gamma-imido)triphosphate and causes 70% inhibition of the light-dependent GTPase activity of transducin in ROS . These results indicate close functional homology between transducin of ROS and the N protein of adenylate cyclase.

J Biol Chem, 1982 Sep 10, 257(17), 10471 - 8
Evidence that human platelet alpha-adrenergic receptors coupled to inhibition of adenylate cyclase are not associated with the subunit of adenylate cyclase ADP-ribosylated by cholera toxin; Smith SK et al.; Exposure of the alpha-adrenergic receptor of the human platelet to agonist prior to solubilization stabilizes a receptor complex of the alpha-adrenergic receptor with the GTP-binding protein(s) which modulates receptor affinity for agonists (Smith, S . K., and Limbird, L . E . (1981) Proc . Natl . Acad . Sci . U . S . A . 78, 4026-4030) . The soluble alpha-adrenergic receptor is characterized by retention of sensitivity to GTP and a faster rate of sedimentation in sucrose gradients than antagonist-occupied or unoccupied receptors . The present studies were undertaken to determine whether the alpha-adrenergic receptor, which is coupled to inhibition of adenylate cyclase, contains the same GTP-binding protein that is involved in activation of adenylate cyclase . The GTP-binding protein that is coupled to activation of adenylate cyclase was labeled with {32P}ADP-ribose using cholera toxin . Incorporation of {32}ADP-ribose into a Mr = 42,000 peptide in human platelet membranes was paralleled by an enhancement of GTP-sensitive catalytic activity in the membranes . However, cholera toxin treatment did not modify alpha-receptor-mediated inhibition of adenylate cyclase or interaction of the alpha-receptor with agonist agents . Moreover, sucrose gradient centrifugation revealed that the {32P}ADP-ribosylated Mr = 42,000 subunit of the stimulatory GTP-binding protein did not appear to associate with the agonist-alpha-receptor complex . These data suggest that the GTP-binding protein that mediates GTP activation of adenylate cyclase in the human platelet membrane is distinct from the GTP-binding protein that modulates alpha-adrenergic receptor affinity for agonist agents and which associates with the receptor in the presence of agonists.

Vopr Med Khim, 1982 Sep-Oct, 28(5), 123 - 7
{Role of phosphodiesterase in the changes of cyclic adenosine-3',5'-monophosphate in the rabbit jejunal mucosa after treatment with cholera enterotoxin}; Iurkiv VA et al.; An increase in content of cAMP in the rabbit jejunum mucosa after treatment with cholerae toxin was due to adenylate cyclase activation . The maximal activation of adenylate cyclase (AC) and increase in the cAMP content in the mucosa were observed within 4 hrs after administration of the cholerae toxin in situ into the lumen of an isolated strip of the rabbit jejunum . Cholerae enterotoxin did not affect the activity of phosphodiesterase (PDE) . It was shown that AC activation did not correlate with an increase in the cAMP content in rabbit jejunum mucosa exposed to cholerae enterotoxin . Absence of the correlation was demonstrated by kinetic characteristics of PDE and by regulatory effect of Ca2+ on the activity of AC and PDE.

Am J Physiol, 1982 Sep, 243(3), H434 - 41
Acetylcholine inhibits positive inotropic effect of cholera toxin in ventricular muscle; Pappano AJ et al.; Acetylcholine (ACh, 10(-6) M) had no effect on basal adenylate cyclase activity (3.4 +/- 0.56 pmol cyclic AMP . min-1 . mg wet wt-1), adenosine 3',5'-cyclic monophosphate (cyclic AMP) content (0.88 +/- 0.09 pmol/mg wet wt), or the force of contraction in paced (2.5 Hz) chick embryo right ventricles superfused with Tyrode solution . After 60-180 min of superfusion in the presence of cholera toxin (5 x 10(-6) g/ml), adenylate cyclase activity (1.7 times), cyclic AMP content (2.4 times), and contractility (2.4 times) had increased significantly above basal levels . ACh reversed the positive inotropic effect of cholera toxin but did not change the increased activity of adenylate cyclase and content of cyclic AMP obtained in cholera toxin . Stimulation of adenylate cyclase by isoproterenol (ISO) was inhibited by ACh in the absence and presence of cholera toxin . ACh did not change guanosine 3',5'-cyclic monophosphate (cyclic GMP) content in the absence or presence of cholera toxin . Cholera toxin has actions on chick embryo ventricle similar to those of the beta-adrenergic agonist, ISO, and the phosphodiesterase inhibitor, isobutylmethylxanthine . The ability of ACh to reverse the positive inotropic effect of cholera toxin without preventing the accumulation of cyclic AMP may involve the prevention or reversal of cyclic AMP-dependent phosphorylation . In this regard, reduction of Ca2+ influx through voltage-sensitive membrane channels may be an essential component of muscarinic inhibition.

Am J Physiol, 1982 Sep, 243(3), C107 - 15
Effect of cholera toxin on cAMP levels and Na+ influx in isolated intestinal epithelial cells; Hyun CS et al.; Freshly isolated chicken intestinal cells contain approximately 20 pmol adenosine 3',5'-cyclic monophosphate (cAMP)/mg cellular protein . Incubation with 3 micrograms/ml cholera toxin (CT) at 37 degrees C induces an elevation of cellular cAMP beginning 10-15 min after initial exposure . The response is linear with time for 40-50 min and causes a six- to eightfold increase over control levels at steady state . Dibutyryl cAMP and agents that increase cAMP production inhibit Na+ influx into the isolated enterocytes . Chlorpromazine completely abolishes the toxin-induced elevation of cAMP in the isolated cells and also reverses the effect on Na+ entry . The data provide evidence for a cAMP-mediated control of intestinal cell Na+ uptake, which may represent the mechanistic basis for the antiabsorptive effect of CT on Na+ during induction of intestinal secretory activity . Studies on the time-dependent effects of chlorpromazine on both intracellular cAMP concentration and Na+ influx suggest that the reactivation of the Na+ transport system after cAMP-induced inhibition is slow relative to the disappearance of cAMP.

Biochemistry, 1982 Aug 31, 21(18), 4474 - 9
Tubulin adenosine diphosphate ribosylation is catalyzed by cholera toxin; Hawkins DJ et al.; Cholera toxin catalyzed the transfer of radioactive label from {adenine-2,8-3H2}NAD+ or ((32P}NAD+ to rat C6 glioma cell membrane and cytosolic proteins . Labeled proteins were resolved by polyacrylamide-NaDodSO4 gel or two-dimensional gel electrophoresis and stained with Coomassie blue, and the gels were subjected to fluorography or autoradiography . Autoradiograms of gels revealed labeled Mr 42000 and 46000-48000 membrane proteins that are putative subunits of the regulatory component (G/F) of the C6 cell hormone-sensitive adenylate cyclase . Cholera toxin also catalyzed the labeling of several cytosolic proteins including a Mr 54000 protein that was observed in autoradiograms of two-dimensional gels to migrate as an acidic satellite relative to Coomassie-stained C6 cell tubulin . Tubulin modified by ADP-ribosylation would undergo an acid shift relative to the stained unmodified tubulin in two-dimensional gels . The data led us to postulate that tubulin undergoes cholera toxin catalyzed ADP-ribosylation . Bovine brain tubulin prepared by three cycles of warm/cold polymerization/depolymerization was incubated with {32P}NAD+, GTP, and cholera toxin and then subjected to two-dimensional gel electrophoresis . Autoradiograms of the gels revealed the presence of {32P}ADP-ribosylated proteins that migrated as acidic satellites relative to the Coomassie-stained brain alpha and beta tubulin . Peptide maps of bovine brain tubulin and the associated {32P}ADP-ribosylated proteins showed a correspondence between the autoradiographic images and the stained peptide fragments . The data demonstrate that cholera toxin catalyzes the ADP-ribosylation of tubulin.

Mol Cell Biochem, 1982 Aug 6, 46(3), 155 - 60
Ganglioside-cholera toxin interactions: a binding and lipid monolayer study; Cumar FA et al.; On t.l.c . plates 125I-cholera toxin binds to a disialoganglioside tentatively identified as GD1b with about 10 times less capacity then to ganglioside GM1 . Binding of labeled toxin to both gangliosides was abolished in presence of excess amounts of unlabeled B subunit . Ganglioside extracts from human or pig intestinal mucosa showed toxin binding to gangliosides GM1 and GD1b . In ganglioside-containing lipid monolayers the penetration of the toxin was independent of the ganglioside binding capacity.

Scand J Gastroenterol, 1982 Aug, 17(5), 695 - 703
5-hydroxytryptamine and cholera secretion . Physiological and pharmacological studies in cats and rats; Cassuto J et al.; The intestinal secretion evoked by close intra-arterial infusion of 5-hydroxytryptamine (5-HT) in cats was inhibited by tetrodotoxin, a drug abolishing action potentials . Furthermore, the intestinal secretion produced by placing a 2-mM 5-HT solution in the intestinal lumen of rats was inhibited by hexamethonium, a ganglionic receptor-blocking agent . These observations strongly indicate that 5-HT-induced secretion is, at least in part, neurally mediated . It was also shown that 5-HT receptors are involved in the pathophysiology of choleraic secretion, since the secretion was inhibited by making the experimental animal tachyphylactic against 5-HT . No effects of 5-HT tachyphylaxis were noted on fluid transport in normal intestines . The results are discussed in relation to a new hypothesis for the pathophysiology of cholera secretion.

Bull Soc Pathol Exot Filiales, 1982 Aug-Oct, 75(4), 345 - 51
{Role of interhuman infection during the cholera epidemic in the People's Republic of the Congo (1978-1979)}; Yala F et al.; The authors make an epidemiologic analysis of the first cholera cases notified by the Congo in 1978 and 1979 . Contrary to the explosive cholera, experiences by Burundi and Zaire during the same period, the cholera, in the Congo, had a very subtle propagation, with the well-know "iceberg" phenomena . Human contacts were very important in the transmission of the disease . The cases encountered were almost exclusively of the family type . Healthy carriers represent a threat to the other countries.

Exp Hematol, 1982 Aug, 10(7), 620 - 7
Inhibition by cholera toxin of clonal growth of murine granulocyte/macrophage progenitor cells in soft agar cultures; Lenz R et al.; Proliferation in vitro of the committed granulocyte-macrophage progenitor cell (CFUC) is inhibited by cholera toxin (CT) in a dose-dependent manner . This inhibition is reduced and counteracted by higher doses of colony stimulating factor (CSF), an obligatory growth stimulator of CFUC . Mixing of CT with its specific receptor, the monosialoganglioside, GM1, before exposure to bone marrow (BM) cells, blocks the toxin's effect, and a restoration of colony formation is achieved . A dose-dependent inhibition of CSF-induced colony formation is also observed in the presence of choleragenoid, the B subunit of the toxin which binds to the specific receptor on the cell surface, but is biologically inactive . Incubation of BM cells with CT prior to cloning in soft agar cultures supplemented with CSF inhibits clonal proliferation of CFUC, whereas a brief in vitro exposure of BM cells to CSF prior to CT protects the cells against subsequent CT inhibition . The ability of CT to inhibit the CSF-induced clonal proliferation of CFUC and the effectiveness of CSF in reducing and even counteracting CT inhibition suggests that CT, by binding to BM cells through the B subunit, might either directly or indirectly interfere with the stimulatory activity of CSF upon its target cells.

Biochim Biophys Acta, 1982 Jul 22, 720(4), 338 - 45
Action of cholera toxin on dispersed acini from rat pancreas . Post-receptor modulation involving cyclic AMP and calcium; Pan GZ et al.; In dispersed acini from rat pancreas, cholera toxin caused a significant increase in cellular cyclic AMP but little or no change in amylase secretion . The presence of a secretagogue that causes mobilization of cellular calcium (e.g., cholecystokinin, carbamylcholine, bombesin or ionophore A23187) caused a substantial increase in the effect of cholera toxin on enzyme secretion . Cholera toxin did not alter calcium transport or the changes in calcium transport caused by other secretagogues, and secretagogues that mobilize cellular calcium did not alter cellular cyclic AMP or the increase in cyclic AMP caused by cholera toxin . These results indicate that in dispersed acini from rat pancreas there is post-receptor modulation of the action of cholera toxin by secretagogues that mobilize cellular calcium and that this modulation is a major determinant of the effect of the toxin on enzyme secretion.

Brain Res, 1982 Jul 15, 243(2), 215 - 24
Cholera toxin and wheat germ agglutinin conjugates as neuroanatomical probes: their uptake and clearance, transganglionic and retrograde transport and sensitivity; Wan XC et al.; Horseradish peroxidase (HRP) conjugates of 6 different lectins and cholera toxin (CTHRP) were quantitatively compared with respect to: (a) their behavior at the injection site and (b) their ability to label, by means of transganglionic and retrograde axonal transport, axon terminals and neurons in the medulla of the rat subsequent to injections of each probe into the anterior two thirds of the tongue . HRP conjugates of wheat germ agglutinin (WGHRP) and CTHRP were more sensitive than any of the other lectin-HRP conjugates . Both were far superior to free-HRP (FHRP) in demonstrating these projections and CTHRP was the most sensitive transganglionic and retrograde probe . Additional experiments demonstrated that this superiority was not an artifact of the volume of material injected into the tongue nor of the injection site area or survival time selection . These experiments demonstrated further that CTHRP and WGHRP remain at the injection site approximately twice as long as FHRP and that their removal from or degradation in retrogradely labeled neurons requires approximately twice as much time as that required for FHRP . These observations, together with earlier studies from this laboratory, suggest the following conclusions: (1) CTHRP and WGHRP are superior in sensitivity to FHRP for studies of neuronal connectivity; and (2) HRP conjugates of ligands such as CTHRP and WGHRP are internalized, transported and/or degraded by mammalian neurons in a manner which differs from that of FHRP, a macromolecule for which neuronal plasma membrane 'receptors' are lacking.

Infect Immun, 1982 Jul, 37(1), 70 - 6
Production and characterization of monoclonal antibodies to cholera toxin; Remmers EF et al.; Monoclonal antibodies against cholera toxin were produced to obtain highly specific antisera to cholera toxin . Fifteen hybridoma cell lines producing monoclonal antibodies specific for the determinants of cholera toxin were derived from the fusion of mouse myeloma cells and spleen cells from mice immunized with cholera toxin . The cell lines were stabilized, examined for specific antibody production, and immortalized by freezing cultured cells and tumor cells which had been grown subcutaneously in mice . All cell lines continued antibody secretion upon thawing . The antibodies produced by the hybridoma cell lines were characterized by determination of the class of light- and heavy-chain components and by determination of specificity for the cholera toxin subunit . All of the antibodies contained the k light chain, 4 contained the mu heavy chain, and the remaining 11 contained the gamma 1 heavy chain . Ten of the monoclonal antibodies are specific for the B subunit of cholera toxin, and three are specific for the A subunit . The remaining two appear to react with both subunits.

J Invest Dermatol, 1982 Jul, 79(1), 42 - 7
Effects of cholera toxin on proliferation of cultured human keratinocytes in relation to intracellular cyclic AMP levels; Okada N et al.; In the culture of epidermal keratinocytes, the cellular growth rate is reported to be accelerated by cholera toxin . The mechanism by which cholera toxin exerts biological effects is thought to result from changes in intracellular cyclic AMP concentrations . But in many reports cyclic AMP elevating agents appeared to inhibit growth of keratinocytes in culture . This study was done to clarify the discrepancy of this problem . Determination of cyclic AMP revealed that cholera toxin over a range of 10-14-10-8 M increased the intracellular concentration of cyclic AMP of cultured keratinocytes about 100-fold over the controls after incubation for 6 hr . When a small number (10(5)) of cells were inoculated in a 60 X 15 mm culture dish, cholera toxin strongly stimulated colony growth . When a relatively larger number (8 X 10(5)) of cells were inoculated in a dish, cholera toxin moderately accelerated cell division, and increased DNA and protein levels of the culture during early days of cultivation . But after about 20 days, of cultivation when the culture reached confluence, cholera toxin decreased both DNA and protein content in a culture dish . The cultures were pulse labeled with 3H-thyrmidine at 12 and 24 hr after the addition of 10-10 M cholera toxin, and its uptake into DNA was determined . In the early days of cultivation the uptake of 3H-thymidine increased after treatment with cholera toxin . But in the late days of cultivation, cholera toxin decreased the rate of 3H-thymidine incorporation into DNA . These results indicated that cholera toxin-cyclic AMP has effects on the proliferation of keratinocytes in culture biphasically according to cellular concentrations in culture.

Brain Res, 1982 Jun 24, 242(2), 233 - 41
Immunocytochemical study on cholera toxin binding sites by monoclonal anti-cholera toxin antibody in neuronal tissue culture; Okada E et al.; An indirect method of immunocytochemistry showed that cholera toxin and its B-subunit served as specific neuronal surface markers in conjunction with monoclonal anti-cholera toxin and FITC labeled anti-mouse Fab . The cell types which get specifically stained in culture were peripheral neurons from dorsal root ganglion cells, superior cervical ganglion cells, and cerebral neurons, all of which were rat tissue, and NGF-treated PC12 cells . Non-neuronal cells, i.e . Schwann cells, fibroblasts and glia cells, were not stained . This method can, therefore, be used to distinguish neurons from non-neuronal cells in neuronal tissue cultures, as in the case of tetanus toxin described in the literature.

Biochemistry, 1982 Jun 22, 21(13), 3231 - 4
Cholera toxin mediated agglutination of ganglioside Gm1 containing phospholipid vesicles and Gm1-coated polystyrene spheres; Dwyer JD et al.; Quasi-elastic laser light scattering is used to examine the cholera toxin mediated agglutination of ganglioside GM1 containing phospholipid vesicles and Gm1-coated polystyrene spheres . We find that the ability of cholera toxin to agglutinate Gm1-containing phospholipid vesicles depends markedly on the lipid composition of the vesicle, with only those composed of short-chain lipids (C14, C16) being appreciably agglutinated . This is interpreted as due to poor mixing of these lipids with the longer chain gangliosides, resulting in lateral separation of the gangliosides in the membrane bilayer . A simple quantitative model, a modification of that developed by von Schulthess et al . {von Schulthess, G . K., Cohen, R . J., Sakato, N., & Benedek, G . B . (1976a) Immunochemistry 13, 955--962}, is developed to describe these agglutination processes . Application of this model to the agglutination of Gm1-coated polystyrene spheres by cholera toxin allows an estimate of a minimum value of 4.5 x 10(4) M-1 for the association constant of cholera toxin for its initial Gm1 receptor.

Biochemistry, 1982 Jun 22, 21(13), 3227 - 31
Subunit arrangement of cholera toxin in solution and bound to receptor-containing model membranes; Dwyer JD et al.; Quasi-elastic laser light scattering (QLS) is used to study the translational frictional properties of cholera toxin and its complex with ganglioside Gm1 receptor containing phospholipid vesicles . These properties are compared to theoretically calculated values for model structures composed of spherical subunits in order to assess the structural configuration of the toxin and its binding geometry on membrane surfaces . The structure for the toxin that best fits the experimental results consists of the five B subunits arranged radially about an elongated A subunit, which extends well above the plane of the B subunits . Binding of cholera toxin to Gm1-containing model membranes results in a complex in which the B subunits are absorbed on the surface while the A subunit penetrates the membrane bilayer.

Gastroenterology, 1982 Jun, 82(6), 1335 - 40
The use of chlorpromazine in the treatment of cholera and other severe acute watery diarrheal diseases; Islam MR et al.; Four hundred and ten patients with severe watery diarrhea; including 316 patients with cholera, were studied in a double-blind, randomized, placebo controlled trial to determine if chlorpromazine (1 mg/kg) would be useful in the management of such patients . All patients were at least 7.5% dehydrated on admission into the study; all received intravenous fluids followed by oral rehydration solution and all received tetracycline . In addition, one-half of the patients received chlorpromazine, 1 mg/kg, orally as a single dose 2 h after admission . Effectiveness of the chlorpromazine was determined by comparing oral therapy failure rates, purging rates, vomiting rates, i.v . fluid requirements and hospitalization time in groups of the patients receiving and not receiving the drug . In children with severe cholera, e.g., with shock on admission or with very high purging rates, chlorpromazine lowered the oral therapy failure rate by about 50% . However, children with less severe cholera, adults with cholera, and patients of all ages with noncholera diarrhea could not be demonstrated to benefit significantly from the drug . In these groups of patients, oral therapy failures were rare irrespective of whether or not chlorpromazine had been given . We, therefore, do not recommend chlorpromazine in the routine management of patients with watery diarrhea, however, it may be useful in treatment of children with severe cholera when added to standard treatment of hydration and tetracycline.

J Cell Biol, 1982 Jun, 93(3), 860 - 5
Internalization and degradation of cholera toxin by cultured cells: relationship to toxin action; Fishman PH; Using anticholeragen antibodies and 125I-protein A, we developed a specific and quantitative assay for measuring choleragen on the surfaces of cultured cells . When neuroblastoma cells containing bound toxin were incubated at 37 degrees C, surface toxin disappeared with a half-life of approximately 2 h and a significant loss was detected by 10 min . When cells were incubated with 125I-choleragen in order to measure toxin degradation, cell-associated radioactivity disappeared with time and a corresponding amount of TCA-soluble label appeared in the culture medium with a half-life of 4-6 h . No degradation was detected until 45 min . Although there was a lag of 15 min before bound choleragen activated adenylate cyclase, the enzyme became maximally activated between 45 and 60 min . Similar results were obtained with Friend erythroleukemia cells . Internalization, degradation, and activation all were blocked when the cells were maintained at 4 degrees C . At 22 degrees C, internalization and activation occurred, albeit at a slower rate, whereas degradation was effectively inhibited . These results indicated that choleragen does not have to be degraded by intact cells in order for it to activate adenylate cyclase . Some internalization of the toxin, however, appears to precede the activation process.

Gut, 1982 Jun, 23(6), 507 - 12
Changes in intestinal alkaline phosphatase activity in cholera toxin-treated rats; Miura S et al.; It is conceivable that brush border enzyme activities of the intestinal mucosa will change when bacterial toxins are exposed to the intestinal microvillous membranes . The effect of cholera toxin on the activity of intestinal alkaline phosphatase in rats was therefore determined in the intestinal mucosa by the histochemical method as well as in intestinal lymph by using lymph fistulated-rats . Activity of intestinal alkaline phosphatase in the intestinal mucosa and lymphatics changed biphasically after the oral administration of cholera toxin to rats . For the first three hours after the administration of cholera toxin it was depressed; it then increased and at eight hours reached a maximum . These changes in the activity of intestinal alkaline phosphatase were prevented by the administration of chlorpromazine, a known inhibitor of adenylate cyclase activity.

Br Med J (Clin Res Ed), 1982 May 8, 284(6326), 1361 - 4
Controlled trial of chlorpromazine as antisecretory agent in patients with cholera hydrated intravenously; Rabbani GH et al.; A randomised controlled trial was conducted to investigate the ability of chlorpromazine to reduce intestinal secretion in cholera . Chlorpromazine had reduced loss of intestinal fluid in animals with diarrhoea induced by cholera toxin, and in a preliminary study the drug had reduced purging in patients with cholera . Forty-six adults with cholera were included in the randomised trial . Of these, 34 were treated with chlorpromazine (1 mg/kg or 4 mg/kg either by mouth or intramuscularly) and 12 served as controls . After treatment with the drug there was a significantly greater reduction in the rate of fluid loss in the treated patients than in the controls during the first (p less than 0.005), second (p less than 0.05), and fourth (p less than 0.01) eight-hour periods, but not during the third eight-hour period; the dose of 4 mg/kg was only marginally more effective than 1 mg/kg . The effect of chlorpromazine was strikingly biphasic, with one peak during the first eight hours and another 24-32 hours after administration . Chlorpromazine also significantly reduced the duration of diarrhoea, frequency of vomiting, and amount of intravenous fluid required . The drug induced mild sedation and no hypotension in these well-hydrated patients . These findings confirm the effectiveness of chlorpromazine in reducing fluid loss in cholera . A sedative effect, however, especially in children, may limit its usefulness and requires further study.

Dig Dis Sci, 1982 May, 27(5), 459 - 66
Effect of somatostatin on diarrhea and on small intestinal water and electrolyte transport in a patient with pancreatic cholera; Ruskone A et al.; The effects of somatostatin on diarrhea and on small intestinal flow of water and electrolytes (slow-marker perfusion technique) in a patient with pancreatic cholera are reported . Continuous intravenous infusion of somatostatin (8 micrograms/kg/hr) suppressed the diarrhea, but a rebound was observed after somatostatin . Infusion of somatostatin at the same dosage decreased the ileal fluid flow rate to within control values . This effect was mainly due to a sharp reduction in the rate fluid entered the jejunum, but was also due to a suppression of the abnormal water and electrolyte secretion in the proximal jejunum . Secretion in the rest of the small bowel remained unchanged . Somatostatin did not noticeably alter the high preinfusion plasma level of prostaglandin E1, but decreased the initially high plasma concentration of vasoactive intestinal peptide to normal values . These results suggest that long-acting somatostatin analogs could be of value in the symptomatic treatment of diarrhea in pancreatic cholera.

Biochim Biophys Acta, 1982 Apr 29, 720(2), 181 - 7
Detergent extraction of cholera toxin and gangliosides from cultured cells and isolated membranes; Hagmann J et al.; Choleragen, when bound to various cultured cells, resisted extraction by Triton X-100 under conditions which retained the cytoskeletal framework of the cells . The resistance (greater than 75% of the bound toxin) was observed in Friend erythroleukemic, mouse neuroblastoma N18 and NB41A and rat glioma C6 cells even though the different cells varied over 1000-fold in the number of toxin receptors . The extent of extraction did not depend on whether the cells were in monolayer culture of in suspension or whether choleragen was found at 0 or 37 degrees C . A similar resistance to extraction was also observed in membranes isolated from toxin-treated cells . Using more drastic conditions and other non-ionic detergents, 90% of the bound choleragen was solubilized from cells and membranes . When rat glioma C6 cells, which bind only small amounts of choleragen, were incubated with the ganglioside GM1, toxin binding was increased and the bound toxin was also resistant to extraction . When these cells were incubated with {3H}GM1, up to 70% of the cell-associated GM1 was extracted under the mild conditions . When the Gm1-labeled cells were incubated with choleragen or its B (binding) component, there was a significant reduction in the solubilization of GM1 . Similar results were obtained with isolated membranes . When choleragen-receptor complexes were isolated from N18 cells labeled with {3H} galactose by immunoadsorption, only labeled GM1 was specifically recovered . These results suggest that it is the choleragen-ganglioside complex that is resistant to detergent extraction.

Acta Physiol Scand, 1982 Apr, 114(4), 573 - 7
The effect of nicotinic and muscarinic receptor blockade on cholera toxin induced intestinal secretion in rats and cats; Cassuto J et al.; The effects of hexamethonium (cholinergic nicotinic receptor antagonist) and atropine (cholinergic muscarinic receptor antagonist) on cholera toxin induced secretion were investigated in denervated segments of the small intestine of rats and cats . While there was no effect of atropine, hexamethonium markedly inhibited choleraic secretion and turned it into a net fluid absorption in many animals . This observation further strengthens our hypothesis that the enteric nervous system is involved in cholera secretion.

Mol Cell Endocrinol, 1982 Apr, 26(1-2), 165 - 76
Stimulation by thyrotropin, cholera toxin and dibutyryl cyclic AMP of the multiplication of differentiated thyroid cells in vitro; Roger PP et al.; Primary cultures of dog thyroid cells have been established . The cells originated from follicles and displayed differentiation characteristics of such cells: iodide trapping and organification, responsiveness of iodide organification and cyclic AMP accumulation to thyrotropin (TSH), induction of a two-dimensional follicular structure by TSH . TSH also stimulated the multiplication of these cells . The effect of TSH was detected with concentrations as low as 100 muU/ml and was reproduced with purified TSH . It was reproduced by cholera toxin (10 ng/ml) and dibutyryl cyclic AMP (10(-5) M) . The data show that TSH, which stimulates the function of thyroid tissue, in vivo and in vitro, activates the multiplication of differentiated dog-thyroid follicular cells in primary culture, which suggests that this trophic effect is, partly at least, mediated by cyclic AMP.

Acta Pharmacol Toxicol (Copenh), 1982 Apr, 50(4), 294 - 9
A study of inhibition of cholera toxin-induced intestinal hypersecretion by neuroleptics; Larsen JJ; Various types of neuroleptics and other agents were examined for inhibitory effect in cholera toxin-induced hypersecretion in ligated loops of mice jejunum . Several agents belonging to the phenothiazine group or thioxanthene group were inhibitory . Only three other neuroleptics, loxapin, haloperidol and penfluridol, inhibited the hypersecretion . Among the miscellaneous agents the calcium antagonist nifedipine was inhibitory . The potencies of the neuroleptics did not correlate with the dopamine antagonistic potencies . The results indicate that the site of antisecretory action of the drugs is to be found outside the central nervous system.

Nouv Presse Med, 1982 Mar 6, 11(11), 859 - 62
{The Verner-Morrison syndrome: endocrine cholera or vipoma? (author's transl)}; Charleux H; This syndrome, also known under the initials W.D.H.A., is due to a single or multiple pancreatic tumour or to micropolyadenomatosis consisting of non-beta islet cells . Malignancy is found in about two-thirds of the cases . The other endocrine glands are rarely involved . The syndrome is more frequent in women than in men . It is characterized by liquid diarrhoea, marked hypokalaemia and absence of gastric hyperacidity . The tumour is mainly diagnosed by echotomography, computerized tomography and arteriography . It can also be located by staged collections of blood along the portal system for hormonal assays . The nature of the tumour can only be ascertained by demonstrating the presence of the responsible hormone, usually the "vaso-intestinal peptide" . Treatment is primarily surgical . Adjuvant treatments include streptozotocine and embolization by superselective catherization in cases of hepatic matastases . The prognosis is sombre since in spite of the various treatments cure can only be achieved in 50% of the patients.

J Gen Microbiol, 1982 Mar, 128(Pt 3), 497 - 502
Entrance of cholera enterotoxin subunits into cells; Tsuru S et al.; Quantitative analysis of the staining of cholera enterotoxin on the surface of cells with specific antibodies against each subunit of cholera toxin, using a Fluorescence-Activated Cell Sorter, showed that not only subunit A but also subunit B penetrates the cell membrane . The detection of each subunit inside the cell was facilitated by the use of saponin, an agent which increases membrane permeability.

Am J Vet Res, 1982 Mar, 43(3), 497 - 8
End-point dilution-fluorescent antibody technique for cloning hog cholera virus; Kresse JI et al.; Hog cholera virus was cloned by incubating selected pretitrated dilutions of the virus on PK-15 cell cultures for 2 hours . After a thorough washing, the coverslip cell cultures were overlaid with medium containing 0.1% hog cholera immune serum to prevent secondary foci . Forty-eight hours later, the cultures were vigorously washed and maintenance medium containing 5% bovine fetal serum was added . When examined by the fluorescent antibody technique 18 hours later, single plaques were observed in some cultures with no evidence of secondary foci . The virus clone subsequently yielded a homogeneous population of hog cholera virus that retained the characteristics of the parent strain; pathogenicity of the virus clone in pigs was demonstrated, and specific immunofluorescence occurred in infected cell cultures stained with fluorescein isothiocyanate-labeled antibody . The method used gave reasonable assurance of the cloned virus' freedom from extraneous agents.

Proc Natl Acad Sci U S A, 1982 Mar, 79(6), 2018 - 22
Selective proliferation of normal human melanocytes in vitro in the presence of phorbol ester and cholera toxin; Eisinger M et al.; Cultures consisting almost entirely of human melanocytes were obtained from epidermal single-cell suspensions by using phorbol 12-myristate 13-acetate (10 ng/ml) in the culture medium . At this concentration, phorbol ester is toxic to human keratinocytes but not to melanocytes . When the seeding density was optimal (0.8-2 x 10(4)/cm2) and the medium contained both phorbol ester and cholera toxin, melanocytes proliferated extensively . Under these conditions, human melanocytes could be passaged serially in vitro and grown in quantity . This cell culture system can thus be used to answer basic questions related to pigment cell biology and may serve as a control for studies of malignant melanocytes.

Cell Biophys, 1982 Mar, 4(1), 25 - 40
Quantitative description of the binding of GM1 oligosaccharide by cholera enterotoxin; Schafer DE et al.; We present a quantitative molecular interpretation of binding between the five B subunits of cholera enterotoxin and the oligosaccharide of ganglioside GM1, based on the currently accepted quaternary structure of the toxin and principles of multiple equilibria . A sequential binding equation is derived and fitted to published binding data obtained by equilibrium dialysis . In one study of binding to reduced toxin (I), intact toxin (II), and isolated B subunits (III) at low concentrations, analysis by the Hill equation suggested that binding was positively cooperative and that there were only four binding sites per toxin molecule; individual affinity constants could not be estimated because of the empirical nature of the Hill equation . Our analysis suggests that the evidence for positive cooperativity is stronger for I and III than for II . Affinity constants for the first binding step are about 2.0-2.1 microM-1 for I and 2.5-2.7 microM-1 for II and III; those for the second binding step are about 3.5-5.0 microM-1 and for I and III, but only 2.5 microM-1 for II . Constants for later binding steps are apparently within the range of 2-7 microM-1 . Predictions of the sequential model at higher ligand concentrations diverge substantially from those of the Hill equation, and are supported by data obtained at higher protein and ligand concentrations . Thus all available equilibrium dialysis data are consistent with a single set of affinity constants and with the hypothesis of five equivalent binding sites.

Biochemistry, 1982 Feb 16, 21(4), 660 - 4
Comparison of the binding of cholera and Escherichia coli enterotoxins to Y1 adrenal cells; Donta ST et al.; The binding of iodinated cholera and Escherichia coli (LT) enterotoxins to Y1 mouse adrenal cells was studied by using saturation analysis (Scatchard) . Each toxin bound to Y1 cells with similar affinity {KA = (1.5--2.0) x 10(9)M-1}, but there appeared to be twice as many receptor sites per cell for E . coli toxin (approximately 4 x 10(5) . Despite the increased binding of E . coli toxin, Y1 cells respond sooner to, and to smaller concentrations of, cholera toxin . The binding of each toxin was inhibited competitively by both toxins, although twice as much E . coli toxin was required to inhibit 50% of the binding of cholera toxin as was needed for either homologous inhibition or the inhibition of E . coli toxin binding by cholera toxin . The B subunits of both toxins were equally effective in competing for the binding of both iodinated toxins . Whereas the A subunits of both toxins had little or no effect on the binding of E . coli toxin, they consistently inhibited 20--40% of the binding of cholera toxin to cells . These results suggest that there are receptor loci on cells for the A subunit and that conformational differences exist between the two toxins that might explain the greater sensitivity of Y1 cells to cholera toxin . A model is suggested in which cholera toxin exhibits a greater degree of multivalent ligand binding than does the E coli toxin, resulting in a more favorable situation for apposition of the A subunit to its receptor or for its insertion into the membrane.

Lancet, 1982 Feb 6, 1(8267), 305 - 8
Intestinal antibody responses after immunisation with cholera B subunit; Svennerholm AM et al.; In about 80% of Bangladeshi volunteers a single oral or intramuscular immunisation with a new cholera toxoid immunogen (B subunit) gave rise to a local intestinal secretory immunoglobulin A (IgA) antitoxin response as measured in intestinal-lavage fluid by enzyme-linked immunosorbent assay methods . The rise in IgA antitoxin titre was similar for both immunisation routes and was comparable to that seen after clinical cholera; however, the response persisted longer after oral than intramuscular immunisation . A second immunisation by either route evoked an antitoxin response which usually closely resembled that seen after the first immunisation.

Biochim Biophys Acta, 1982 Feb 2, 714(2), 337 - 43
Plasma membrane-associated component(s) that confer(s) cholera toxin sensitivity to adenylate cyclase; Pinkett MO et al.; Incubation of crude normal rat kidney membranes with activated cholera toxin in the presence of DTP, ATP and NAD results in a 10--20 fold stimulation of adenylate cyclase activity . Optimal choleragen activation f the cyclase is shown to be dependent upon the presence of a plasma membrane-associated reconstituting activity, which can be dissociated from the membranes by washing with 10 mM potassium phosphate buffer, pH 7.5, containing 5 mM EDTA and 0.1 mM dithiothreitol . choleragen-catalyzed ADP ribosylation of plasma membrane substrate proteins also requires the presence of reconstituting activity factor . Sephadex G-150 filtration of solubilized reconstituting activity shows a peak activity eluting in the region corresponding to a protein with a molecular weight of approx . 13,000 . Reconstituting activity is eluted from DEAE-cellulose at a salt concentration of 40--100 mM KCl . This active factor is not destroyed by trypsin treatment or by boiling for 30 min . These observations indicate that an endogenous membrane-associated factor, along with GTP, may be involved in modulating the ability of choleragen to activate adenylate cyclase.

J Trop Med Hyg, 1982 Feb, 85(1), 27 - 9
Efficacy of short course antibiotic prophylaxis in controlling cholera in contacts during epidemic; Khan MU; During an epidemic of cholera, vaccination has limited applicability in controlling its spread . It has been seen that one out of every five to 10 V . cholerae-infected people develops diarrhoea severe enough to require hospital treatment . Most health authorities are concerned with this severely ill group in whom the majority of deaths occur . During the cholera epidemic of 1975 in Dacca two doses of tetracycline were administered to all family contacts of index cases . The control group of cholera cases did not receive the drug . The families were re-visited after 10-12 days and history of any diarrhoea and hospitalization was obtained . It was found that the subsequent diarrhoea or cholera cases occurring among the cholera contacts within 10-12 days were not different between the treated (13.5%) and the untreated (14.4%) groups . The occurrence of severe cases requiring hospitalization was, however, significantly reduced in the treated group (8.0% to 4.5%) . In view of the emergence of V . cholera strains resistant to tetracycline, antibiotic sensitivity testing of epidemic strains would be needed before use of tetracycline for protecting cholera contacts as an immediate control measure.

Eur J Biochem, 1982 Feb, 122(2), 333 - 7
Conformational changes in subunit A of cholera toxin following the binding of ganglioside to subunit B; van Heyningen SU; 1 . Cholera toxin has been labelled with the fluorescent probe 4-chloro-7-nitrobenzofuran (Nbf-Cl) in both subunits, and the labelled subunits separated by gel-permeation . They retained their biological activities . 2 . Addition of ganglioside GM1 (which binds to subunit B only) to either labelled subunit did not alter the fluorescence of the Nbf probe . 3 . Whole toxin was reconstituted using labelled subunit A and unlabelled subunit B . Addition of ganglioside GM1 to the reconstituted toxin enhanced the fluorescence by about 90%, but did not change the wavelength . This enhancement reached a maximum when the ganglioside to toxin ratio was about 1 to 1 . Ganglioside GD1b (which does not bind) did not affect the fluorescence . 4 . These results suggest that the binding of ganglioside to subunit B alters the environment of the Nbf probe bound to subunit A, presumably by a conformational change.

In Vitro, 1982 Feb, 18(2), 87 - 91
Modified nutrient medium MCDB 151, defined growth factors, cholera toxin, pituitary factors, and horse serum support epithelial cell and suppress fibroblast proliferation in primary cultures of rat ventral prostate cells; McKeehan WL et al.; Nutrient medium WAJC 401 containing 5 micrograms/ml insulin . 10 ng/ml EGF, 10 ng/ml cholera toxin, 50 micrograms/ml pituitary extract, 1 microgram/ml prolactin, 1 microM dexamethasone, and 5% horse serum supports the rapid proliferation of rat ventral prostate epithelial cells in primary culture . The same medium suppresses the growth of prostate fibroblasts.

Proc Natl Acad Sci U S A, 1982 Feb, 79(3), 850 - 4
Cyclic AMP-mediated control of meiosis: effects of progesterone, cholera toxin, and membrane-active drugs in Xenopus laevis oocytes; Schorderet-Slatkine S et al.; Progesterone depressed rapidly (50% at 1 min) and persistently cyclic AMP (cAMP) concentration that had been elevated by cholera toxin in Xenopus laevis oocytes . cAMP remained below 1 pmol per oocyte (mean basal level) for approximately 1 hr and thereafter rose to approximately 120% of control values, while germinal vesicle (nucleus) breakdown did not occur . In the absence of cholera toxin, progesterone treatment for 6 hr maintained cAMP concentration below the basal level (but not lower than 80%), and germinal vesicle breakdown occurred . Experiments in the presence of phosphodiesterase inhibitors suggested that progesterone modulates adenylate cyclase activity . The maturation promoting factor, which is formed after 3-5 hr of progesterone treatment and provokes germinal vesicle breakdown after its injection into untreated oocytes, also decreased cAMP concentration, an observation that may explain its "autoamplification." Nonsteroidal inducers of meiosis reinitiation (e.g., propranolol, methoxyverapamil, mersalyl) diminished the cholera toxin-mediated accumulation of cAMP, in contrast to compounds devoid of meiotic-inducing capacity and antagonist to progesterone action, such as gammexane (an inositol analogue) and 5'-deoxy-S-(2-methylpropyl)-5'-thioadenosine (a methylase inhibitor), that increased the nucleotide level . The fine control, suggested by the effects of small changes in cAMP levels, gives evidence of great sensitivity to a critical determinant governing meiotic cell division.

Naunyn Schmiedebergs Arch Pharmacol, 1982 Feb, 318(3), 181 - 4
Alpha 2-adrenoceptors inhibit the cholera-toxin-induced intestinal fluid accumulation; Nakaki T et al.; The effects of adrenoceptor agonists and antagonists on the cholera-toxin-induced intestinal fluid accumulation and the mucosal levels of cAMP were investigated in vivo . Cholera toxin produced a marked fluid accumulation . Adrenaline inhibited the effect of the toxin in a dose-dependent manner . An alpha 2-adrenoceptor blocking agent yohimbine antagonized the effect of adrenaline . The alpha 1-adrenoceptor blocking agents prazosin and phenoxybenzamine failed to antagonize the effect of adrenaline . A high dose of a beta-adrenoceptor blocking agent pindolol did not antagonize the effect of adrenaline . Yohimbine or pindolol alone did not produce any effects on the toxin-induced fluid accumulation . However, prazosin and phenoxybenzamine per se inhibited the toxin-induced fluid accumulation . An alpha 2-selective agonist clonidine was slightly more potent than adrenaline, and was about 100-fold more potent than the alpha 1-selective agonist methoxamine in inhibiting the cholera-toxin-induced intestinal secretion . Clonidine, adrenaline and methoxamine failed to reduce the mucosal levels of cAMP, while these alpha-adrenoceptor agonists inhibited the toxin-induced fluid accumulation in the same preparations . These results suggest that the stimulation of alpha 2-adrenoceptors inhibit the cholera-toxin-induced intestinal secretion without reducing the whole mucosal levels of cAMP.

J Biol Chem, 1982 Jan 10, 257(1), 20 - 3
Requirements for cholera toxin-dependent ADP-ribosylation of the purified regulatory component of adenylate cyclase; Schleifer LS et al.; The requirements for cholera toxin-catalyzed ADP ribosylation of the purified regulatory component of adenylate cyclase are described . In addition to the toxin, this reaction is dependent on or is facilitated by NAD, GTP, phospholipid, and a factor found associated with plasma membranes from several sources . Factor activity is heat-labile and protease-sensitive but is unaffected by treatment with N-ethylmaleimide . Gel filtration indicates that the factor behaves as a monodisperse species with a Stokes radius of 3.2 nm . The factor thus appears to be a protein that is distinct from any of the known components of adenylate cyclase . Factor activity was also detected in the cytoplasm of S49 cells . The cytoplasmic factor was smaller (Stokes radius = 2.0 nm) than the membrane-derived factor, and it was inactivated in the presence of sodium cholate . The initial rate of activation of the regulatory component of adenylate cyclase by toxin was found to be linearly related to the amount of factor present in the reaction . This has allowed the quantitation and partial purification (33-fold from detergent extracts) of the factor from turkey erythrocyte membranes.

Brain Res, 1982 Jan 7, 231(1), 33 - 50
Horseradish peroxidase (HRP) conjugates of cholera toxin and lectins are more sensitive retrogradely transported markers than free HRP; Trojanowski JQ et al.; Horseradish peroxidase (HRP) conjugates of 8 different lectins (wheat germ agglutinin, ricinus communis I and II, peanut agglutinin, lens culinaris, soybean agglutinin, limulus polyhemus, ulex europaeus I) and cholera toxin (CT) or free HRP (FHRP) were individually injected into the submandibular gland (SMG) or anterior chamber (AC) of the eye and the retrogradely labeled neurons in the superior cervical ganglion (SCG) were quantitated . The effect of using 3 different cross-linking reagents (glutaraldehyde, p-benzoquinone and periodic acid) on the results obtained with HRP conjugates of wheat germ agglutinin (WG) was also examined . The results in 100 rats demonstrated the superior sensitivity of ligand-HRP conjugates over FHRP as retrogradely transported markers . After SMG injections, HRP conjugates of CT, WG and soybean agglutinin were 20-50 times more sensitive than FHRP . After AC injections, HRP conjugates of CT and WG consistently yielded labeled SCG neurons while FHRP failed to do so even when the amount of FHRP injected was increased 10-fold . The sensitivity of HRP conjugates of WG was similar after SMG injections using each of the 3 cross-linking reagents, but AC injections of conjugates produced with p-benzoquinone yielded twice as many labeled SCG neurons as the other WG conjugates . Mixtures of conjugates with and without FHRP were no more sensitive than the most sensitive individual ligand-HRP conjugates, except after SMG injections of a conjugate mixture with FHRP . Additional experiments demonstrated the specificity of the ligand-"receptor" interaction of WG and CT and that the superior sensitivity of these ligand-HRP conjugates does not depend on the tissue destruction produced by the injection procedure.

Graefes Arch Clin Exp Ophthalmol, 1982, 219(6), 272 - 8
Fine structural studies of ciliary processes after treatment with cholera toxin or its B subunit; Mishima H et al.; Delivery of 2 micrograms of cholera toxin (CT), a specific, irreversible activator of adenyl cyclase, via the blood causes dilation of capillaries and stromal edema of the ciliary processes . These morphologic changes occur within 3 h, are maximal at 12 to 24 h, then gradually return to normal by 72 h . In the late phase of hypotony, ultrastructural changes in the ciliary epithelia, similar to Greeff vesicles, are due to a "paracentesis effect" from hypotony, caused by decreased aqueous flow through the eye . Delivery of 2 micrograms of the B subunit of CT (Sub-B) causes very mild capillary dilation and stromal edema of ciliary processes . These changes reach their peak at 3 h, then return to normal at 24 h . No significant damage occurred to the pigmented or non-pigmented epithelium with either agent . No hemorrhage, invasion of inflammatory cells or appearance of fibrin exudates in the ciliary processes could be detected.

Trans R Soc Trop Med Hyg, 1982, 76(3), 373 - 7
Role of water and sanitation in the incidence of cholera in refugee camps; Khan MU et al.; The purpose of this study was to determine the prevalence of cholera in two groups: (i) people using covered latrine and piped water; (ii) people using uncovered surface latrine and pond and tubewell water . The study population consisted of cholera cases admitted to the ICDDR, B hospital from three refugee camps . In the one camp with sanitation facilities, the cholera rate was 1.6 per 1,000, whereas in the two camps without facilities the rates were 4.0 and 4.3 per 1,000 . Following demolition of the camps, the cholera rates decreased significantly in the camps geographical zones . Cholera was not totally eliminated, even in the one camp with sanitation facilities, suggesting that health education, as well as proper sanitation, is necessary to eradicate cholera.

Int Arch Allergy Appl Immunol, 1982, 68(3), 242 - 6
Effects of parenteral keyhole limpet hemocyanin or cholera toxin on intestinal immune response to keyhole limpet hemocyanin; Hamilton SR et al.; This study evaluated whether subcutaneous priming with either keyhole limpet hemocyanin (KLH), a protein antigen lacking toxic properties, or cholera toxin (CT), whose toxic activity is known to modulate immune responses, would enhance or suppress the local intestinal IgA response to KLH . In rabbits given KLH into chronically isolated ileal loops, subcutaneous priming and boosting with the same antigen resulted in increased serum and loop fluid IgG anti-KLH, but loop fluid IgA anti-KLH was not statistically significantly different from controls . With subcutaneous administration of CT, loop fluid IgA anti-KLH and serum IgG anti-KLH showed a suggestion of an earlier rise than in controls, but were not significantly different . The failure of subcutaneous KLH or CT to enhance local intestinal IgA immune response to KLH indicated that the feasibility of parenteral priming must be determined individually for each antigen to which intestinal immunity is desired.

Cell Tissue Res, 1982, 223(2), 241 - 53
Ultracytochemistry of cholera-toxin binding sites in ciliary processes; Mishima H et al.; Cholera toxin reduces the rate of aqueous humor in concentrations (10-11M) that do not disturb the morphology of the aqueous-humor forming epithelial cells of the ciliary processes of the rabbit eye . The search for an endogenous mediator of aqueous-humor formation comparable to cholera toxin in its mode of operation prompted us to map the distribution of cell surface receptors for cholera toxin in the ciliary processes of the eyes of rabbits . Cytochemical studies were carried out with the use of conjugates of cholera toxin to fluorescein isothiocyanate (CT-FITC) and to horseradish peroxidase (CT-HRP), and of the B subunit of cholera toxin to horseradish peroxidase (B-HRP) . Multiple fluorescent CT-FITC binding sites were observed on the outer nonpigmented epithelial layer near the crests of the processes . Processes incubated with CT-HRP in vitro showed surface staining of 30-40% of the nonpigmented epithelial cells . A prominent reaction product was observed along the basal and lateral plasma membranes of these cells . In vivo studies carried out after arterial infusion of B-HRP showed a reproducible dense reaction product between the apical surfaces of the pigmented epithelium (PE) and of the nonpigmented epithelium (NPE) facing each other . Aggregations of reaction product were observed with the electron microscope in the extracellular space between the apices of PE and NPE . The apical plasma membrane of the endothelium of the blood vessels near the crests of the ciliary processes was stained after either in vivo or in vitro exposure to peroxidase conjugates . These findings indicate that the cell-surface receptors which mediate the action of cholera toxin on aqueous humor formation are very likely localized in the apical plasma membranes of the epithelium of the ciliary processes.

Am J Physiol, 1982 Jan, 242(1), G47 - 51
Migrating action-potential complex activity in absence of fluid production is produced by B subunit of cholera enterotoxin; Sinar DR et al.; The myoelectric response and fluid output from in vivo rabbit ileal loops injected with B subunits of purified cholera enterotoxin are compared with the response to the purified cholera holotoxin . Migrating action-potential complex (MAPC) frequency is similar after injection of purified cholera toxin or B subunits . In contrast, fluid output after B-subunit injection is significantly (P less than 0.05) less than fluid output after purified cholera toxin injection . Specific antiserum to the holotoxin incubated with holotoxin at equivalence significantly decreased both MAPC activity (P less than 0.05) and fluid output (P less than 0.001) from the purified toxin . Purified cholera toxin and B subunits, modified by reaction with 2-nitrophenylsulfonyl chloride to produce monomeric B fractions with decreased GM1 ganglioside-binding properties, produced significantly (P less than 0.05) less fluid output and MAPC activity . Binding of the toxin or B subunits in the aggregated form is essential for the expression of MAPC activity or fluid output . These results suggest that the B subunit of cholera toxin produces MAPC activity in the rabbit ileum in the absence of fluid production . Furthermore, previous assumptions that MAPC activity is linked to fluid secretion should be reconsidered . It appears that the gut responses to cholera toxin, fluid production, or MAPC activity are produced by separate mechanisms.

Zh Mikrobiol Epidemiol Immunobiol, 1982, (11), 29 - 33
{Biochemical and immunochemical characteristics of a new oral, chemical cholera bivalent vaccine and results of a trial of the preparation on volunteers}; Dzhaparidze MN et al.; Oral cholera vaccine contains 45% of O-antigen (serovars Ogawa and Inaba in equal parts) and at least 9 serologically active proteins; of these, toxoid (about 60% of the total amount of protein) and 5 enzymes have been identified: neuraminidase, proteinase, ribonuclease, phospholipase and ATPase . The safety, absence of reactogenicity and definite immunological effectiveness of the preparation in the primary immunization of volunteers have been shown.

J Cell Biochem, 1982, 20(4), 359 - 67
Cholera toxin B-subunit protects mammalian cells from ricin and abrin toxicity; Delfini C et al.; The glycoproteins ricin and abrin intoxicate cells by inhibiting protein synthesis . Pretreatment of HeLa cells with cholera toxin partially protects them from ricin and abrin activity . The involvement in this phenomenon of the various effects of cholera toxin, namely, redistribution of membrane receptors elicited from protomer B and increasing cyclic AMP concentrations induced by protomer A, were studied . Substances able to enhance cyclic AMP concentrations do not affect ricin and abrin activity, while protomer B alone protects cells . In addition, the effects of several lectins on ricin or abrin toxicity were examined . Almost complete prevention of ricin or abrin activity was obtained using concanavalin A (Con A) and wheat germ agglutinin (WGA) . Conversely, neither succinyl Con A nor Ulex europeus agglutinin (UEA) affected the cellular response . Both protomer B of cholera toxin and Con A did not alter the binding of ricin or abrin; they seem to protect cells by altering membrane structure.

J Cyclic Nucleotide Res, 1982, 8(4), 267 - 75
Dopamine and bromocriptine inhibit cyclic AMP accumulation in the anterior pituitary: the effect of cholera toxin; Cronin MJ et al.; Although dopamine clearly inhibits prolactin release from the anterior pituitary gland, the biochemical events occurring subsequent to the activation of the dopamine receptor are poorly understood . We have shown that dopamine reduces the concentration of cyclic AMP within 5 min in primary cultures of rat anterior pituitary cells . This action could be blocked with pretreatment of the cells with the dopamine antagonist spiperone . The long-lasting dopamine agonist bromocriptine also reduced cyclic AMP accumulation in parallel with the inhibitory effect of this drug on prolactin release . Cholera toxin enhanced prolactin release and cyclic AMP content of the anterior pituitary cells, but did not totally annul the efficacy of dopamine or bromocriptine to inhibit prolactin secretion and cyclic AMP accumulation.

Digestion, 1982, 24(3), 176 - 82
Streptozotocin treatment in pancreatic cholera (Verner-Morrison) syndrome; Pignal F et al.; A case of pancreatic cholera (Verner-Morrison syndrome) associated with a pancreatic endocrine tumor and hepatic metastases is presented . VIP and HPP plasma levels, initially elevated, were accurately followed in various conditions: during corticosteroid therapy, after pancreatic tumor excision, during and after streptozotocin therapy (1.5 g/m2) by repeated intraarterial route) . Only streptozotocin therapy resulted in a reduction of the stool volume with concomitant decrease in VIP plasma levels . However, the size of the hepatic metastases was unchanged and HPP plasma levels remained elevated . It is suggested that VIP represents the tumoral secretion and HPP a marker of the residual malignant tissue.

J Membr Biol, 1982, 64(3), 225 - 31
Properties of rat erythrocyte membrane cytoskeletal structures produced by digitonin extraction: digitonin-insoluble beta-adrenergic receptor, adenylate cyclase, and cholera toxin substrate; LeVine H 3rd et al.; Rat erythrocyte plasma membranes have been extracted exhaustively with digitonin at low temperature, and the residual, detergent-extracted membrane cytoskeletal material is compared to that prepared with Triton X-100 with respect to protein, glycoprotein, phospholipid, and cholesterol content . Digitonin, a weaker detergent than Triton X-100, solubilizes only 26% of the phospholipids and none of the cholesterol . SDS-polyacrylamide gel electrophoresis reveals that differences between the proteins extracted by the two detergents are primarily quantitative . In terms of functional preservation, digitonin retains in the cytoskeleton 28% of the beta-adrenergic receptor binding activity (with the balance accounted for in the supernatant), greater than 90% of the adenylate cyclase and greater than 90% of the 45,000 mol wt polypeptide cholera toxin substrate . The cytoskeletal-associated beat-adrenergic receptor retains binding properties for antagonist and agonist which are identical to those of the native membrane receptor . The digitonin-extracted cytoskeleton containing the beta-adrenergic receptor may provide a useful vehicle for the reconstitution of a hormone-sensitive adenylate cyclase.

Connect Tissue Res, 1982, 10(2), 173 - 86
Activation of human synovial cells by cholera enterotoxin: correlation of morphological responses with adenylate cyclase activities, and the reversing effects of hyaluronidase; Clarris BJ et al.; Previously described morphological changes in human synovial cell cultures due to cholera enterotoxin (CT) were studied in relation to activation of adenylate cyclase . A single pulse of CT at nanomolar concentration or less induced at least two-fold activation of adenylate cyclase, which persisted for 7 days or more . The enzyme hyaluronidase was found to cause a rapid reversal of the morphological effects of CT . There was also a reduction in adenylate cyclase activity but only with hyaluronidase concentrations greater than those required to produce maximum reversal of the CT-induced morphological changes . Removal of hyaluronidase was followed by reappearance of the CT-associated morphological effects and a slower reactivation of adenylate cyclase . The mechanism by which hyaluronidase produces the observed changes in synovial cells is not known, but might be related to the dispersal of hyaluronic acid gels bound to the surface of these cells.

Comp Biochem Physiol C, 1982, 73(2), 383 - 8
Cholera toxin: mitogen for Harderian glands of golden hamsters; Hoffman RA et al.; 1 . Within 2 days, a single injection of 10 micrograms cholera toxin induces a 6-20-fold increase in mitotic activity in the Harderian glands of male and female golden hamsters . 2 . Neither hypophysectomy nor ovariectomy had any influence on this response . 3 . The cellular proliferation does not appear to involve cAMP nor is it the result of stress, nor a release from an early mitotic block . 4 . Nuclear polyploidy increases within a few hours after treatment . 5 . DNA density per unit area increases within 24 hr and is maintained for at least 3 days.

J Immunol, 1981 Dec, 127(6), 2461 - 4
Cellular dissemination of priming for a mucosal immune response to cholera toxin in rats; Pierce NF et al.; Using CT as the test antigen, we sought 1) to learn whether primary immunization at 1 mucosal site caused priming of distant nonstimulated mucosae, 2) to study the role of migrating memory cells in the dissemination of mucosal priming, and 3) to compare disseminated priming with priming that occurs at the site of initial immunization . CT given i.c . or i.d . caused priming in tracheal and nonexposed enteric mucosae; i.t . immunization, however, did not cause detectable enteric priming . Adoptive transfer of immune TDLs showed that priming was conveyed by migrating memory cells . These appeared to be of 2 types: those that recirculated briefly before settling in MALT, and those that continued to recirculate until recruited by antigen to the site of mucosal challenge . Both types were required for secondary responses at mucosae distant from the site of priming . The time-course of disseminated mucosal priming resembled that of priming at the site of initial CT exposure, both lasting at least 16 wk . Disseminated priming persisted better in jejunal than tracheal mucosa, suggesting that the subgroup of memory cells that did not continue to recirculate settled preferentially in jejunal MALT . Disseminated priming supported smaller challenge responses than priming at the site of initial CT exposure did, suggesting that sessile memory cells also contributed to the latter process . These observations extend the concept of a "common mucosal immune system" to include cellular dissemination of mucosal priming, but also show quantitative differences between local and disseminated priming that probably reflect the patterns of distribution of migrating and sessile memory cells.

Arch Fr Pediatr, 1981 Dec, 38 Suppl 1, 823 - 8
{Intestinal response to glucose in experimental cholera in malnourished, adult and growing rats (author's transl)}}; Mansour AB et al.; We examined the influence of malnutrition on intestinal response to glucose in experimental cholera . Water, electrolytes, and glucose fluxes were directly measured, in isolated ligated loops of rat jejunum . Intestinal loops were filled with Ringer added with either mannitol (30 mM), glucose (30 mM), cholera toxin (TC), or a combination of these three components . The results are as follows: 1 . Malnutrition has an effect on basal absorption rate, intestinal response to TC and also to glucose . 2 . The secretory effect of cholera toxin is altered by the type of malnutrition, the animal's age and the presence of cholera toxin . 3-0 methyl-glucose, a non-metabolised glucose analogue, has essentially the effect of glucose . The effects produced on water fluxes are also found for Na, K, Cl, HCO3 and glucose fluxes . The relationships between water, glucose, and Na fluxes are not influenced by the different experimental conditions . These results indicate that the intestinal response to glucose in experimental cholera is strongly dependent on the type of malnutrition, the animal's age and the presence of cholera toxin.

C R Seances Acad Sci III, 1981 Nov 16, 293(10), 563 - 6
{Ultrastructural visualization of binding and internalization of cholera and tetanuts toxins}; Montesano R et al.; In monolayer cultures of a liver line, a population of non-coated membrane microinvaginations is preferentially involved in both the initial binding and subsequent internalization of colloidal gold-labelled cholera and tetanus toxins . These two toxins thus appear to be internalized via another pathway than most other polypeptide ligands studied, which enter cells via coated plasma membrane invaginations.

J Biol Chem, 1981 Nov 10, 256(21), 11177 - 81
Lipid insertion of cholera toxin after binding to GM1-containing liposomes; Tomasi M et al.; The technique of hydrophobic photolabeling with photoreactive lipids was used to study the topology of interaction of cholera toxin with liposomes containing galactosyl-N-acetylgalactosaminyl-{N-acetyl neuraminyl}-galactosyl glucosyl ceramide (GM1) . The toxin appears to locate itself superficially on the lipid bilayer . The interaction is mediated only by the gamma beta 5 part . On reduction of the disulfide bridge joining the alpha and gamma subunits, the alpha subunit penetrates deeply into the lipid bilayer . The mere binding of cholera toxin to GM1 is not sufficient to allow the insertion of the enzymatically active alpha subunit in the membrane . Some processing, which may involve a modification of covalent bonds of the toxin molecule (such as that caused by reduction) appears to be necessary . The specific reduction of the alpha-gamma disulfide bond on the external surface of the membrane as a prerequisite for the membrane penetration of the alpha subunit is discussed.

Biochim Biophys Acta, 1981 Nov 5, 677(3-4), 408 - 16
Mn2+-uncoupling of the catecholamine-sensitive adenylate cyclase system of rat reticulocytes . Parallel effects on cholera toxin-catalyzed ADP-ribosylation of the system; Limbird LE et al.; High concentrations of Mn2+ interfere with functional interactions between the GTP-binding regulatory protein (G) and the catalytic moiety (C) of adenylate cyclase without perturbing interactions between receptor (R) and component G in rat reticulocyte membranes . The ability of cholera toxin to ADP-ribosylate component G and to enhance GTP-stimulated adenylate cyclase activity also appears to be correlated with the efficacy of the communication of component G with the adenylate cyclase system . Thus, increasing the concentration of Mn2+ in rat reticulocyte membrane during in vitro incubations causes a parallel loss of Gpp(NH)p-stimulated adenylate cyclase activity, cholera toxin-catalyzed {32P}ADP-ribosylation of the 42 000 Mr subunit of component G and cholera toxin-catalyzed enhancement of GTP-sensitive adenylate cyclase activity . Removal of Mn2+ by washing the membranes completely restores the sensitivity of adenylate cyclase activity . Removal of Mn2+ by washing the membranes completely restores the sensitivity of adenylate cyclase to all effectors, including cholera toxin . The data suggest that exposure of membranes to Mn2+ provides a useful tool for reversibly uncoupling catecholamine-sensitive adenylate cyclase systems . The data also suggest that the extent of cholera toxin-catalyzed ADP.-ribosylation of membrane substrates, i.e., the G component may rely on functional communication among the various components of the adenylate cyclase system . A corollary of the latter is that the amount of {32P}ADP-ribose-product detected in a membrane may reflect both the quantity and coupling efficiency of component G.

Brain Res, 1981 Nov 2, 223(2), 381 - 5
Conjugates of horseradish peroxidase (HRP) with cholera toxin and wheat germ agglutinin are superior to free HRP as orthogradely transported markers; Trojanowski JQ et al.; The relative sensitivity of horseradish peroxidase (HRP) conjugates of cholera toxin (CTHRP) and wheat germ agglutinin (WGHRP) as orthogradely transported markers was compared with that of free HRP (FHRP) following unilateral, intravitreal injections of each probe in the rat . Both CTHRP and WGHRP labeled all of the recognized retinal efferents of the rat and were 30-50 times more sensitive than FHRP . In additional experiments the 'receptors' specificity of CTHRP and WGHRP was also demonstrated.

Biochem J, 1981 Nov 1, 199(2), 457 - 60
The hydrophobicities of cholera toxin, tetanus toxin and their components; Ward WH et al.; 1 . Charge-shift electrophoresis showed that cholera toxin and its subunits have no hydrophobic surfaces . 2 . Amino-acid composition and sequence data suggested that the proteins have no masked hydrophobic regions . 3 . The A subunit of cholera toxin may interact with polar molecules in the membrane to exert its effect inside the cell . 4 . The only hydrophobic part of tetanus toxin was the H-chain.

J Cell Biol, 1981 Nov, 91(2 Pt 1), 410 - 3
Cholera toxin can catalyze ADP-ribosylation of cytoskeletal proteins; Kaslow HR et al.; Cholera toxin catalyzes transfer of radiolabel from {32P}NAD+ to several peptides in particulate preparations of human foreskin fibroblasts . Resolution of these peptides by two-dimensional gel electrophoresis allowed identification of two peptides of Mr = 42,000 and 52,000 as peptide subunits of a regulatory component of adenylate cyclase . The radiolabeling of another group of peptides (Mr = 50,000 to 65,000) suggested that cholera toxin could catalyze ADP-ribosylation of cytoskeletal proteins . This suggestion was confirmed by showing that incubation with cholera toxin and {32P}NAD+ caused radiolabeling of purified microtubule and intermediate filament proteins.

Infect Immun, 1981 Nov, 34(2), 341 - 6
In vitro formation of hybrid toxins between subunits of Escherichia coli heat-labile enterotoxin and those of cholera enterotoxin; Takeda Y et al.; Heat-labile enterotoxin (LT) was purified from cells of enterotoxigenic Escherichia coli isolated from a patient with traveller's diarrhea . Purified LT was separated into A and B subunits by treatment with 6 M urea solution in 0.1 M propionic acid (pH 4.0) . Biologically active toxin was reconstituted from isolated A and B subunits of LT . Hybrid toxins with biological activity were obtained in vitro from the A subunit of cholera enterotoxin and B subunit of LT, and from the A subunit of LT and B subunit of cholera enterotoxin . The hybrid toxins show a similar toxicity to that of the parent toxins from which the A subunits were derived . The in vitro formations of the hybrid toxins were confirmed by polyacrylamide gel disk electrophoresis.

Gut, 1981 Nov, 22(11), 958 - 63
Release of vasoactive intestinal polypeptide from the cat small intestine exposed to cholera toxin; Cassuto J et al.; During a four hour observation period vasoactive intestinal polypeptide (VIP) is released in increasing amounts from the feline small intestine exposed to cholera toxin . As VIP is known to be located almost exclusively in the intestinal nerves, the present findings strongly suggest that cholera toxin activates the enteric nervous system . The findings of this and other studies performed in this laboratory lead to the proposal that the choleraic secretion is, at least in part, secondary to the activation of intramural nervous reflexes in the gut.

Gut, 1981 Nov, 22(11), 953 - 7
Effect of cholera toxin on ileal water and solute transport after resection of the proximal small intestine in the rat; Townsend WF et al.; Intestinal adaptation after extensive small bowel resection results in mucosal hypertrophy and an increased capacity of the remaining small intestine to absorb solutes and water . We tested the ability of the adapted rat ileum to respond to a secretory stimulus, cholera toxin . Six weeks after 50% jejunal resection (short gut) or sham operation water and solute transport were measured in a 16 cm segment of ileum before and after exposure to cholera toxin in a single pass in vivo perfusion system . During the control periods absorption of glucose, acetate and water per unit length of intestine was significantly greater in short gut animals (P less than 0.05 to 0.001) . After exposure to cholera toxin absorption of glucose and acetate was significantly reduced in both groups (P less than 0.05 to 0.01) . Sodium and chloride secretion and net change in water movement in response to cholera toxin were significantly greater (P less than 0.05 to 0.01) in short gut animals . Generally the differences between short gut and sham operation animals disappeared when the data were normalised for mucosal weight . Chloride secretion per gram mucosa was less in short gut animals (P less than 0.001) . The data indicate that the adapted small bowel is not only capable of enhanced absorption but also of enhanced net secretion in response to cholera toxin . The changes reflect the increased number of enterocytes per unit length of intestine after intestinal adaptation.

Zh Mikrobiol Epidemiol Immunobiol, 1981 Oct, (10), 101 - 4
{Production of an erythrocytic ganglioside diagnostic reagent for detecting cholera enterotoxin and toxoid in indirect hemagglutination tests}; Dolmatova LA et al.; The studies described in this work have indicated that cholera enterotoxin and its components (cholerogenoid and subunit B) can be detected in amounts of 0.25, 0.28 and 0.6 microgram of protein per 1 ml, respectively, by means of erythrocytes sensitized with gangliozide-containing complex . The conditions for erythrocyte sensitization have been established . The methods of cholerogen titration in the passive hemagglutination test by means of the erythrocytic gangliozide diagnostic reagent and in Craig's skin test have been shown to correlate . Similarly, the passive hemagglutination test is supposed to be suitable for detecting other bacterial toxins interacting with gangliozides.

Biull Eksp Biol Med, 1981 Oct, 91(10), 432 - 4
{Sodium 2,3-dithiopropanesulfate blockade of the effect of cholera enterotoxin on adenylate cyclase and the concentration of cyclic 3',5'-adenosine monophosphate in the small intestine mucosa of the rabbit}; Iurkiv VA et al.; Activation of adenylate cyclase in membrane preparations of the rabbit small intestine mucosa by cholera enterotoxin in the presence of sodium 2,3-dithiopropanesulfate (DTPS) is similar to that in the presence of dithiothreitol (DTT) . Both DTT and DTPS do not influence the activity of adenylate cyclase without cholera enterotoxin . DTPS activates cyclic 3,5-AMP phosphodiesterase of mucosa homogenates (K0.5 = 10(-5) M) . Combined administration of cholera enterotoxin and DTPS in situ into isolated loops of the rabbit small intestine decreases the activating effect of cholera enterotoxin on adenylate cyclase and diminishes the content of cyclic AMP in the mucosa . The destruction of disulfide bonds of cholera enterotoxin by DTPS is assumed to lower its ability to penetrate the mucosal cells of the small intestine.

Lab Invest, 1981 Oct, 45(4), 372 - 7
Effect of cholera toxin on secretion of mucin by explants of guinea pig trachea; Adler KB et al.; Cholera toxin (CT) elicits a dosage-dependent increase in mucin secretion by explants of guinea pig trachea . Concomitantly, the mucin in goblet cells of the mucosa and submucosal glands is depleted . This effect is realized in the absence of cell injury, as assessed morphologically and by the assay of culture medium for the release of acid phosphatase . Mucosal concentrations of cyclic AMP increase after exposure to CT . However, the stimulatory effects on secretion appear to be independent of the cyclic nucleotide, as exogenous dibutyryl cyclic AMP and cyclic GMP fail to increase secretion, and theophylline, a phosphodiesterase inhibitor, also is ineffective . The stimulatory effect of CT is decreased by preincubation of the explants with inhibitors of microtubules (nocodazole) and microfilaments (cytochalasin D) in a dosage-dependent manner . Addition of the calcium chelator, ethylene glycol bis(beta-aminoethyl ether)-N,N'-tetraacetic acid, with CT also inhibits the secretory response . CT appears to stimulate mucin secretion by tracheal epithelial cells by a mechanism independent of cyclic nucleotide activation but requires intact microtubules, microfilaments, and exogenous calcium ions.

Cancer Res, 1981 Oct, 41(10), 4075 - 9
Differential sensitivity of normal and chemically transformed epithelial cells to cholera toxin; Niles RM et al.; We have been studying the regulation of growth by cyclic adenosine 3':5'-monophosphate (cyclic AMP) and other factors in untransformed (K16) and chemically transformed (W8) rat liver epithelial cells . Initially, we found that 8-bromocyclic adenosine 3':5'-monophosphate was a more potent inhibitor of cell replication in K16 than in W8 cells . In addition, the phosphodiesterase inhibitor 1-methyl-3-isobutylxanthine (MIX) caused marked growth inhibition in K16 but not in W8 cells . Through the use of cholera toxin (CT) with or without MIX, we elevated intracellular cyclic AMP levels in a quantifiable fashion . With CT alone or combined with MIX, we observed a dose-dependent morphological change in W8 cells, which consisted of extensive "process" formation . K16 morphology was not altered at any concentration of CT +/- MIX tested . K16 cell growth was only marginally inhibited by CT alone, but markedly inhibited by CT plus MIX . W8 cell growth was moderately inhibited by CT alone or combined with MIX . Analysis of cyclic AMP levels revealed that, at all concentrations of CT +/- MIX and at all time periods tested, W8 cells produced significantly more cyclic AMP than K16 cells . It appears that morphological changes and growth inhibition are not necessarily linked and that MIX may inhibit K16 cell replication by means other than its ability to increase intracellular cyclic AMP levels.

Int J Gynaecol Obstet, 1981 Oct, 19(5), 403 - 7
The significance of cholera outbreak in the prognosis of pregnancy; Ayangade O; Five hundred sixty-one persons were treated in a comprehensive cholera unit during the 1979-1980 cholera outbreak at Ile-Ife . Sixty-one pregnant cholera patients were identified and followed up . Compared to the general female population, all female cholera patients in the 15-29 year age group show significantly more resistance to the disease than those aged 30 years and above . The pregnant cases, as well as all reproductive-years age groups, showed significantly less mortality than both the non-pregnant patients and those at both extremes of age . Our findings show that pregnancy does not render the woman more susceptible and may, in fact, render her less susceptible after the first trimester, when prognosis brightens for both the mother and the fetus.

J Clin Lab Immunol, 1981 Sep, 6(2), 185 - 92
Effects of cholera toxin on the lymphoid system II . Selective augmentation of delayed footpad reaction in mice; Tsuru S et al.; Effects on immune responses to sheep erythrocytes (SRBC) were examined at various intervals after an intravenous injection of 1 microgram of cholera toxin in DDD and AKR mice . Delayed footpad reaction was augmented by pretreatment with cholera toxin 1, 7 or 10 days before immunization in both strains . The delayed reaction was not suppressed even in the presence of a prominent antibody production . In mice given cholera toxin, macrophage migration inhibition was not positive 5 days after immunization, but became weakly positive 10 days after immunization . Antibody production against SRBC especially of IgG class, was facilitated, when cholera toxin was given one day before immunization, on the other hand, antibody production was suppressed irrespective of immunizing routes and mouse strains . Similar results were observed in guinea pigs which were injected with 1 microgram of cholera toxin 0 or 7 days before immunization with bovine gamma globulin in complete or incomplete Freund's adjuvant . Erythema was augmented strikingly by cholera toxin, while macrophage migration inhibition was affected scarcely.

J Clin Lab Immunol, 1981 Sep, 6(2), 181 - 3
Effects of cholera toxin on lymphoid system . I . Reduction of thymus cells after cholera toxin injection; Tsuru S et al.; One microgram of cholera toxin was injected intravenously into DDD and AKR mice and its effects on lymphoid tissues were examined at various intervals . A remarkable reduction of thymus cells were revealed from days 1 to 11 and from days 1 to 7 in DDD and AKR mice, respectively . Only slight effects were detected on the numbers of spleen cells and peripheral blood leukocytes in both strains throughout the observation period.

Am J Physiol, 1981 Sep, 241(3), G248 - 52
Reversal of cholera toxin-induced secretion in rat ileum by luminal berberine; Swabb EA et al.; The effect of luminal berberine hydrochloride concentration on cholera toxin-induced water and electrolyte secretion and on normal water and electrolyte transport was determined in vivo in the cannulated, perfused rat ileum using {14C}polyethylene glycol as a nonabsorbable marker . Berberine reduced the cholera toxin-induced secretion of water, Na, Cl, and calculated residual ion (primarily HCO3) in a concentration-dependent manner . The effect of berberine on cholera toxin-induced ileal secretion was evident 60-80 min after exposure and was reversed 60-80 min after removal of berberine from the perfusate . Mild changes in mucosal histology (villous tip edema) due to cholera toxin were also reversed by berberine . Berberine did not significantly alter normal ileal water and electrolyte transport.

S Afr Med J, 1981 Jul 18, 60(3), 87 - 90
The spread of cholera in South Africa; Kustner HG et al.; The current cholera epidemic in the RSA began in October 1980 and is part of the seventh pandemic . Initial investigation of the epidemic revealed a virtually closed system of water supply, which explained the distribution of the early cases . The spread of cholera in the RSA is examined and local factors contributing to cholera transmission are discussed . Attempts are being made to prevent cholera from becoming endemic in the RSA and long-term improvements in health facilities in the susceptible areas of the country are being undertaken.

J Biol Chem, 1981 Jul 10, 256(13), 6573 - 6
Cycloheximide potentiation of prostaglandin E1- and cholera toxin-stimulated cyclic AMP accumulation in NG108-CC15 neuroblastoma-glioma hybrid cells; Moylan RD et al.; Previous studies have demonstrated that catecholamine responsiveness in a variety of cells can be altered by inhibitors of RNA and protein synthesis . The neuroblastoma-glioma hybrid, NG108-CC15, which lacks catecholamine-stimulated accumulation of cyclic AMP, was investigated to determine if the responsiveness to prostaglandin E1 (PGE1) could be modified by inhibitors of protein synthesis . Cycloheximide in a time-dependent manner potentiated the ability of prostaglandin E1 to stimulate accumulation of intracellular cyclic AMP . However, the alpha-adrenergic inhibition of the prostaglandin response was not affected by cycloheximide . Withdrawal of norepinephrine following a long-term incubation resulted in a potentiation of subsequent PGE1-stimulated cyclic AMP accumulation . Cycloheximide enhanced this norepinephrine withdrawal effect . Our previous studies have shown that cholera toxin induces refractoriness to beta-adrenergic agonists in C6-2B rat astrocytoma cells and that cycloheximide blocked this action of cholera toxin . In an analogous manner cholera toxin caused refractoriness to subsequent prostaglandin-stimulated cyclic AMP production in NG108-CC15 cells, and cycloheximide reduced cholera toxin-induced prostaglandin refractoriness . Thus cycloheximide potentiates the prostaglandin stimulatory effect, has no effect on the ability of alpha-agonists to inhibit the prostaglandin response, increases the stimulatory effect of PGE1 after norepinephrine withdrawal, and reduces cholera toxin-induced PGE1 refractoriness . these observations suggest that PGE1-stimulated cyclic AMP accumulation in NG108-CC15 cells contains components which are regulated by de novo protein synthesis.

Brain Res, 1981 Jul, 227(4), 539 - 49
Cholera-toxin binding to cells of developing chick retina analyzed by fluorescence-activated cell sorting; Rathjen FG et al.; The occurrence of gangliosides on nerve cells of the developing retina was studied by fluorescence-activated cell analysis and sorting, using fluorescent cholera toxin as marker . This toxin binds to GM1; neuraminidase converts several other gangliosides into GM1 . Without pretreatment by this enzyme weak binding of toxin is detected at later stages of development, whereas pretreatment leads to considerable toxin binding at earlier stages . The number of cells binding toxin as well as the amount bound per cell increase with developmental age of the retina . Cells binding a given amount of toxin vary strongly in size . Cell sorting was used to separate postmitotic cells from proliferating cells . Proliferating cells have little binding capacity, while postmitotic cells bind relatively large amounts of toxin . Localization of gangliosides which bind toxin in the developing retina was studied in cryostat sections . At an early stage (day 6) toxin binding is localized in the inner layer of the developing retina which contains the ganglion and other postmitotic cells, but is not found in the outer layer of matrix cells . At later stages complex staining patterns evolve with binding predominantly in the nerve fiber layers.

Biull Eksp Biol Med, 1981 Jul, 92(7), 17 - 20
{Spin probe study of the effect of cholera toxin on enterocyte brush border membranes}; Iurkiv VA et al.; The physical state of the enterocyte brush border membranes during stimulation of cholera diarrhea was examined by electron paramagnetic resonance with the use of spin probes localized in different areas of the lipid bilayer and the spin label covalently bound with protein SH-groups . In vivo experiments have disclosed a slight increase in bilayer fluidity during cholera intoxication . In experiments in vitro the binding of cholera toxin with the enterocyte brush border membranes on addition of 3',5'-AMP (up to 5 X 10(-5) M) did not affect the physical state of the lipid of protein areas of the membrane.

Brain Res, 1981 Jul, 227(4), 622 - 7
Differential effects of cyclic AMP and cholera toxin on nerve growth factor-induced neurite outgrowth from adrenal medullary chromaffin and pheochromocytoma cells; Ziegler W et al.; The extension of neurites from adrenal medullary chromaffin cells and PC 12 cells upon addition of nerve growth factor (NGF) has been proposed to be mediated by cyclic AMP . It is shown here that substances increasing intracellular cyclic AMP levels have a reverse effect on NGF-induced neurite outgrowth of these two related cell types . Hence, cyclic AMP is not generally involved in neurite outgrowth from NGF responsive cells . Furthermore, it is concluded that PC 12 cells cannot always be considered as a suitable model for adrenal medullary chromaffin cells.

Biochim Biophys Acta, 1981 Jun 11, 675(1), 46 - 61
Distinct effects of the C-terminal octapeptide of cholecystokinin and of a cholera toxin pretreatment of the kinetics of rat pancreatic adenylate cyclase activity; Svoboda M et al.; (1) The kinetic parameters of rat pancreatic adenylate cyclase were evaluated, using GTP, p{NH}ppG or GTP gamma S as nucleotide activator, cholecystokinin as peptide hormone, and GDP beta S and dibutyryl cyclic GMP as inhibitors of guanosine triphosphate and CCK-8, respectively . The time courses of activation and the degree of activation at steady state (EA/ETOT) were compatible with a simple two-state model of activation-deactivation based on a pseudo-monomolecular activation process (rate constant kappa+1), and a deactivation process (rate constant kappa off) that included, depending on the activating nucleotide, the hydrolysis of GTP (rate constant kappa 2) and/or the dissociation of the intact nucleotide (rate constant kappa-1), so that EA/ETOT = kappa+1/(kappa+1 + kappa 2 + kappa-1) . (2) The hormone CCK-8 increased the value of kappa+1 with GTP dose-dependently, from 0.2 to 10.9 min-1 . The value of kappa-1 increased 0.01 to 0.3 min-1 but the value of kappa 2 was unaltered at 7 min-1, so that EA/ETOT increased 15-fold, from 4% to 61% . (3) A cholera toxin pretreatment at 30 micrograms/ml allowed also a large increase in EA/ETOT with GTP (up to 51%) but the underlying mechanism was different . It consisted of a 14-fold decrease in the kappa off value of the GTP-activated enzyme (from 7 min-1 to 0.5 min-1) that corresponded to a reduction in GTPase activity . When testing the system with p{NH}ppG, two added effects of the cholera toxin pretreatment were observed: a 4-fold increase in the value of kappa+1 (from 0.2 to 0.8 min-1) and the occurrence of a significant 0.3 min-1 value for kappa-1.

J Biol Chem, 1981 Jun 10, 256(11), 5481 - 8
Structure-function studies of cholera toxin and its A and B protomers . Modification of tryptophan residues; De Wolf MJ et al.; The tryptophan residues on cholera toxin and its A and B protomers have been modified by reaction with 2-nitrophenylsulfenyl chloride and 2,4-dinitrophenylsulfenyl chloride . Modification of the tryptophan residues of cholera toxin results in complete loss of toxicity measured in a skin permeability assay . Modification of cholera toxin and its B protomer results in the complete loss of binding activity toward membrane receptors, the ganglioside galactosyl-N-acetylgalactosaminyl-{N-acetylneuraminyl}-galactosylceramide (GM1), and the oligosaccharide moiety of the ganglioside GM1 . Modification of cholera toxin and its A protomer results in a complete loss of the ADP-ribosylation activity exhibited by their native counterparts . Modification of the A protomer results in no apparent change in its physical properties by sedimentation velocity in the ultracentrifuge or by gel filtration chromatography . Modification of the B protomer, either directly or when it remains a component part of the holo toxin structure, results in a change in its sedimentation value and its elution from gel filtration columns . The changes are compatible with a conversion of the B protomer from a pentameric moiety in aqueous solvents to its existence as a monomer unit, i.e . to the individual polypeptide chains comprising the native B pentamer . Thiolysis of the 2,4-dinitrophenylsulfenyl chloride derivative of the B protomer reaggregates the individual-polypeptide chains but does not return its ability to interact with GM1.

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