Prostaglandins and cholera: the occurrence of prostagland in-like smooth muscle contracting substances in cholera diarrhoea.

Prostaglandin-like smooth muscle contracting substances were identified in acid lipid extracts of diarrhoeal faeces taken from patients presenting clinical symptoms of cholera. Five out of the eleven cases studied were bacteriologically confirmed as cholera. The PG-like activity in the individual samples ranged from 3-27 mg PGE2 equivalents /ml of diarrhoeal stool. Parallel bioassay and thin-layer chromatographic analysis of the extracts indicated that about 60% of the activity was due to an F-type PG, 15% to an E-type PG and the rest to unidentified lipid substanc(s). It is proposed that increased PG production in cholera (indicated by these findings) may at least contribute to the disease.     

Prostaglandins and cholera: The occurrence of prostaglandin-like smooth muscle contracting substances in cholera diarrhoea

Prostaglandin-like smooth muscle contracting substances were identified in acid lipid extracts of diarrhoeal faeces taken from patients presenting clinical symptoms of cholera. Five out of the eleven cases studied were bacteriologically confirmed as cholera. The PG-like activity in the individual samples ranged from 3 – 27 ng PGE₂ equivalents /ml of diarrhoeal stool. Parallel bioassay and thin-layer chromatographic analysis of the extracts indicated that about 60% of the activity was due to an F-type PG, 15% to an E-type PG and the rest to unidentified lipid substance(s). It is proposed that increased PG production in cholera (indicated by these findings) may at least contribute to the disease.     

Vibrio cholerae: cholera toxin

The bacterial protein toxin of Vibrio cholerae, cholera toxin, is a major agent involved in severe diarrhoeal disease. Cholera toxin is a member of the AB toxin family and is composed of a catalytically active heterodimeric A-subunit linked with a homopentameric B-subunit. Upon binding to its receptor, GM0(1), cholera toxin is internalized and transported in a retrograde manner through the Golgi to the ER, where it is retrotranslocated to the cytosol. Here, cholera toxin reaches its intracellular target, the basolaterally located adenylate cyclase which becomes constitutively activated after toxin-induced mono-ADP-ribosylation of the regulating G(S)-protein. Elevated intracellular cAMP levels provoke loss of water and electrolytes which is manifested as the typical diarrhoea. The cholera toxin B-subunit displays the capacity to fortify immune responses to certain antigens, to act as a carrier and to be competent in inducing immunological tolerance. These unique features make cholera toxin a promising tool for immunologists.     

Properties of cholera toxin- and NaF-stimulated adenylate cyclase from mouse thymocytes.

Kinetic parameters of mouse thymocyte adenylate cyclase activity were determined. NaF and cholera toxin stimulated adenylate cyclase. Stimulation by either agent did not change the pH or Mg2+ optima relative to control (unstimulated cyclase). The Km value for ATP of adenylate cyclase stimulated by NaF was significantly reduced from control. By contrast, cholera toxin treatment did not change the Km relative to control. Adenylate cyclase, when stimulated by NaF, had an optimum for Mn2+ alone, or Mn2+ in combination with Mg2+, at least twice that of control. In contrast, cyclase activity prepared from cells treated with cholera toxin remained unchanged with regard to these divalent cations when compared to control. Addition of NaF to adenylate cyclase prepared from cells treated with cholera toxin resulted in a significant reduction (30%) in activity suggesting that both NaF and cholera toxin were acting on the same cyclase. NaF inhibition of cholera toxin-stimulated activity was shown to be a direct interaction of fluoride on the stimulated cyclase enzyme. This inhibition appeared to be immediate and independent on pH, Mg2+ or ATP concentrations. Although NaF inhibition was lost when Mn2+ was present in the reaction mixture, the activity expressed by addition of NaF to cyclase prepared from cholera toxin-treated cells was much less than by addition of NaF to control. As observed with cholera toxin stimulation alone, activity expressed by the inhibited enzyme (cholera toxin treated + NaF) exhibited a Km for ATP and an optimum for Mn2+ alone or in combination with Mg2+ similar to control.     

Purification of a protein cofactor required for ADP-ribosylation of the stimulatory regulatory component of adenylate cyclase by cholera toxin

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.     

Inhibition of cyclic AMP-mediated intestinal hypersecretion by pituitary extracts from rats pretreated with cholera toxin

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.     

Biosynthesis and metabolism of prostaglandins in the small intestine of rats exposed to cholera enterotoxin

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.     

The effects of cholera toxin, pertussis toxin, sodium fluoride and alpha-interferon on prostaglandin production by the guinea-pig endometrium

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

Effect of cholera toxin on triacylglycerol lipase activity and triacylglycerol content of rat heart

This study was performed to reexamine the effect of cholera toxin on total and intracellular alkaline lipoprotein lipase (LPL) activity in rat heart. In addition, the relationship between intracellular triacylglycerol (TG)lipase activity and TG content of cardiac tissue was determined in cholera toxin treated rats. One intravenous injection of cholera toxin increased total LPL activity significantly above control activity 4 h following treatment. After 16 h, total enzyme activity in hearts of cholera toxin treated rats was 2.4-fold above control levels and remained significantly above the control activity up to the 24-h time point. Intracellular alkaline TG lipase activity was increased 24%, 59%, 2.1-fold, and 2.1-fold above control levels measured 0.5, 8, 16, and 24 h following cholera toxin treatment, respectively. Heart TG content fell significantly following cholera toxin treatment, with a maximal reduction seen 8 h following agent injection. At that time, TG was 0.61 mumol/g, a reduction of 63% below the control concentration of 1.8 mumol/g. A negative relationship between myocardial intracellular TG lipase activity and TG concentration of r = -0.83 was highly significant (P less than 0.001). These findings indicate that cholera toxin injection can increase total cardiac LPL activity and show that 70% of this increased activity is in the intracellular fraction. The highly significant relationship between enzyme activity and TG content support our working hypothesis that the intracellular TG lipase (LPL) is playing a role in regulating cardiac TG content.     

O1群霍乱Ogawa血清型结合疫苗免疫学效果研究

霍乱是经粪口传播的烈性传染病,相应的疫苗研究已逾百年,但目前还没有理想的疫苗。实验中以霍乱O1群小川血清型的脂多糖为目标抗原,用不同方法制备了其四种霍乱结合疫苗,通过小鼠模型验证了各结合物的免疫学效果。结果显示,不同结合物免疫学效果不一,其中增大多糖分子量后制备的结合物免疫效果较好,氨还原法制备的结合物多针免疫后也可诱导特异性抗体产生,而且具有针对小川和稻叶两种血清型的杀弧菌活性。     

Indomethacin inhibits cholera toxin-induced cyclic AMP accumulation in Chinese hamster ovary cells

Abstract Indomethacin was examined for its capacity to inhibit increases in adenosine-3',5'-monophosphate (cAMP) concentrations in Chinese hamster ovary (CHO) cells treated with cholera toxin. When added to the culture medium 1 h prior to cholera toxin (100 ng/ml), indomethacin (500 μg/ml) exhibited maximum protection against the typical increase in cAMP. Application of indomethacin at the same time as cholera toxin or up to 3 h after the toxin progressively decreased the drug's capacity to block further increases in cAMP. The drug appeared to block adenylate cyclase activity because addition of forskolin to drug-treated cells did not elicit a cAMP response. Binding of ¹²⁵I-labeled cholera toxin to indomethacin-treated cells was also reduced by at least 50%. These data indicate that indomethacin's inhibitory effect on cAMP formation in cholera toxin-treated cells could be explained by its capacity to alter adenylate cyclase activity and cholera toxin binding.     

Expression of cholera toxin B subunit oligomers in transgenic potato plants

A gene encoding the cholera toxin B subunit protein (CTB), fused to an endoplasmic reticulum (ER) retention signal (SEKDEL) was inserted adjacent to the bi-directional mannopine synthase P2 promoter in a plant expression vector containing a bacterial luciferase AB fusion gene (luxF) linked to the P1 promoter. Potato leaf explants were transformed by Agrobacterium tumefaciens carrying the vector and kanamycin-resistant plants were regenerated. The CTB-SEKDEL fusion gene was identified in the genomic DNA of bioluminescent plants by polymerase chain reaction amplification. Immunoblot analysis indicated that plant-derived CTB protein was antigenically indistinguishable from bacterial CTB protein, and that oligomeric CTB molecules (Mr 50 kDa) were the dominant molecular species isolated from transgenic potato leaf and tuber tissues. Similar to bacterial CTB, plant-synthesized CTB dissociated into monomers (Mr 15 kDa) during heat or acid treatment. The maximum amount of CTB protein detected in auxin-induced transgenic potato leaf and tuber tissues was approximately 0.3% of total soluble plant protein. Enzyme-linked immunosorbent assay methods indicated that plant-synthesized CTB protein bound specifically to GM1-ganglioside, the natural membrane receptor of cholera toxin. In the presence of the SEKDEL signal, CTB protein accumulates in potato tissues and is assembled into an oligomeric form that retains native biochemical and immunological properties. The expression of oligomeric CTB protein with immunological and biochemical properties identical to native CTB protein in edible plants opens the way for preparation of inexpensive food plant-based oral vaccines for protection against cholera and other pathogens in endemic areas throughout the world     

Characterization of the cholera toxin receptor on Balb/c 3T3 cells as a ganglioside similar to, or identical with, ganglioside GM1. No evidence for galactoproteins with receptor activity.

Balb/c 3T3 cells contain a large number [(0.8-1.6) x 10(6)] of high-affinity (half-maximal binding at 0.2 nM) binding sites for cholera toxin that are resistant to proteolysis, but are quantitatively extracted with chloroform/methanol. The following evidence rigorously establishes that the receptor is a ganglioside similar to, or identical with, ganglioside GM1 by the galactose oxidase/NaB3H4 technique on intact cells was inhibited by cholera toxin. (2) Ganglioside GM1 was specifically adsorbed from Nonidet P40 extracts of both surface- (galactose oxidase/NaB3H4 technique) and metabolically ([1-14C]palmitate) labelled cells in the presence of cholera toxin, anti-toxin and Staphylococcus aureus. (3) Ganglioside GM1 was the only ganglioside labelled when total cellular gangliosides separated on silica-gel sheets were overlayed with 125I-labelled cholera toxin, although GM3 and GD1a were the major gangliosides present. In contrast no evidence for a galactoprotein with receptor activity was obtained. Cholera toxin did not protect the terminal galactose residues of cell-surface glycoproteins from labelling by the galactose oxidase/NaB3H4 technique. No toxin-binding proteins could be identified in Nonidet P40 extracts of [35S]-methionine-labelled cells by immunochemical means. After sodium dodecyl sulphate/polyacrylamide-gel electrophoresis none of the major cellular galactoproteins identified by overlaying gels with 125I-labelled ricin were able to bind 125I-labelled cholera toxin. It is concluded that the cholera toxin receptor on Balb/c 3T3 cells is exclusively ganglioside GM1 (or a related species), and that cholera toxin can therefore be used to probe the function and organisation of gangliosides in these cells as previously outlined [Critchley, Ansell, Perkins, Dilks & Ingram (1979) J. Supramol. Struct. 12, 273-291].     

Antidiabetic potential of Citrus sinensis and Punica granatum peel extracts in alloxan treated male mice

An investigation on the effects of four different concentrations of peel extract from Citrus sinensis (CS) or Punica granatum (PG) in male mice revealed the maximum glucose lowering and antiperoxidative activities at 25 mg/kg of CS and 200 mg/kg of PG. In a separate experiment their potential was evaluated with respect to the regulation of alloxan induced diabetes mellitus. While a single dose of alloxan (120 mg/kg) increased the serum levels of glucose and alpha-amylase activity, rate of water consumption and lipid peroxidation (LPO) in hepatic, cardiac and renal tissues with a parallel decrease in serum insulin level, administration of 25 mg/kg of CS or 200 mg/kg of PG was found to normalize all the adverse changes induced by alloxan, revealing the antidiabetic and anti peroxidative potential of test fruit peel extracts. Subsequent phytochemical analysis indicated that the high content of total polyphenols in the test peels might be related to the antidiabetic and antiperoxidative effects of the test peels.     

Orientation of cholera toxin bound to model membranes.

The orientation of cholera toxin bound to its cell-surface receptor, ganglioside GM1, in a supporting lipid membrane was determined by electron microscopy of negatively stained toxin-lipid samples. Image analysis of two dimensional crystalline arrays has shown previously that the B-subunits of cholera toxin orient at the membrane surface as a pentameric ring with a central channel (Reed, R. A., J. Mattai, and G.G. Shipley. 1987. Biochemistry. 26:824-832; Ribi, H. O., D. S. Ludwig, K. L. Mercer, G. K. Schoolnik, and R. D. Kornberg. 1988. Science (Wash, DC). 239:1272-1276). We recorded images of negatively stained cholera toxin and isolated B-pentamers oriented perpendicular to the lipid surface so that the pentamer ring is viewed from the side. The pentamer dimensions, estimated from the average of 100 molecules, are approximately 60 by 30 A. Images of side views of whole cholera toxin clearly show density above the pentamer ring away from the lipid layer. On the basis of difference maps between averages of side views of whole toxin and B-pentamers, this density above the pentamer has been identified as a portion of the A-subunit. The A-subunit may also extend into the pore of the pentamer. In addition, Fab fragments from a monoclonal antibody to the A-subunit were mixed with the toxin prior to binding to GM1. Density from the Fab was localized to the region of toxin above the pentamer ring confirming the location of the A-subunit.(ABSTRACT TRUNCATED AT 250 WORDS)     

Actions of cholera toxin on dispersed Chief cells from guinea pig stomach

Although much is known about the actions of cholera toxin on intestinal and extra-gastrointestinal tissues, almost nothing is known about the interaction of this toxin with cells in the stomach. In the present study, we prepared 125I-labeled cholera toxin (1900 Ci/mmol) and examined the binding of this radioligand to dispersed Chief cells from guinea pig stomach. Moreover, we examined the actions of cholera toxin on cellular cAMP and pepsinogen secretion from Chief cells. Binding of 125I-labeled cholera toxin could be detected within 5 min, was maximal by 60 min, and was increased by increasing the radioligand or cell concentrations. Inhibition of binding by unlabeled toxin indicated a dissociation constant of 3 nM and 8.7 X 10(5) cholera toxin receptors per Chief cell. In contrast to the rapidity of binding, a cholera toxin-induced increase in cAMP and pepsinogen secretion was not detected until 30-45 min of incubation. A 3 to 6-fold increase in cAMP and pepsinogen secretion was observed with maximal concentrations of cholera toxin. Binding of 125I-labeled cholera toxin and the toxin's actions on cAMP and pepsinogen secretion were inhibited by the B subunit of the toxin. Binding was not altered by other agents that have been shown to stimulate pepsinogen secretion (carbachol, CCK-8, secretin, vasoactive intestinal peptide, prostaglandin E1, or forskolin). These data indicate that Chief cells from guinea pig stomach possess a specific class of cholera toxin receptors. Binding of cholera toxin to these receptors causes an increase in cellular cAMP that stimulates pepsinogen secretion.     

Human platelets are defective in processing of cholera toxin.

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.     

Epidemiology of cholera in travellers,and conclusions for vaccination recommendations.

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.     

Studies of the ligand binding to cholera toxin, I. The lipophilic moiety of sialoglycolipids.

The fixation of cholera toxin by ganglioside GGtet1 is dependent on the nature of the carbohydrate as well as the lipid moiety of the glycolipid. The role of the lipid in binding to the toxin investigated with synthetic ganglioside analogues (gangliosidoides). The interaction between glycolipid and toxin was followed by precipitate formation, by inhibition of toxicity and in polyacrylamide gel electrophoresis. For specific precipitation, an aliphatic hydrocarbon chain at least 14 C-atoms in length is required. Some of the gangliosidoides form high molecular weight complexes with cholera toxin at lower molar ratios of ligand to protein than the natural compound. None of the synthetic gangliosidoides equalled natural ganglioside in its ability to inhibit the effects of the toxin in vivo, but some did show considerable inhibitory activity ih monosialo-gangliotetraose or corresponding sialo-glycolipids prevents the easy degradation of the B-protein of cholera toxin into protein subunits by sodium dodecylsulfate.     

Lipoprotein lipases and stress hormones: studies with glucocorticoids and cholera toxin

The intravenous injection of cholera toxin in rats 17 h prior to experimentation results in increased levels of insulin and corticosterone in the blood. This is accompanied by a rise in lipoprotein lipase activity in muscle and a decrease in adipose tissue. Pre- and postheparin blood levels of the enzyme are increased, representing the higher overall muscle activity. Hepatic lipase is decreased by cholera toxin treatment. These enzyme changes are accompanied by increased levels of non-esterified fatty acids, ketone bodies and unesterified cholesterol in the blood, whereas triacylglycerol levels are lowered. The lipoprotein triacylglycerol secretion is not affected by cholera toxin, suggesting increased triacylglycerol removal from the blood. On the other hand the unesterified cholesterol removal may be decreased due to the decreased hepatic lipase activity. Administration of excess glucocorticoid 2 days prior to blood and tissue sampling also resulted in a rise in lipoprotein lipase, a decrease in hepatic lipase activity and an increase of non-esterified fatty acids. In contrast to the effect of cholera toxin, the triacylglycerol levels were increased. Adrenalectomy, whether by inhibition of 11-beta-steroid hydroxylase or by surgical intervention, did not abolish the choleratoxin effects. It is concluded that corticosterone increase is not essential to the cholera toxin effects. Corticosterone itself probably causes an increase of cyclic AMP and/or Ca2+ levels, as is discussed.