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 ng PGE₂ equivalents /ml of diarrhoeal stool. Paralled 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.     

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.     

Part II: diffraction from two-dimensional cholera toxin crystals bound to their receptors in a lipid monolayer

The structure of cholera toxin (CTAB₅) bound to its putative ganglioside receptor, galactosyl-N-acetylgalactosaminyl (N-acetyl-neuraminyl) galactosylglucosylceramide (GM₁), in a lipid monolayer at the air-water interface has been studied utilizing grazing incidence x-ray diffraction. Cholera toxin is one of very few proteins to be crystallized in two dimensions and characterized in a fully hydrated state. The observed grazing incidence x-ray diffraction Bragg peaks indicated cholera toxin was ordered in a hexagonal lattice and the order extended 600-800 Å. The pentameric binding portion of cholera toxin (CTB₅) improved in-plane ordering over the full toxin (CTAB₅) especially at low pH. Disulfide bond reduction (activation of the full toxin) also increased the protein layer ordering. These findings are consistent with A-subunit flexibility and motion, which cause packing inefficiencies and greater disorder of the protein layer. Corroborative out-of-plane diffraction (Bragg rod) analysis indicated that the scattering units in the cholera layer with CTAB₅ shortened after disulfide bond reduction of the A subunit. These studies, together with Part I results, revealed key changes in the structure of the cholera toxin-lipid system under different pH conditions.     

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.     

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.     

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.     

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.     

Upregulation of PG synthesis on sulfur-starvation for PS I in Chlamydomonas

Sulfur(S)-starvation was previously shown to induce the degradation of an acidic lipid in chloroplasts, sulfoquinovosyl diacylglycerol (SQDG), to yield a major internal S-source in a green alga, Chlamydomonas reinhardtii. We here found that the synthesis of phosphatidylglycerol (PG), the other acidic lipid in chloroplasts, is activated to elevate its content up to a level that just compensates for the loss of SQDG. Similar activation of PG synthesis was also observed in an SQDG-deficient mutant under S-replete conditions, which led us to propose that upregulation of PG synthesis under S-starved conditions occurs through direct sensing of SQDG-loss, but not of S-starvation. Moreover, thylakoid membranes isolated from S-starved cells were reduced in photosystem I activity on treatment with phospholipase A₂ that specifically broke down PG, which suggested a critical role of PG that is increased under S-starved conditions in the maintenance of the photosystem I activity.     

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.     

Fluorescently labeled liposomes for monitoring cholera toxin binding to epithelial cells

Vibrio cholerae, the causative agent for cholera, expresses a toxin required for virulence consisting of two subunits: the pentameric cholera toxin B (CTB) and cholera toxin A (CTA). CTB is frequently used as an indicator of the presence of pathogenic V. cholerae and binds to the G_M1 ganglioside on the surface of epithelial cells. To study V. cholerae virulence (CTB expression) in the presence of human epithelia, we devised an inexpensive, simple, and rapid method for quantifying CTB bound on epithelial surfaces in microtiter plates. G_M1 ganglioside was incorporated into the lipid bilayer of liposomes both encapsulating the fluorescent dye sulforhodamine B (SRB) and with SRB tagged to lipids in the bilayer (BEGs). In addition, G_M1-embedded liposomes encapsulating SRB only (EGs) and with SRB in their bilayers only (BGs) were synthesized. The three types of liposomes were compared with respect to their efficacy for both visualizing and quantifying CTB attached to the surface of Caco-2 cells. The BEGs were the most effective overall, providing both visualization under a fluorescence microscope and quantification after lysis in a microtiter plate reader. A limit of detection corresponding to 0.28 μg/ml applied CTB was attained for the on-cell assay using the microtiter plate reader approach, whereas as low as 2 μg/ml applied CTB could be observed under the fluorescence microscope.     

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.     

The sites on the regulatory component of adenylate cyclase which are ADP-ribosylated by cholera toxin

In rat liver membranes cholera toxin ADP-ribosylated two polypeptides (M_r 42000 and 47000) in the regulatory component of adenylate cyclase. L-arginine methyl ester specifically inhibited both the activation of adenylate cyclase and ADP-ribosylation by cholera toxin, suggesting that cholera toxin modified arginine, or arginine-like, residues. A hydrolysis-resistant analogue of GTP (β, γ-imidoguanosine 5′-triphosphate, p(NH)ppG) bound to the regulatory protein in an essentially irreversible manner. Pretreatment with the analogue failed to inhibit the labelling of polypeptides by cholera toxin showing that the sites for ADP-ribosylation were different from those at which guanyl nucleotides were bound.     

Coordinate regulation of adenylate cyclase, protein kinase, and specific enzyme synthesis by cholera toxin in hormonally unresponsive hepatoma cells

Since none of the hormones which activate adenylate cyclase in other tissues have been found to activate adenylate cyclase or to induce tyrosine aminotransferase in cultured Reuber hepatoma cells (H35), despite the stimulatory effects of cyclic AMP derivatives on the latter enzyme, we tested the ability of cholera toxin to influence these processes. At low concentrations cholera toxin was found to mimic the ability of cyclic AMP derivatives to selectively stimulate the synthesis of the aminotransferase. Adenylate cyclase and protein kinase activity were also enhanced, but only after a lag period as in other systems. Specific phosphorylation of endogenous H₁ histone was also shown to be increased by cholera toxin treatment. The increase in tyrosine aminotransferase activity is due to an increase in de novo synthesis as shown by radiolabeling experiments utilizing specific immunoprecipitation. The activity of another soluble enzyme induced by dibutyryl cyclic AMP, PEP carboxykinase, was also stimulated by exposure of H35 cells to cholera toxin. Combinations of cholera toxin and dexamethasone led to greater than additive increases in the activity of both the aminotransferase and carboxykinase. Close coupling of cyclic AMP production with protein kinase activation and enzyme induction was suggested by the observation that the ED₅₀ values for the stimulation of adenylate cyclase, cyclic AMP production, protein kinase, and tyrosine aminotransferase activities were found to be the same (5–7 ng/ml) within experimental error. The results indicate that the adenylate cyclase system in H35 cells is functionally responsive and they support the suggestion that activation of protein kinase is functionally linked to induction of specific enzymes.     

Analysis of transmembrane dynamics of cholera toxin using photoreactive probes

Using sodium dodecyl sulfate--polyacrylamide gel electrophoresis and autoradiography, we have shown that ¹²⁵I-labeled cholera toxin binds to Newcastle disease virus. Pretreatment of Newcastle disease virus with “cold” cholera toxin (at 37°C for 30 minutes) inhibits the binding of ¹²⁵I-labeled toxin in a subsequent incubation (at 37°C for 30 minutes). These results suggest that cholera toxin binds to Newcastle disease virus in a specific manner. The precise receptor for toxin is unknown in Newcastle disease virus but it is presumed to be the ganglioside GM₁. We have previously shown that the photoreactive probe 12-(4-azido-2-nitrophenoxy)stearoylgucosamine[1-¹⁴C] labels the membrane proteins of Newcastle disease virus. Since the reactive group of the probe, ie, N₃, resides within the membrane bilayer, studies were initiated to determine which, if any, of the subunits of cholera toxin cross the membrane of Newcastle disease virus and become radioactively labeled upon photoactivation of the probe at 360 nm. After a 15-minute incubation of cholera toxin with Newcastle disease virus containing the photoreactive probe, irradiation effected the ¹⁴C-labeling of the active A₁ subunit of cholera toxin. Irradiation of cholera toxin in solution with an equivalent amount of probe but without virus resulted in no labeling of toxin subunits.     

Gravimetric antigen detection utilizing antibody-modified lipid bilayers

Lipid bilayers containing 5% nitrilotriacetic acid (NTA) lipids supported on SiO₂ have been used as a template for immobilization of oligohistidine-tagged single-chained antibody fragments (scFvs) directed against cholera toxin. It was demonstrated that histidine-tagged scFvs could be equally efficiently coupled to an NTA-Ni²⁺-containing lipid bilayer from a purified sample as from an expression supernatant, thereby providing a coupling method that eliminates time-consuming protein prepurification steps. Irrespective of whether the coupling was made from the unpurified or purified antibody preparation, the template proved to be efficient for antigen (cholera toxin) detection, verified using quartz crystal microbalance with dissipation monitoring. In addition, via a secondary amplification step using lipid vesicles containing G_M1 (the natural membrane receptor for cholera toxin), the detection limit of cholera toxin was less than 750 pM. To further strengthen the coupling of scFvs to the lipid bilayer, scFvs containing two histidine tags, instead of just one tag, were also evaluated. The increased coupling strength provided via the bivalent anchoring significantly reduced scFv displacement in complex solutions containing large amounts of histidine-containing proteins, verified via cholera toxin detection in serum.     

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)     

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。     

Low Temperature Development Induces a Specific Decrease in trans-Delta-Hexadecenoic Acid Content which Influences LHCII Organization

Lipid and fatty acid analyses were performed on whole leaf extracts and isolated thylakoids from winter rye (Secale cereale L. cv Puma) grown at 5°C cold-hardened rye (RH) and 20°C nonhardened rye (RNH). Although no significant change in total lipid content was observed, growth at low, cold-hardening temperature resulted in a specific 67% (thylakoids) to 74% (whole leaves) decrease in the trans-Δ³-hexadecenoic acid (trans-16:1) level associated with phosphatidyldiacylglycerol (PG). Electron spin resonance and differential scanning calorimetry (DSC) indicated no significant difference in the fluidity of RH and RNH thylakoids. Separation of chlorophyll-protein complexes by sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that the ratio of oligomeric light harvesting complex:monomeric light harvesting complex (LHCII₁:LHCII₃) was 2-fold higher in RNH than RH thylakoids. The ratio of CP1a:CP1 was also 1.5-fold higher in RNH than RH thylakoids. Analyses of winter rye grown at 20, 15, 10, and 5°C indicated that both, the trans-16:1 acid levels in PG and the LHCII₁:LHCII₃ decreased concomitantly with a decrease in growth temperature. Above 40°C, differential scanning calorimetry of RNH thylakoids indicated the presence of five major endotherms (47, 60, 67, 73, and 86°C). Although the general features of the temperature transitions observed above 40°C in RH thylakoids were similar to those observed for RNH thylakoids, the transitions at 60 and 73°C were resolved as inflections only and RH thylakoids exhibited transitions at 45 and 84°C which were 2°C lower than those observed in RNH thylakoids. Since polypeptide and lipid compositions of RH and RNH thylakoids were very similar, we suggest that these differences reflect alterations in thylakoid membrane organization. Specifically, it is suggested that low developmental temperature modulates LHCII organization such that oligomeric LHCII predominates in RNH thylakoids whereas a monomeric or an intermediate form of LHCII predominates in RH thylakoids. Furthermore, we conclude that low developmental temperature modulates LHCII organization by specifically altering the fatty composition of thylakoid PG.     

Calcium transport affinity,ion competition and cholera toxin effects on cytosolic Ca concentration

Summary The physiological relevance of an apparent ionophore activity of cholera toxin towards Ca²⁺ has been examined in several different systems designed to measure affinity, specificity, rates of ion transfer, and effects on intracellular ion concentrations. Half-maximal transfer rates across porcine jejunal brush-border vesicles were obtained at a concentration of 0.20 M Ca²⁺. When examined in the presence of competing ions the transfer process was blocked by very low concentrations of La³⁺ or Cd²⁺. Sr²⁺, Ba²⁺ and Mg²⁺ were relatively inefficient competitors for Ca²⁺ transport mediated by cholera toxin. The relative affinities observed would be compatible with a selectivity for Ca²⁺ transfer at physiological ion concentrations, as well as an inhibition of this ionophore activity by recognized antagonists of cholera toxin such as lanthanum ions. Entry rates of Ca²⁺ into brush-border vesicles exposed to cholera toxin were large enough to accelerate the collapse of a Ca²⁺ gradient generated by endogenous Ca, Mg-ATPase activity. The treatment of isolated jejunal enterocytes with cholera toxin caused a significant elevation in cytosolic Ca²⁺ concentrations as measured by Quin-2 fluorescence. This effect was specifically prevented by prior exposure of the cholera toxin to excess ganglioside G_M1. We conclude that cholera toxin has many of the properties required for promoting transmembranes Ca²⁺ movement in membrane vesicles and appears to be an effective Ca²⁺ ionophore in isolated mammalian cells.     

Global dynamics of cholera models with differential infectivity

A general compartmental model for cholera is formulated that incorporates two pathways of transmission, namely direct and indirect via contaminated water. Non-linear incidence, multiple stages of infection and multiple states of the pathogen are included, thus the model includes and extends cholera models in the literature. The model is analyzed by determining a basic reproduction number R₀ and proving, by using Lyapunov functions and a graph-theoretic result based on Kirchhoff’s Matrix Tree Theorem, that it determines a sharp threshold. If R₀?1, then cholera dies out; whereas if R₀>1, then the disease tends to a unique endemic equilibrium. When input and death are neglected, the model is used to determine a final size equation or inequality, and simulations illustrate how assumptions on cholera transmission affect the final size of an epidemic.