Role of neuraminidase from cholera vibrions in the pathogenesis of experimental cholera]

A study was made of a possible effect of neuraminidase of cholera vibrios on cholera pathogenesis. It was shown that in intraintestinal injection of cholera vibrios of the El Tor biotype to nursling rabbits neuraminidase could be revealed in their intestine 5 to 8 hours after the infection. Addition of neuraminidase to the weakly cholerogenic strain cholera vibrios intensified its cholerogenic action in infection of the animals. The antineuraminidase serum administered to the infected rabbits prevented clinical manifestations of experimental cholera, although it failed to always eliminate the cholerogenic syndrome (revealed during autopsy). At the same time neuraminidase did not influence the capacity of cholerogen to produce the cholerogenic syndrome. The authors consider that the action of the enzyme should occur at the early stages of the pathogenic process, and could be associated with creation of conditions for the attachement of cholera causative agent to the intestinal wall or for the action of their exotoxin.     

Toxins of Vibrio cholerae

Surveyed in the paper are published data on properties, biological activity, genetic determinants and action mechanisms of recently known toxins produced by different strains of Vibrio cholerae irrespectively of their capacity for the synthesis of choleric toxin--the main virulence factor. Their possible importance both for the general clinical pattern of cholera provoked by cholerogenic agents and as independent virulence factors causing diarrhea without cholera is elucidated. The sets and levels of expression of additional toxins can differ for different pathogenic clones and they can correspondingly condition degrees of their epidemic and etiological safety.     

Inhibiting effect of cholera vibrio neuraminidase in Rauscher mouse leukemia]

A study was made of the inhibitory effect of neuraminidase of cholera vibrio in mouse Rauscher leukemia. It was shown that the processing of cells of the spleen of mice suffering from leukemia with neuraminidase (in a dose of 50 units/ml and more) significantly inhibited the capacity of these cells to induce leukemia in their injection to BALB/c mice. In the mentioned concentration neuraminidase injected repeatedly parenterally produced no therapeutic effect in Rauscher leukemia.     

Hemagglutinating activity of vibrio cholerae enterotoxin

Abstract The hemagglutinating activity and carbohydrate specificity of cholera toxin (cholera enterotoxin) was studied using hemagglutination and hemagglutination inhibition. Hemagglutination was obtained with cholera toxin at >108 μg/ml for human types A, B, and O erythrocytes, >216 μg/ml for chicken erythrocytes, and >865 μg/ml for sheep erythrocytes. When the erythrocytes were treated with either neuraminidase or pronase, the hemagglutinating activity of cholera toxin was enhanced about 8- to 32-fold. Hemagglutination of pronase-treated human type B erythrocytes induced by cholera toxin was inhibited by lactose, galactose, melibiose and l -arabinose. Lactose was the most effective of the mono-, di-, and polysaccharides used as inhibitors, being a slightly better inhibitor than galactose, and much more potent than melibiose. These results suggest that cholera toxin is a bacterial lectin specific for galactose and/or lactose.     

Vibrio cholerae metalloproteinase degrades intestinal mucin and facilitates enterotoxin-induced secretion from rat intestine

Mucin secretion in situ from rat intestinal loops was promoted more effectively by dialysed crude cholera filtrate than by an equivalent amount of purified enterotoxin. The filtrate could be rendered inactive by incubation with mixed gangliosides or passage through a GM1-affinity column, which indicated that the secretory action of the filtrate depended upon its enterotoxin component. In an effort to explain the greater potency of the filtrate, we established the presence of a metalloproteinase in the filtrate and demonstrated that this enzyme was capable of degrading purified rat intestinal mucin. Sufficient degradation occurred to cause a substantial decrease in viscosity (57% in 120 min). Biochemical analysis of the mucin before and after exposure to filtrate revealed a rise in the combined percentage of serine, threonine and proline (53-58%), suggesting that poorly glycosylated areas (which are less abundant in these amino acids) were being partly removed from the mucin. The carbohydrate composition was essentially unaltered. Inhibition of the filtrate metalloproteinase by Zincov and alpha 2-macroglobulin significantly (P less than 0.005) reduced the ability of cholera filtrate to degrade mucin or to stimulate mucin secretion from rat intestinal slices in vitro. Purified cholera enterotoxin added to enterotoxin-depleted filtrate was a more potent secretagogue (secretory stimulant) in intestinal loops than an equivalent amount of enterotoxin alone. We therefore propose that mucin secretion induced by cholera filtrate is caused by cholera enterotoxin, but that degradation of the protective epithelial mucus layer by a constituent metalloproteinase may assist the toxin by allowing increased access to mucosal GM1 receptor sites.     

Inhibitory effect of combined administration of neuraminidase and interferon in mice with Rauscher leukemia

A study was made of the effect of cholera vibrio neuraminidase and mouse interferon on the development of Rauscher's leukemia in BALB/c mice. It was shown that administration of 1500 units interferon to mice 4 hours before and 5 days after inoculation of Rauscher's leukemia virus prolonged the animals' lifespan. Preliminary treatment of the virus-containing material with neuraminidase prevented the death of one third of the experimental animals. Meanwhile the remainder of the animals inoculated with the same virus showed a longer lifespan. Combined use of both the preparations proved the most effective, enabling the prevention from death of 90% of the experimental animals. The mechanism of antiviral action of neuraminidase and interferon is discussed.     

Cells Persistently Infected with Newcastle Disease Virus III. Thermal Stability of Hemagglutinin and Neuraminidase of a Mutant Isolated from Persistently Infected L Cells

Data were obtained which indicated the possible cause of the defective elution from erythrocytes of the mutant virus (NDV(pi)) isolated from L cells persistently infected with the Herts strain of Newcastle disease virus (NDV(o)). The chicken erythrocyte receptors for the mutant and wild-type viruses were equally sensitive to the action of Vibrio cholera filtrate neuraminidase; this suggests that the failure of NDV(pi) to elute from chicken erythrocytes is not due to a specific neuraminidase-resistant receptor for this virus on the erythrocyte membrane. There was no difference in the enzyme content of the intact virions of NDV(o) and NDV(pi) when tested with a soluble substrate, indicating that the inefficient elution of NDV(pi) was not due to a reduced enzyme content. The neuraminidase activity of intact NDV(pi) virions was significantly more stable at 55 C than the enzyme of NDV(o) virions, whereas the dissociated enzymes of the two viruses were inactivated at the same rate. On the basis of these findings, it seems likely there is a structural difference between the two viruses. The neuraminidase protein of the mutant NDV(pi) may be incorporated into the viral envelope in such a manner that it is prevented from reacting with the substrate in the erythrocyte membrane, although it can react with a soluble substrate. The hemagglutinin activity of both intact and disrupted NDV(pi) was significantly more resistant to thermal inactivation than that of the wild-type NDV(o). This finding suggests a genetic difference in the hemagglutinin protein of the two viruses.     

Stimulation of Neurite Outgrowth in Neuroblastoma Cells by Neuraminidase: Putative Role of GM1 Ganglioside in Differentiation

Treatment of three neuroblastoma cell types in culture with neuraminidase resulted in enhanced neurite outgrowth. These included the mouse Neuro-2A and rat B104 and B50 lines. The morphological changes depended on the presence of exogenous Ca2+ and were accompanied by modest but statistically significant increases in 45Ca2+ influx. Neuraminidase-stimulated neuritogenesis was blocked by the B subunit of cholera toxin (cholera B) and anti-GM1 antibody, a finding suggesting the effect was due to an increased amount of GM1 on the cell surface. Cholera B also blocked the increase in 45Ca2+ influx. The mouse N1A-103 line, previously characterized as "neurite minus," did not respond to neuraminidase with either neurite outgrowth or enhanced Ca2+ influx. These results point to an influence of GM1 on neuritogenesis in cells with differentiation potential and suggest a mechanism involving modulation of Ca2+ flux.     

A tissue culture color test for the titration of neutralizing antibodies against sendai virus (parainfluenza 1)

Summary A color test for the detection and titration of neutralizing antibodies against Sendai virus is described. Non-specific inhibitors should be removed by treatment with cholera filtrate R.D.E. In contrast to both the haemagglutination inhibition test and the complement fixation test, which do not allow a differentiation between mumps and Sendai virus infection because of cross reactions, the color test is assumed to be sufficiently specific for Sendai virus infection.     

A New Screening Method of Viral-neuraminidase Inhibitors

A new and practical method for the screening of neuraminidase inhibitors (NI) by means of the viral hemagglutination (HA)-dehemagglutination(deHA) reactions was suggested. The best conditions for the HA and deHA reactions were investigated. Existence of strong inhibition activity on the viral deHA has been recognized in the culture filtrates of some strains of actinomycetes. All of these deHA inhibitors showed NI activity that is not specified to the strain of the test viruses. About 0.25 mg/ml of the preparation obtained from the culture filtrate of the strongest actinomycetes, No. 289, inhibited the liberation of neuraminic acid from bovine submaxillary mucin by 80 HA units/ml of influenza A Fukuoka/1/70 (H₃N₂) virus up to 80%.     

Fowl Cholera Immunization in Turkeys: I. Efficacy of Various Cell Fractions of Pasteurella multocida as Vaccines1

Cell fractions of Pastuerella multocida (P-1059) were tested as vaccines against fowl cholera in turkeys. These fractions were culture filtrate, cell wall, and cytoplasm. A second culture filtrate preparation made from cells grown on blood-agar rather than the standard medium was also tested along with a "combination" preparation made by recombination of the cell fractions. Each preparation was tested in three vehicles: saline, alum (0.5%), and Freund Incomplete Adjuvant (50%). The turkeys vaccinated with these preparations were challenged by exposure to an experimental epornitic of fowl cholera. The combination fraction appeared to be the most promising vaccine when compared to the protective action of the commercial bacterin included in the test as a positive control.     

Binding of bacterial toxins to glycoproteins in the envelopes of rainbow trout eggs

Summary The ability of the vitelline and fertilization envelopes of rainbow trout eggs to trap toxins was investigated using cholera enterotoxin B and staphylococcal enterotoxin B in cytochemical or immunocytochemical experiments. Extracts from both envelopes were investigated by immunoblot analysis to identify toxin-binding proteins after SDS-PAGE. Binding studies of cholera enterotoxin B to vitelline envelopes and fertilization envelopes revealed a greater reactive intensity in the former. Treatment with neuraminidase enhanced the reactive intensity (or deposit) in the vitelline envelope and fertilization envelope outermost layers, with more conspicuous reactivity in the former. Cytochemical experiments showed that exogenous ganglioside GM₁ considerably enhanced cholera enterotoxin B binding to vitelline and fertilization envelopes. This enhancement was shown by an intense reactivity following the occurrence of new binding sites on the vitelline envelope inner surface and the inner wall of the zona radiata, a simultaneous extreme reduction in the reactivity of the vitelline envelope outermost layer, and a striking increase in reactive products in the fertilization envelope outermost layer. The surface region of the vitelline or fertilization envelope outermost layer was the binding site for staphylococcal enterotoxin B, and neuraminidase treatment caused a considerable reduction of reactive products in these areas. Immunoblot analysis of cholera enterotoxin Bor staphylococcal enterotoxin B-binding substances in extracts from the vitelline envelopes or fertilization envelopes demonstrated that the great majority of the binding substances are glycoproteins. The present results suggest that glycoproteins constituting the vitelline envelope or fertilization envelope may contribute to the protection of the egg itself or the embryo by trapping noxious toxins.     

The use of a CHO cell culture in studying Vibrio cholerae grown under different conditions]

Studies of biological activity of cholera vibrios in cultures of chinese hamster ovary cells (CHO) have revealed their strong dependence on culture conditions. Elongation of CHO cells is caused only by choleragenic strains. Under stationary conditions of culture the vibrios were found to release haemolisin into the medium and had a cytotoxic effect. Most of cytotoxic supernatants exhibited a neuraminidase activity. Proteolytic activity was less dependent on the vibrio culture conditions. Strains with a high proteolytic activity caused rounding of the CHO cells.     

Cycloheximide potentiation of prostaglandin E1- and cholera toxin-stimulated cyclic AMP accumulation in NG108-CC15 neuroblastoma-glioma hybrid cells

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.     

Immunofluorescence method of detecting cholera enterotoxin

The quantitative immunofluorescent assay for the determination of cholera enterotoxin is proposed. The assay is based on the selective sorption of cholera enterotoxin by gangliosides incorporated into polyacrylamide granules. The preliminary treatment of gangliosides with neuraminidase enhances the sensitivity of this assay. The assay permits the detection of cholerigen in an amount of 20 ng.     

Cholera vibrios lectins as main pathogenicity and persistence factors (biotechnological aspects of use)

Literature data and results of our studies of lectins are analyzed in the review. All the leading pathogenicity factors of cholera vibrios that possess enzymatic activity--cholera toxin, hemolysin, neuraminidase, chitinase have several lectin domains, that determine not only their pathogenetic role but also open perspectives for their use in medical practice. At the same time the variable receptor profile of cholera vibrios cells of various biovars and epidemical significance established with hemagglutination inhibition reaction by carbohydrates could be used to develop new principles of testing and typing of cholera vibrios.     

Structural analysis and functional role of the carbohydrate component of somatostatin receptors.

SRIF receptors are membrane-bound glycoproteins. To structurally identify the carbohydrate components of SRIF receptors, solubilized rat brain SRIF receptors were subjected to lectin affinity chromatography. Solubilized SRIF receptors specifically bound to wheat germ agglutinin-lectin affinity columns but not to succinylated wheat germ agglutinin. This finding, as well as the ability of the solubilized receptor to interact with a Sambucus nigra L. lectin affinity column suggested that sialic acid residues are associated with SRIF receptors. The inability of the receptor to bind to concanavalin A, Dolichus biflorus agglutinin, Ulex europeaus I, and Jacalin lectin affinity columns suggests that high mannose, N-acetylgalactosamine, fucose, and O-linked carbohydrates are not associated with receptor. To investigate the functional role of the carbohydrate groups in brain SRIF receptors, specific sugars were selectively cleaved from SRIF receptors and the subsequent effect on the specific high affinity binding of the agonist [125I]MK 678 to SRIF receptors was determined. Treatment of the receptor with endoglycosidase D did not affect the specific binding of [125I] MK 678 to the solubilized SRIF receptors, consistent with the finding from lectin affinity chromatography that high mannose-type carbohydrate structures were not associated with SRIF receptors. Treatment of solubilized SRIF receptors with peptide-N-glycosidase F and endoglycosidases H and F reduced [125I]MK 678 binding to SRIF receptors indicating that either hybrid, or a combination of hybrid and complex N-linked carbohydrate structures, have a role in maintaining the receptor in a high affinity state for agonists. Treatment of solubilized SRIF receptors with neuraminidase from Vibrio cholera abolished high affinity agonist binding to the receptors, whereas treatment of the receptor with neuraminidase from Newcastle disease virus did not affect [125I]MK 678 binding to the receptor. These findings suggest that sialic acid residues in an alpha 2,6-configuration have a role in maintaining the SRIF receptor in a high affinity conformation for agonists. This is further indicated by studies on SRIF receptors in the pituitary tumor cell line, AtT-20. Treatment of AtT-20 cells in culture with neuraminidase (V. cholera) greatly reduces high affinity [125I] MK 678 binding sites, but did not alter the maximal ability of SRIF to inhibit forskolin-stimulated cAMP accumulation in intact AtT-20 cells. This finding suggests that the desialylated SRIF receptor is functionally active and remains coupled to GTP-binding proteins, but exhibits a reduced affinity for agonists. Treatment of AtT-20 cell membranes with neuraminidase from V. cholera was also able to greatly reduce the affinity of SRIF receptors for [125I]MK 678.(ABSTRACT TRUNCATED AT 400 WORDS)     

Effects of indomethacin on intestinal secretion, prostaglandin E and cyclic AMP: evidence against a role for prostaglandins in cholera toxin-induced secretion.

The effects of indomethacin on intestine mucosal cAMP, intestinal fluid secretion, and mucosal and fluid PGE were studied in rabbits in vivo following challenge with cholera toxin. Indomethacin had no effect on cholera toxin-induced fluid secretion or cAMP accumulation. Inhibition of PGE synthesis was achieved by the administration of two but not one injection of indomethacin. These studies provide evidence against a role for PGE in mediating cholera toxin-induced secretion and point out the need to measure prostaglandin levels when using prostaglandin synthetase inhibitors in vivo.     

Perspectives on antiviral use during pandemic influenza.

Antiviral agents could potentially play a major role in the initial response to pandemic influenza, particularly with the likelihood that an effective vaccine is unavailable, by reducing morbidity and mortality. The M2 inhibitors are partially effective for chemoprophylaxis of pandemic influenza and evidence from studies of interpandemic influenza indicate that the neuraminidase inhibitors would be effective in prevention. In addition to the symptom benefit observed with M2 inhibitor treatment, early therapeutic use of neuraminidase inhibitors has been shown to reduce the risk of lower respiratory complications. Clinical pharmacology and adverse drug effect profiles indicate that the neuraminidase inhibitors and rimantadine are preferable to amantadine with regard to the need for individual prescribing and tolerance monitoring. Transmission of drug-resistant virus could substantially limit the effectiveness of M2 inhibitors and the possibility exists for primary M2 inhibitor resistance in a pandemic strain. The frequency of resistance emergence is lower with neuraminidase inhibitors and mathematical modelling studies indicate that the reduced transmissibility of drug-resistant virus observed with neuraminidase inhibitor-resistant variants would lead to negligible community spread of such variants. Thus, there are antiviral drugs currently available that hold considerable promise for response to pandemic influenza before a vaccine is available, although considerable work remains in realizing this potential. Markedly increasing the quantity of available antiviral agents through mechanisms such as stockpiling, educating health care providers and the public and developing effective means of rapid distribution to those in need are essential in developing an effective response, but remain currently unresolved problems.     

Novel aromatic inhibitors of influenza virus neuraminidase make selective interactions with conserved residues and water molecules in the active site.

The active site of type A or B influenza virus neuraminidase is composed of 11 conserved residues that directly interact with the substrate, sialic acid. An aromatic benzene ring has been used to replace the pyranose of sialic acid in our design of novel neuraminidase inhibitors. A bis(hydroxymethyl)pyrrolidinone ring was constructed in place of the N-acetyl group on the sialic acid. The hydroxymethyl groups replace two active site water molecules, which resulted in the high affinity of the nanomolar inhibitors. However, these inhibitors have greater potency for type A influenza virus than for type B influenza virus. To resolve the differences, we determined the X-ray crystal structure of three benzoic acid substituted inhibitors bound to the active site of B/Lee/40 neuraminidase. The investigation of a hydrophobic aliphatic group and a hydrophilic guanidino group on the aromatic inhibitors shows changes in the interaction with the active site residue Glu275. The results provide an explanation for the difference in efficacy of these inhibitors against types A and B viruses, even though the 11 active site residues of the neuraminidase are conserved.