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R A Finkelstein

Publications and source records attributed to R A Finkelstein.

At least 73 records · Page 4Linked to original sources
PubMed1983

Antigenic and structural variations in the cholera/coli family of enterotoxins.

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.

Antigens, Bacterial↗
PubMed1983

Observations on the Texas Star-SR avirulent A-B+ mutant candidate cholera vaccine.

Comprehensive controlled vaccine trials have revealed that neither conventional cholera vaccines nor cholera toxoid administered parenterally are effective in inducing durable immunity against cholera. The only truly effective means of immunization against cholera as yet demonstrated conclusively only in volunteers, are (a) recovery from the disease itself and (b) that observed following oral administration of living attenuated mutants. In both instances, the host is presented with a consortium of vibrio products at the important local level. Studies in our laboratory have resulted in the isolation from cholera vibrios of a soluble haemagglutinin (HA), which has inherent protease activity, and which appears to participate in adherence of the vibrios to the intestinal epithelium. Current observations indicate that this HA/lectin/protease (a) is produced in both a cell-associated and a soluble form in vivo and (b) that it hydrolyzes fibronectin, secretory immunoglobulin A (sIgA), and mucin, three host proteins which may be significant in specific and non-specific resistance. The Texas Star-SR mutant has been shown to have a mild side effect in volunteers in that, independent of the dose administered about 20% of the recipients manifest one or a few non-inconveniencing loose movements. It is possible that the mere act of intestinal colonization may alter fluid transport in the gut and that this could be a model for other diarrheal disease.

Cholera↗
PubMed1982

Characterization and distribution of the hemagglutinins produced by Vibrio cholerae.

Examination of the distribution of cell-associated and soluble hemagglutinins (HA) produced by Vibrio cholerae revealed the existence of four different HAs. A cell-associated mannose-sensitive HA (MSHA) was produced only by the El Tor biotype. This was evident with all El Tor strains examined. It appears to be responsible for the HA biotyping differentiation of El Tor from classical biotype V. cholerae. The MSHA had no apparent divalent ion requirement; it was inhibited by D-mannose and D-fructose; and it was active on all human (A, B, O) and all chicken erythrocytes tested. Spontaneous MSHA- mutants of El Tor strains were selected by cosedimentation of MSHA+ parent bacteria with erythrocytes. An L-fucose-sensitive HA was detected transiently in early log-phase growth with two of the four classical strains examined and with MSHA- mutants of El Tor biotype strains 3083, 26-3, and 17. MSHA- mutants also expressed another cell-associated HA in late log-phase cultures. A "soluble" HA was detected in late log-phase cultures of all strains tested. This HA was not inhibitable by any sugars tested; it required CA2+ ions for maximum activity; and it was active on some chicken erythrocytes but not others.

Animals↗
PubMed1982

Comparison of heat-labile enterotoxins from porcine and human strains of Escherichia coli.

Heat-labile enterotoxins (LTs) from porcine EWD299) and human (H74-114) enterotoxigenic strains of Escherichia coli were isolated by a single-step galactose elution procedure. Although both strains had similar amounts of LT in their whole-cell lysates, H74-114 yielded a smaller quantity of purified LT than did EWD299. Immunodiffusion studies with specific antisera revealed that although the two LTs shared major antigenic determinants each also had unique antigens. Both also had shared and unique specificities in comparison with the cholera enterotoxin (choleragen). Differences also exist in the apparent molecular weights of their B-subunit oligomers (coligenoid) as well as in the B-subunit monomers. The monomer molecular weights are 11,500 for EWD299 porcine LT and 12,700 for H74-114 human LT. The results suggest that either this isolated human LT has a tetrameric coligenoid or it moves differently in sodium dodecyl sulfate gels for other reasons. The A-subunits of both LTs were similar in size (28,000 daltons), and both LTs were activated by mild proteolytic processing. Amino acid analysis showed a threefold increase in the level of tryptophan and two- and fourfold decreases in the levels of glutamic acid and methionine, respectively, in H74-114 LT compared with EWD299 LT. These structural and antigenic differences may prove to be significant in immunoprophylaxis of the cholera-coli family of enterotoxins. Further studies to define the extent of evolutionary drift of these toxins are needed.

Amino Acids↗
PubMed1982

Protection in rabbits induced by the Texas Star-SR attenuated A-B+ mutant candidate live oral cholera vaccine.

The avirulent, A-B+, streptomycin-resistant mutant designated Texas Star-SR, isolated from a virulent, hypertoxinogenic, colonizing strain of Vibrio cholerae (Ogawa serotype, El Tor biotype) and administered intragastrically or intraduodenally in adult rabbits, has been found to induce substantial immunity to subsequent challenge (in ligated intestinal loops) with virulent wild-type cholera vibrios (of both homologous and heterologous biotype and serotype). Significant resistance to challenge with one strain of human heat-labile enterotoxin (LT)-producing Escherichia coli was also demonstrated, but resistance against two other human LT-producing strains was either nil or marginal under these experimental conditions. Significant, but not striking, resistance against challenge with purified choleragen was obtained, whereas protection against a bolus challenge of purified porcine LT was not statistically significant.

Animals↗
PubMed1982

Purification and characterization of the soluble hemagglutinin (cholera lectin)( produced by Vibrio cholerae.

The soluble hemagglutinin (HA) (cholera lectin) produced by Vibrio cholerae strain CA401 was purified to apparent homogeneity by a sequence of ammonium sulfate fractionation, gel filtration, and preparative isoelectric focusing. Soluble HA activity was found to focus at three different pIs, 6.3, 5.3, and 4.7. Each of the factors migrated as a large-molecular-weight protein in sodium dodecyl sulfate-polyacrylamide gel electrophoresis under normal conditions, and each, upon heating in sodium dodecyl sulfate was found to dissociate into 32,000-molecular-weight subunits. Treating the samples with a reducing agent did not affect their mobility. Each gave a reaction of immunological identity with antiserum prepared against the others. Thus, there are apparently three distinct pH isotypes of soluble HA which exist as noncovalently associated polymers of 32,000-molecular-weight subunits. Electron microscopy of purified preparations revealed long filamentous polymers. The molecule does not stain as a glycoprotein; it is hydrophobic; it is inactivated during incubation at 25, 37, or 60 degrees C; and it has significant protease activity. The protease activity likewise focused at pH values of 6.3 and 5.3 to 4.7, and it was inhibited by antiserum against the HA. However, whereas the HA is active at 4 degrees C, the protease is not. The soluble HA is, therefore, a bifunctional molecule capable of mediating hemagglutination and proteolysis. Its amino acid composition is reported. Fab fragments of antibody against the purified HA inhibited attachment of heterologous serotype-biotype V. cholerae to infant rabbit small bowel.

Bacterial Proteins↗
PubMed1981

Ability of an avirulent mutant of Vibrio cholerae to colonize in the infant mouse upper bowel.

Vibrio cholerae strain 3083 (biotype El Tor, serotype Ogawa) and Texas Star-SR (SR), a mutant derived from 3083 that produces the B (binding) but not the A (toxic) subunit of choleragen, were compared in their abilities to: (i) associate with the infant mouse upper bowel; (ii) survive and multiple there; and (iii) induce diarrhea. Vibrios labeled with 35SO4 were used to determine association with the upper bowel and ability to multiply. The parental strain associated significantly better than SR, although viable mutant cells were found in the infant mouse intestine 16 to 18 h after challenge. Addition of exogenous toxin enhanced the rate at which labeled SR (but not 3083) was cleared, further suggesting that SR associates less well with the upper bowel. Both SR and 3083 multiplied in the upper bowel but, due perhaps to slight net killing during the first 3 h and its more rapid rate of clearance, SR achieved a population size only 10% that of 3083 by 8 h postchallenge. Strain 3083 elicited diarrhea in infant mice but SR did not, even after 10 successive passages through the infant mouse intestine. Strain SR was slightly temperature sensitive at 37 and 40 degrees C. Its potential use as a live vaccine is discussed.

Animals↗
PubMed1981

Assmilation of iron by pathogenic Neisseria spp.

Iron assimilation by Neisseria meningitidis and Neisseria gonorrhoeae has been shown to be important to their growth and virulence. Iron acquisition in vitro was studied in an agar diffusion assay employing the iron-binding protein conalbumin. The ability of various iron compounds to alter the growth-inhibitory effect of conalbumin was investigated. On an equimolar iron basis, citrate-containing iron compounds were most effective; hemin was slightly less effective; ferrous sulfate and ferrous ammonium sulfate were even less effective; and the ferric compounds, ferric nitrate, ferric chloride, and ferric dextran (Imferon), were least effective. The results suggested that, as with Escherichia coli and certain other bacteria, the Neisseria spp. may utilize a citrate-mediated iron transport system. Microbial siderophores were also tested for their ability to relieve the growth-inhibitory effect of conalbumin. Two phenolate siderophores and Desferal enhanced growth inhibition in the deferrated form, but were inactive in the ferrated form. Several trihydroxamates of the ferrichrome family and coprogen were inactive in either the deferrated or ferrated forms. Of the 12 different siderophores tested, only the dihydroxamates (schizokinen, arthrobactin, and aerobactin) were stimulatory, but then only in the ferrated forms. Apparently, even though those siderophores could be utilized as specific iron transport agents by the Neisseria spp., they could not compete with conalbumin for iron under these assay conditions.

Conalbumin↗
PubMed1981

Siderophore production by pathogenic Neisseria spp.

Previous studies have established the importance of iron acquisition to the growth and virulence of Neisseria meningitidis and Neisseria gonorrhoeae. Although preliminary evidence that the Neisseria spp. produce siderophores has been presented, the exact mechanism of iron acquisition has remained obscure. Siderophore production by N. gonorrhoeae and N. meningitidis was induced in two different low-iron media. The iron-reactive siderophores, "gonobactin" and "meningobactin," were partially purified by ion exchange chromatography followed by extraction with phenol-chloroform-ether or by gel filtration. The compounds were of low molecular weight, their synthesis was repressed by iron in the medium, and they appeared to be hydroxamic acids since they were stimulatory for Arthrobacter flavescens JG-9 (a hydroxamate auxotroph) and gave a positive Csáky reaction for bound hydroxylamine. In the iron form, the compounds had an absorption maximum of approximately 420 nm. Although meningobactin stimulated growth of the gonococcus in low-iron media and vice versa, the homologous activity was more marked, indicating that the compounds, though similar, were probably not identical. As determined by A. flavescens assay the meningococcus produced three to five times more siderophore than did the gonococcus; however, the amount of siderophore present in the culture fluids of even the meningococcus was 100- to 1,000-fold lower than the concentration of hydroxamate siderophores reported to be produced by Bacillus megaterium or Aerobacter aerogenes. Virulent, colony type 1 N. gonorrhoeae produced significantly more gonobactin than did the avirulent colony type 3 gonococci.

Biological Assay↗
PubMed1981

Effect of siderophores on virulence of Neisseria gonorrhoeae.

The virulence of Neisseria gonorrhoeae for chicken embryos can be modified in a predictable manner by the addition of microbial siderophores to the inoculum. "Meningobactin" and "gonobactin," siderophores isolated from iron-limited cultures of meningococci and gonococci, respectively, enhance the virulence of the relatively avirulent colony type 3 (T3) organisms, but have essentially no effect on the virulence of T1 organisms. Both of these compounds were found previously to stimulate in vitro growth of the pathogenic Neisseria spp. under conditions made iron limiting by the addition of conalbumin, the transferrin counterpart of chickens. Similarly, ferrated schizokinen and arthrobactin, both dihydroxamate siderophores which stimulated growth in iron-limited conditions in vitro, also enhanced virulence of T3 organisms, whereas desferrioxamine B mesylate (Desferal), a trihydroxamate previously shown to be inhibitory in vitro, decreased the virulence of the T1 colony form. This was due to the iron-binding function of the molecule, as the iron-saturated form, ferrioxamine B mesylate, did not affect virulence. An additional trihydroxamate siderophore, ferrichrome A, which was inactive on Neisseria spp. in either the deferri- or ferrated forms in vitro, likewise did not affect virulence in the chicken embryo model. The neisserial siderophores were more effective than the other microbial siderophores in enhancing virulence of T3 gonococci. The results add to the evidence that the ability to acquire iron is an important determinant of virulence.

Animals↗
PubMed1981

Subunit number and arrangement in Escherichia coli heat-labile enterotoxin.

The Escherichia coli heat-labile enterotoxin (LT) was found to have the same subunit structure as cholera toxin, namely, one A subunit and five B subunits. Reaction with a bisimidate generated all the possible cross-linked derivatives of A5B: B,2B ... 5B and A, AB ... A5B. The isolated B component, coligenoid, contained five B subunits and showed some tendency of polymerize: with a bisimidate it became covalently connected into the set B ... 5B with lesser amounts of 6B ... 10B, etc. The subunit formulas of two independently prepared samples of LT were both proved to be A5B by cross-linking, but their B pentamers migrated at different rates on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, indicating that they have different conformations. The faster (R) form could be converted to a diffuse slower (C) form by incubating it at 50 degrees C or at 37 degrees C with 0.2 M galactose, which is the terminal sugar of ganglioside GM1, the natural receptor for LT. Cholera toxin resembled the R form more than the C form of LT.

Bacterial Toxins↗
PubMed1980

Properties of homogeneous heat-labile enterotoxin from Escherichia coli.

Recently, the heat-labile enterotoxin (LT) of Escherichia coli has been purified to homogeneity and partially characterized (Clements and Finkelstein, Infect. Immun. 24:760-769, 1979). This study extends our observations on the physicochemical properties of LT. The toxin has an isoelectric point of pH 8.0, as compared with choleragen and choleragenoid, which have isoelectric points of pH 6.75 and 7.75, respectively. Sedimentation equilibrium measurements established an approximate molecular weight for LT of 91,440. LT had an even more marked affinity than choleragen for agarose-containing matrixes in gel filtration. Of several mono- and disaccharides tested, only galactose and lactose were highly efficient in removing 125I-labeled LT from agarose-containing columns. LT dissociated into subunits (designated A and B) during gel filtration in the presence of 5 M guanidine. These subunits were immunologically distinct and possessed unique and shared antigenic determinants to the corresponding A and B subunits of choleragen. During gel filtration of LT at pH 6.5 and room temperature, a spontaneously occurring toxoid of LT, analogous to choleragenoid, was discovered and designated "coligenoid." This product contains only the B subunits of the toxin. A partial amino acid sequence of the B subunit of LT revealed a remarkable homology to the primary structure of cholera toxin B. Within the first 20 amino acids of the two chains, only 5 differ, and these differences may be attributable to single base substitutions.

Amino Acids↗
PubMed1979

Selection and characteristics of a Vibrio cholerae mutant lacking the A (ADP-ribosylating) portion of the cholera enterotoxin.

After mutagenesis with nitrosoguanidine and selection by immuno-halo techniques, an avirulent mutant, designated Texas Star-SR, which produces no detectable A (active; ADP-ribosylating) region of the cholera enterotoxin (choleragen) but produces the B region (choleragenoid) in amounts similar to the hypertoxinogenic wild-type parent Vibrio cholerae (biotype E1 Tor serotype Ogawa), has been isolated. The mutant retains the colonizing ability, motility, prototrophy, and serologic characteristics of the parent. In relevant intestinal experimental models, it has been shown to be avirulent and to induce protection against challenge with virulent cholera vibrios. The mutant appears to be suitable for further evaluation in volunteers as a candidate living enteric vaccine against cholera and related enterotoxic enteropathies.

Adenosine Diphosphate Sugars↗
PubMed1979

Isolation and characterization of homogeneous heat-labile enterotoxins with high specific activity from Escherichia coli cultures.

The heat-labile enterotoxin (LT) has been isolated in homogeneous form with high specific activity from three sources: cell-free supernatant, NaCl extract, and whole-cell lysates of an enterotoxigenic Escherichia coli strain. In vitro immunological assays were used in lieu of tedious and highly variable bioassays to recognize fractions with activity. This revealed that the major portion of the LT remained adherent to columns containing agarose, from which it could be eluted quantitatively in practically homogeneous form by galactose. Isolated LT has remarkable similarities to the cholera enterotoxin (choleragen) in both subunit structure and amino acid composition, although there are also notable differences in these two enterotoxins, which are related immunologically and by mode of action. Unlike choleragen, in which the A region is totally nicked, E. coli LT, depending on its source, is activated by proteolytic processing. The activity of LT is equivalent to that of choleragen in bioassays on adrenal cells, in rabbit skin, and in rabbit ileal loops, especially when, depending on the source of material, the LT has been activated by treatment with trypsin. The whole-cell lysate is the richest source of LT.

Amino Acids↗
PubMed1979

Purification and characterization of a hemolysin produced by Vibrio cholerae biotype El Tor: another toxic substance produced by cholera vibrios.

A thermolabile direct hemolysin from an El Tor cholera vibrio strain has been isolated and partially characterized as a simple protein of ca. 20,000 molecular weight. In addition to its hemolytic activity, the hemolysin is cytotoxic, cardiotoxic, and rapidly lethal. In these respects it resembles the thermostable direct hemolysin/cytotoxin/cardiotoxin/lethal toxin of Vibrio parahaemolyticus and certain other bacterial hemolysins, although there are other significant differences. Because identical diseases are produced by both hemolytic and nonhemolytic cholera vibrios, the El Tor hemolysin may be presumed to be pathogenetically irrelevant. These observations raise the question of "When is a toxic substance also a toxin?"

Adrenal Glands↗