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H R Kaslow

Publications and source records attributed to H R Kaslow.

11 recordsLinked to original sources
PubMed1992

Pertussis toxin and target eukaryotic cells: binding, entry, and activation.

Pertussis toxin, a protein virulence factor produced by Bordetella pertussis, is composed of an A protomer and a B oligomer. The A protomer consists of a single polypeptide, termed the S1 subunit, which disrupts transmembrane signaling by ADP-ribosylating eukaryotic G-proteins. The B oligomer, containing five polypeptides, binds to cell receptors (most likely containing carbohydrate) and delivers the S1 subunit. Current knowledge suggests that expression of ADP-ribosyltransferase activity in target eukaryotic cells arises after 1) nucleotides and membrane lipids allosterically promote the release of the S1 subunit; and 2) the single disulfide bond in the S1 subunit is reduced by reductants such as glutathione. This model suggests conditions for the proper use of the toxin as an experimental reagent.

Animals↗
PubMed1992

Detection of antibodies inhibiting the ADP-ribosyltransferase activity of pertussis toxin in human serum.

Bordetella pertussis produces a protein virulence factor termed pertussis toxin. Many candidate pertussis vaccines are based on the rationale that an immune response that neutralizes the virulence activities of this toxin, which are thought to arise from its catalytic ADP-ribosyltransferase activity, would be beneficial. The report describes two methods that quantify the inhibition of this activity by human serum. One, termed a direct assay, involves an initial incubation of toxin with serum, a second incubation that activates the toxin, and a third incubation that measures the ADP-ribosyltransferase activity of the mixture. The other assay, termed a plate assay, involves immobilization of the toxin, exposure of the immobilized toxin to serum and washing of the plate, and then activation and assay of the toxin's ADP-ribosyltransferase activity. The plate assay may be more selective than the direct assay in terms of identifying antibodies that neutralize the toxin in vivo. Sera from controls, selected patients presenting with cough, and vaccinated infants were first analyzed by the direct assay. In contrast to sera from controls, sera from several of the patients and vaccinated infants strongly inhibited activity. Dose-response curves of inhibition were determined for samples from three vaccinated infants by both the direct and plate assays. One of the samples had a dose-response curve of a different shape and thus differed not only in titer but also in functional characteristics. A comparison of inhibition of ADP-ribosyltransferase activity and neutralization in a CHO cell assay indicated that there was incomplete agreement between the two assays. Taken together, these results indicate that measurement of inhibition of ADP-ribosyltransferase activity by human serum is practical and may be useful in the evaluation of responses to pertussis vaccines.

Adolescent↗
PubMed1991

Monoclonal antibodies against the enzymatic subunit of both pertussis and cholera toxins.

A synthetic peptide corresponding to amino acids 6-17 of the A subunit of pertussis toxin was synthesised and used for the immunization of Balb/c mice and the subsequent production of monoclonal antibodies (MAbs). This peptide contains a region of eight amino acids which is homologous to a region in the cholera toxin A subunit. The properties of two of the resultant MAbs are described. Both of the antibodies (CP7-3003F7, an IgG3 and CP7-3004G6X1, an IgG1) react in an ELISA with the peptide and with intact pertussis toxin, pertussis toxin A subunit and cholera toxin A subunit, but do not react significantly with pertussis toxin B subunit, intact cholera toxin, or cholera toxin B subunit. Competition ELISA assays in which the peptide, the intact toxins and the toxin subunits were compared with respect to their ability to inhibit the binding of the MAbs to peptide-coated ELISA plates demonstrated that only pertussis toxin A subunit was as active, on a molar basis, as the peptide. Western blot analyses of the holotoxins confirmed that both MAbs were reactive only with the toxin A subunits. The MAbs were unable to neutralize the activity of cholera toxin or pertussis toxin in a Chinese hamster ovary (CHO) cell assay. Both were also unable to neutralize either the ADP-ribosylation activity or the NAD-glycohydrolase activity of the pertussis toxin A subunit. The significance of these results with respect to the role of this conserved site in the activity of these two toxins is discussed.

Amino Acid Sequence↗
PubMed1991

Evaluation of antibodies elicited by immunization with pertussis toxin.

The monoclonal antibody termed 1B7 neutralizes pertussis toxin in vivo in cell culture systems and can also passively protect mice from a challenge with live Bordetella pertussis (9). It has been suggested that most other independently derived neutralizing monoclonal antibodies recognizing the S1 subunit apparently recognize the same epitope as 1B7, and that the S1 subunit contains only one immunodominant protective epitope (1). These antibodies have been termed Class A antibodies (8) and inhibit the ADP-ribosyltransferase but not the NAD glycohydrolase activity of the toxin (7). We are testing the hypothesis that immunization with inactivated preparations of pertussis toxin that lead to protection are associated with the production of Class A antibodies. If true, then identification of Class A antibodies in sera might provide a serological correlate of protection. If false, then development of assays designed to detect the important protective antibodies are necessary. Our initial results suggest that Class A antibodies are not the predominant neutralizing antibody in mice immunized with vaccines containing formalin-treated pertussis toxin.

Animals↗
PubMed1991

The molecular engineering of pertussis toxoid.

The demand for a safer pertussis vaccine has led to the development of acellular vaccine products. We have sought to manufacture a component vaccine based upon the genetic inactivation of pertussis toxin derived by recombinant DNA technology and protein engineering. Rational site-directed mutagenesis of the S1 subunit of pertussis toxin has resulted in an enzymatically-deactivated polypeptide which retains its immunogenic potential. Mutagenic analysis of the other subunits of this toxin has permitted a delineation of the structural determinants involved in its recognition of cellular receptors. The in vitro assembly of holotoxin species possessing selectively engineered subunits may facilitate the production of a molecularly-defined genetic toxoid for pertussis prophylaxis.

Genetic Engineering↗
PubMed1979

Interconversion between multiple glucose 6-phosphate-dependent forms of glycogen synthase in intact adipose tissue.

We have tested the hypothesis that interconversion between multiple glucose-6-P-dependent forms of glycogen synthase helps regulate glycogen synthesis in adipose tissue. Our results indicate that interconversion of glycogen synthase in adipose tissue involves primarily dependent forms and that these interconversions were measured better by monitoring the activation constant (A0.5) for glucose-6-P than measuring the -: + glucose-6-P activity ratio. Insulin decreased and epinephrine increased the A0.5 for glucose-6-P without significant change in the activity ratio. Insulin consistently decreased the A0.5 in either the presence or absence of glucose, indicating that the insulin-promoted interconversion did not require increased hexose transport. Isoproterenol increased the A0.5 for glucose-6-P, while methoxamine was without effect, indicating beta receptors mediate adrenergic control of interconversion between glucose-6-P-dependent forms. The changes in the A0.5 produced by incubations with insulin or epinephrine were mutually reversible. We conclude that 1) glycogen synthesis in adipose tissue is catalyzed by multiple glucose-6-P-dependent forms of glycogen synthase, 2) hormones regulate glycogen metabolism by promoting reversible interconversions between these forms, and 3) there is no evidence that a glucose-6-P-independent form of glycogen synthase exists in intact adipose tissue.

Adipose Tissue↗
PubMed1979

Adaptations of glycogen metabolism in rat epididymal adipose tissue during fasting and refeeding.

It is well documented that adipose tissue glycogen content decreases during fasting and increases above control during refeeding. We now present evidence that these fluctuations result from adaptations intrinsic to adipose tissue glycogen metabolism that persist in vitro: in response to insulin (1 milliunit/ml), [3H]glucose incorporation into rat fat pad glycogen was reduced to 10% of control after a 3-day fast; incorporation increased 6-fold over fed control on the 4th day of refeeding following a 3-day fast. We have characterized this adaptation with regard to alterations in glycogen synthase and phosphorylase activity. In addition, we found that incubation of fat pads from fasted rats with insulin (1 milliunit/ml) increased glucose-6-P content, indicating that glucose transport was not the rate-limiting step for glucose incorporation into glycogen in the presence of insulin. In contrast, feeding a fat-free diet resulted in dramatic increases in glycogen content of fat pads without a concomitant increase in glucose incorporation into glycogen in response to insulin (1 milliunit/ml). Thus, fasting and refeeding appeared to alter insulin action on adipose tissue glycogen metabolism more than this dietary manipulation.

Adipose Tissue↗
PubMed1978

Genetic evidence that cholera toxin substrates are regulatory components of adenylate cyclase.

Cholera toxin, using [32P]NAD+ as substrate, specifically radiolabels at least two proteins in plasma membranes of wild type S49 mouse lymphoma cells. The toxin-specific substrates are detectable by sodium dodecyl sulfate-polyacrylamide gel electrophoresis as bands corresponding to molecular weights of 45,000 and a doublet of 52,000 to 53,000. Membranes of two other cell types exhibit similar patterns of radiolabeled bands specifically produced by incubation with cholera toxin: the "uncoupled" variant S49 cell, which possesses adenylate cyclase activity unresponsive to hormones, and the HTC4 rat hepatoma cell, which lacks detectable catalytic adenylate cyclase activity but contains components of the cyclase system necessary for regulation by guanyl nucleotides and NaF. Little or no toxin-specific radiolabeling is observed in membranes of a fourth cell type, the adenylate cyclase activity-deficient S49 variant, which functionally lacks components of the cyclase system involved in cholera toxin action and regulation by guanyl nucleotides and NaF. The toxin-specific labeling pattern is not observed in membranes prepared from wild type S49 cells previously treated with cholera toxin in culture. One or both of the toxin substrates thus appears to be involved in regulation of adenylate cyclase by guanyl nucleotides and fluoride ion.

Adenylyl Cyclases↗
PubMed1978

Reconstitution of cholera toxin-activated adenylate cyclase.

Reconstitution of adenylate cyclase activity responsive to stimulation by guanylyl-5'imidodiphosphate or NaF may be achieved by mixing dilute Lubrol 12A9-solubilized extracts of wild-type S49 membranes with membranes of an adenylate cyclase-deficient variant. Experiments using N-ethylmaleimide to inactivate components of the adenylate cyclase system indicate that distinct components from both wild-type detergent extracts and adenylate cyclase-deficient membranes are essential for reconstitution. These results and conclusions confirm those of E. M. Ross and A. G. Gilman [J. Biol. Chem. (1977) 252, 6966-6969]. Detergent extracts of cholera toxin-treated wild-type membranes yield a reconstituted adenylate cyclase as responsive to GTP as to guanylyl-5'-imidodiphosphate whereas, in the absence of cholera toxin treatment, GTP has little or no effect. Cholera toxin-treated adenylate cyclase-deficient membranes and Lubrol 12A9 extracts from them, however, fail to yield a reconstituted adenylate cyclase that responds to GTP with an increase in cyclase activity. Because treatment of the adenylate cyclase-deficient variants with cholera toxin is without effect on the reconstituted cyclase, we propose that the cholera toxin substrate is absent or altered in the adenylate cyclase-deficient phenotype.

Adenylyl Cyclase Inhibitors↗