Biomedical subjects

G A Merrill

Publications and source records attributed to G A Merrill.

5 recordsLinked to original sources

PubMed1992

Immunological evidence for a conformational difference between recombinant bovine rhodanese and rhodanese purified from bovine liver.

Rhodanese has been utilized as a model enzyme for the study of protein structure-function relationships. The enzyme has recently been cloned and the recombinant enzyme is now available for investigation. However, prior to use in structure-function studies, the recombinant enzyme must be shown to have the same structure and activity as the bovine liver enzyme used in the previous studies. An immunological study of the conformations of these enzyme conformers is described. Three antibodies (two monoclonal and one polyclonal, site-directed antibody) were shown to detect distinct and nonoverlapping epitopes. The epitopes of the monoclonal antirhodanese antibodies (R207 and MAB11) were mapped to the same CNBr digest fragment of the amino terminal domain of rhodanese, and the epitope of the site-directed antibody prepared against the interdomain tether sequence of rhodanese (PAT-T1) was mapped to that region of rhodanese (residues 142-156). The rhodanese conformers were studied by monitoring the accessibility of the epitopes recognized by each antibody in each conformer using an indirect ELISA. None of the antibodies could detect its epitope on the purified liver enzyme. Two of the antibodies (R207 and PAT-T1) could also not detect their epitopes on the recombinant enzyme. However, MAB11 did detect a conformational difference between the natural and recombinant rhodanese conformers, indicating the conformational difference is localized in the first 73 amino acids of rhodanese. This difference presumably reflects the difference in the histories of the two enzymes and may be due to differences in enzyme folding, differences in the purification procedures, and differences in storage conditions--all of which could influence the final conformation of the enzyme.

Amino Acid Sequence↗

PubMed1988

Detection of time-dependent and oxidatively induced antigens of bovine liver rhodanese with monoclonal antibodies.

Rhodanese has been extensively utilized as a model protein for the study of enzyme structure-function relationships. An immunological study of conformational changes occurring in rhodanese as a result of oxidation or thermal inactivation was conducted. Three monoclonal antibodies (MABs) to rhodanese were produced. Each MAB recognized a unique epitope as demonstrated by binding of the MABs to different proteolytic fragments of rhodanese. While none of the MABs significantly bound native, soluble, sulfur-substituted bovine rhodanese, as indicated in indirect enzyme-linked immunosorbent assay experiments, each MAB was immunoadsorbed from solution by soluble rhodanese as a function of the time rhodanese was incubated at 37 degrees C. Thus, as rhodanese was thermally inactivated, conformational changes resulted in the expression of three new epitopes. Catalytic conformers demonstrated different rates of thermally induced antigen expression. Each MAB also recognized epitopes expressed when rhodanese was immobilized on microtiter plates at 37 degrees C. Two conformers resulting from oxidation of rhodanese by hydrogen peroxide were identified immunologically. All MABs recognized rhodanese that was oxidized with peroxide and allowed to undergo a secondary cyanide-dependent reaction which also resulted in a fluorescence shift and increased proteolytic susceptibility. Only one MAB was capable of recognizing an epitope expressed when rhodanese was oxidized with peroxide and then separated from the reactants to prevent induction of the secondary conformational changes.

Animals↗

PubMed1987

Involvement of endogenous opiates in glucose-stimulated hyperinsulinism of canine endotoxin shock. Inhibition by naloxone.

Hyperinsulinism has been associated with infection and endotoxin shock in rodents, dogs, and humans. In dogs with Escherichia coli-induced endotoxin shock, this hyperinsulinism was in response to glucose administration. To determine the role of endogenous opiates in endotoxin-induced glucose-stimulated hyperinsulinism, plasma beta-endorphin, Met-enkephalin, Leu-enkephalin, insulin, and glucose concentrations were measured for 6 h in fasted, anesthetized dogs given LD70 of E. coli endotoxin; endotoxin and glucose; endotoxin, glucose, and naloxone (an opiate antagonist); glucose and naloxone; or glucose alone. Plasma endogenous opiate immunoreactivity was elevated in dogs that received endotoxin, regardless of the presence of glucose or naloxone. The elevation of plasma Met-enkephalin and beta-endorphin preceded the onset of hyperinsulinism, but the elevation of plasma Leu-enkephalin did not. Plasma insulin was elevated 100-fold by 360 min in dogs given endotoxin and glucose. The magnitude of this hyperinsulinism was markedly reduced by naloxone, supporting the hypothesis that endogenous opiates are involved in the development of the glucose-stimulated hyperinsulinism associated with endotoxin shock. Interestingly, naloxone, given in conjunction with glucose, appeared to have a stimulatory effect on insulin secretion.

Animals↗

PubMed1983

A day at Boho.

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Delivery of Health Care↗

PubMedDate not supplied

Staphylococcal food poisoning associated with an Easter egg hunt.

Staphylococcal contamination of intact, hard-boiled eggs resulted in the food poisoning of an estimated 300 children out of 850 who had participated in an Easter egg hunt. Enterotoxigenic staphylococci that were isolated from the Easter eggs matched that obtained from an infected cook who prepared the eggs three to five days before the hunt and which he left unrefrigerated. Experimental studies demonstrated that heated eggs can absorb 2 mL of contaminated cool water through intact eggshells. When water was inoculated with pathogenic staphylococci at even low contamination levels, rapid growth and enterotoxin production within cooked eggs could be easily duplicated. This is the first large outbreak of its type; safeguards can and should be employed to prevent future ones.

Adolescent↗