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Biomedical subjects

T D Bartley

Publications and source records attributed to T D Bartley.

At least 19 recordsLinked to original sources
PubMed1991

Contribution of the B oligomer to the protective activity of genetically attenuated pertussis toxin.

An enzymatically deficient recombinant S1 subunit, in which Arg-9 was replaced by Lys, was combined with native B oligomer to form a mutant holotoxin molecule. This molecule exhibited decreased leukocytosis-promoting and histamine-sensitizing activities compared with those of the native toxin, supporting the view that the B oligomer is not responsible for these activities. The protective activity of this genetically attenuated pertussis toxin was compared with that of B oligomer alone. The mutant pertussis toxin and B oligomer were similarly capable of protecting mice against a respiratory infection with Bordetella pertussis, suggesting that the B oligomer makes a significant contribution to the protection afforded by the genetically attenuated holotoxin.

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↗
PubMed1990

Isolation and structural characterization of three isoforms of recombinant consensus alpha interferon.

Recombinant DNA-derived consensus alpha interferon was expressed in Escherichia coli and purified. Isoelectric focusing of this purified protein indicated the presence of three isoelectric subforms of pI 6.1, 6.0, and 5.7. These three subforms were preparatively separated by isoelectric focusing using Immobiline polyacrylamide gel and did not exhibit apparent differences in biological activity and tertiary structure. The pI 5.7 subform could also be separated from the pI 6.1 and 6.0 subforms by reverse-phase HPLC. Automated N-terminal amino acid sequence analysis of the pI 6.1 and 6.0 subforms yielded sequences corresponding to the methionyl and des-methionyl forms of the protein, respectively. Sequence analysis of the pI 5.7 subform indicated that its N terminus is blocked. To further determine the structure of the blocking moiety in the pI 5.7 subform, a blocked N-terminal tryptic peptide was isolated from HPLC peptide mapping of the S-carboxymethylated derivative. Results obtained from mass spectroscopic and amino acid analyses of this peptide suggest that it is blocked with an acetyl group at the N-terminal cysteine residue.

Amino Acid Sequence↗
PubMed1989

Pertussis holotoxoid formed in vitro with a genetically deactivated S1 subunit.

The cytotoxicity of pertussis toxin, a multisubunit exotoxin produced by Bordetella pertussis, is believed to be due to the ADP-ribosyltransferase activity of the S1 subunit. We have previously described the recombinant expression of each of the five individual pertussis toxin subunits in Escherichia coli and the production of an enzymatically deficient form of the S1 subunit by site-directed mutagenesis. We now report the in vitro assembly of holotoxin from native pertussis toxin B oligomer and recombinant S1 subunits, the latter purified and refolded from insoluble inclusion bodies. Holotoxin assembled with recombinant S1 of authentic amino acid sequence was indistinguishable from native pertussis toxin in its electrophoretic migration and ability to elicit a cytopathic response in cultured Chinese hamster ovary cells; in contrast, holotoxin assembled with the genetically deactivated analog of recombinant S1 displayed greatly diminished cytopathicity. These results verify that the in vitro cytopathic effects of pertussis toxin are the result of the enzymatic activity of the S1 subunit and illustrate the potential for constructing complex quaternary protein structures in vitro from insoluble, unfolded polypeptides derived from expression in recombinant systems.

Animals↗
PubMed1988

Structural characterization of recombinant consensus interferon-alpha.

Recombinant consensus interferon-alpha is derived from genetically modified Escherichia coli containing a synthetic gene constructed from a consensus of interferon sequences. The purified and biologically active protein has been subjected to detailed structural characterization including sequence determination and peptide isolation and identification. The homogeneous consensus interferon-alpha preparation contains two chromatographically indistinguishable homologous polypeptides with one containing an extra methionyl residue at the amino terminus. The delineated amino acid sequence of the protein is identical to that expected from the coding sequence of the gene. Correct oxidation of the molecule has been confirmed with two intramolecular disulfide linkages observed at Cys(1)-Cys(99) and Cys(29)-Cys(139).

Amino Acid Sequence↗
PubMed1984

A method for the detection and differentiation of cellulase components in polyacrylamide gels.

Endoglucanase and exoglucanase components of cellulase can be detected and differentiated after polyacrylamide gel electrophoresis by performing activity stains. Endoglucanase activity was visualized in carboxymethyl cellulose agar replicas of gels by staining with Congo red. General beta-1,4-glucanase activity was located by soaking the gel in a solution of NaBH4-reduced cellulooligosaccharides, and detecting the formation of reducing sugars by reaction with triphenyl tetrazolium chloride. Endoglucanases are active in both assays, while exoglucanases can be distinguished by their activity in the cellulo-oligosaccharide assay only. This methodology has facilitated the purification and characterization of cellulase components from Trichoderma reesei and Microbispora bispora.

Cellulase↗
PubMed1982

Membrane filter technique for the isolation of Yersinia enterocolitica.

A membrane filter procedure was developed for the isolation of Yersinia enterocolitica from aquatic environments. Primary differentiation was based on the fermentation of sorbitol, the absence of lysine decarboxylase and arginine decarboxylase-dihydrolase activities, and the production of urease. Sodium deoxycholate was incorporated as an inhibitor of background organisms. The presumptive identification of Y. enterocolitica was accomplished in 50 h, and the rate of identity confirmation of typical colonies was 88%. The mean recovery rate of 15 strains from phosphate buffer suspensions was 91%, and quantitative recovery was demonstrated for low populations of the organism in both laboratory-prepared and naturally occurring mixed cultures. The technique was used to isolate 33 strains of Y. enterocolitica from 15 of 27 river water samples and from prechlorinated sewage effluent. Nine (27%) of the isolates were rhamnose positive, and only five (15%) were serotypable. Two isolates were identified as serotype O:4 (or O:4,32), two were O:17, and the fifth was O:40.

Animals↗
PubMed1976

Long-term follow-up of sequential aortocoronary venous grafts.

In 1972 we reported myocardial revascularization of 130 patients using multiple sequential aortocoronary anastomoses to a single saphenous vein ]1]. Of the 122 survivors described in that report, 121 (99%) have been followed an additional 3 years. Twelve deaths occurred during the interval. The 110 currently followed patients represent 290 anastomoses; 54 have been studied angiographically since operation. Angiographic patency at 3 years in the studied group (18) was 70%. These figures exceed our follow-up data for 135 patients revascularized during the same period using individual vein grafts. Comparison of life table survival curves demonstrates this result. We believe the improved patency and decreased operating time that have resulted from employing this technique have outweighed the likelihood of a proximal stenosis causing closure of the whole graft system. We continue to use this technique in combination with internal mammary artery grafts in the management of multiple-vessel coronary disease. Good early results using this technique have been reported by other authors [2, 3, 5].

Adult↗