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Biomedical subjects

J A Thomas

Publications and source records attributed to J A Thomas.

At least 19 recordsLinked to original sources
PubMed1995

Protein sulfhydryls and their role in the antioxidant function of protein S-thiolation.

Protein S-thiolation/dethiolation, i.e., the oxidation of protein sulfhydryls to mixed disulfides and their reduction back to sulfhydryls, is an early cellular response to oxidative stress (1-5). This response may be elicited by oxidative phenomena of diverse origins, and the few cases that have been studied extensively give a limited insight into the metabolic roles and the molecular mechanism of the process. Much of our current understanding arose from experiences with isolated proteins containing "reactive" sulfhydryls (6, 7), but recent experiments at the cellular level have begun to reveal interesting insights that suggest a complexity and importance not appreciated previously (8). This article will discuss the current status of experiments that relate to both the role of the cellular process and to the reactivity of selected proteins that participate in the cellular processes. The discussion will center on the role of glutathione, a molecule of central interest in every aspect of protein S-thiolation and dethiolation.

Animals↗
PubMed1995

Use of the rectus abdominis myocutaneous flap in treating advanced carcinomas of the penis.

OBJECTIVE: To examine the methods used to obtain skin closure after radical surgery for carcinomas of the penis, with special reference to the rectus abdominis myocutaneous flap. PATIENTS AND METHODS: Over a 20 year period, 21 patients underwent surgery for carcinomas of the penis. Seven had advanced tumours, requiring radical surgery for complete local tumour control. The resulting wounds were repaired with scrotal skin transfer in three patients and with a rectus abdominis myocutaneous flap in the other four. RESULTS: These methods of repair were a 100% reliable, all wounds being healed within 10 days. CONCLUSIONS: Scrotal skin transfer is a simple and effective method of achieving wound closure. Where removal of the scrotum is required along with curative or palliative surgery, and after ilio-inguinal lymphadenectomy, a rectus abdominis myocutaneous flap is useful for skin closure.

Aged↗
PubMed1995

Induction of latent membrane protein expression in in vitro Epstein-Barr virus-infected leukaemic B lymphocytes by interleukin 4 and antibodies to CD40.

Chronic lymphocytic leukaemia (CLL) B cells are clones representing the mature B cell phenotype. On infection with Epstein-Barr virus (EBV) CLL cells express the EB nuclear antigen (EBNA) complex but unlike EBV-infected normal B cells they do not express LMP nor do they proliferate or immortalize. Furthermore, EBV-CLL rapidly die by apoptosis in culture. In the present study we have used the B cell growth factors interleukin 4 and antibodies to CD40 to induce activation and proliferation of EBV-infected CLL cells. Although cell numbers did not significantly increase, apoptosis was partially inhibited in CLL cells which expressed increased levels of CD23 and were activated to immunoglobulin-secreting lymphoblasts. Expression of LMP was induced by interleukin (IL)-4 and anti-CD40 in all five EBV-infected CLL samples examined. However, this did not enhance cell proliferation or induce immortalization. Further analysis showed that LMP could be detected 4-5 days after EBV infection, and that both IL-4 and anti-CD40 could independently induce LMP but that their effect was additive. These results indicate that LMP expression is dependent on B cell activation processes and that in some circumstances full latent viral gene expression is not sufficient to cause B cell immortalization.

Antibodies↗
PubMed1994

S-thiolation of glyceraldehyde-3-phosphate dehydrogenase induced by the phagocytosis-associated respiratory burst in blood monocytes.

Chemical oxidants can induce the covalent binding of low molecular weight thiols to reactive sulfhydryls on proteins (S-thiolation). We found that stimulation of the respiratory burst of human blood monocytes resulted in S-thiolation of several proteins, most prominently one of 38 kDa. This purified protein was identified as glyceraldehyde-3-phosphate dehydrogenase (GAPDH) by enzyme activity, immunoblotting, and amino acid analysis. After stimulation of the respiratory burst, S-thiolation of GAPDH gradually increased, and cytosol GAPDH activity decreased; so that at 60 min, GAPDH activity was reduced by approximately 40%. Activity was restored by the addition of the sulfhydryl-reducing agent dithioerythritol. H2O2 appeared to be particularly important in mediating S-thiolation during the respiratory burst. Exposure of monocytes to H2O2 induced concentration-dependent S-thiolation of GAPDH and a concomitant decrease in enzyme activity. The addition of respiratory burst stimuli to lymphocytes, which lack a full respiratory burst, had no effect on GAPDH S-thiolation or activity; but H2O2 induced S-thiolation of lymphocyte GAPDH and inhibition of enzyme activity. Stimulation of monocytes from three patients with chronic granulomatous disease resulted in no respiratory burst, S-thiolation of GAPDH, or inactivation of GAPDH activity. The thiols covalently bound to purified S-thiolated GAPDH were removed by dithioerythritol and were identified as glutathione and cysteine; glutathione was predominant. These results indicate that during the respiratory burst in monocytes, low molecular weight thiols can bind to specific cytosolic proteins, including GAPDH. It is possible that S-thiolation of cytosolic proteins serves to modulate cellular metabolic events during phagocytosis.

Ethylmaleimide↗
PubMed1994

Rapid dendritic cell recruitment is a hallmark of the acute inflammatory response at mucosal surfaces.

Immunohistochemical analysis of challenge sites such as skin and the peritoneal cavity has identified neutrophils as virtually the sole cellular participants in acute bacterial inflammation, peak influx occurring 24-48 h in advance of mononuclear cell populations associated with adaptive immunity. This study challenges the general applicability of this paradigm. We demonstrate here that the earliest detectable cellular response after inhalation of Moraxella catarrhalis organisms is the recruitment of putative class II major histocompatibility complex-bearing dendritic cell (DC) precursors into the airway epithelium, the initial wave arriving in advance of the neutrophil influx. Unlike the neutrophils which rapidly transit into the airway lumen, the DC precursors remain within the epithelium during the acute inflammatory response where they differentiate, and develop the dendriform morphology typical of resident DC found in the normal epithelium. During the ensuing 48-h period, these cells then migrate to the regional lymph nodes. No comparable DC response was observed after epidermal or intraperitoneal challenge, and it may be that mucosal surfaces are unique in their requirement for rapid DC responses during acute inflammation. We hypothesize that the role of the DC influx during acute inflammation may be surveillance for opportunistic viruses, and that this covert protective mechanism is operative at a restricted number of mucosal tissue sites.

Animals↗
PubMed1994

Protein S-thiolation in hepatocytes stimulated by t-butyl hydroperoxide, menadione, and neutrophils.

In order to examine potentially important S-thiolated proteins, 35S-labeled hepatocytes were exposed to oxidative stress. A similar group of S-thiolated proteins including carbonic anhydrase III was observed in cells treated with t-butyl hydroperoxide, menadione, or stimulated neutrophils. The radioactive thiols bound to hepatocyte proteins were identified by HPLC and more than 85% was glutathione. In menadione-treated hepatocytes, proteins were gradually S-thiolated over 30 min and 25% of the cellular glutathione pool became protein-bound. In t-butyl hydroperoxide-treated cells, S-thiolation was more transient and 11% of the glutathione was protein-bound. Neutrophil-treated hepatocytes had nearly the same amount of protein S-thiolation (8% after 25 min). Two major proteins that were S-thiolated in untreated hepatocytes did not increase during any form of oxidative stress. In neutrophil-treated hepatocytes protein S-thiolation was not accompanied by either formation of glutathione disulfide or a measurable change in the total amount of glutathione. In both t-butyl hydroperoxide-and menadione-treated cells there was extensive formation of glutathione disulfide and in menadione-treated cells a significant increase in the total hepatocyte glutathione pool was observed. This result suggests that protein S-thiolation may occur by mechanisms that do not result from thiol/disulfide exchange between glutathione disulfide and protein sulfhydryls. It is suggested that a thiyl radical intermediate is important in neutrophil-mediated protein S-thiolation.

Animals↗
PubMed1994

S-thiolation of individual human neutrophil proteins including actin by stimulation of the respiratory burst: evidence against a role for glutathione disulfide.

Protein S-thiolation, a reversible modification of protein sulfhydryls resulting in formation of mixed-disulfides, was studied in human neutrophils stimulated with phorbol diester to produce superoxide anion. Rapid S-thiolation of several proteins was examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Glutathione was identified as the primary protein-bound thio by HPLC chromatography, contributing considerably more than 85% of the total. Minor amounts of homocysteine and/or cysteine were also detected as protein-bound thiols. During the first 30 min after stimulation, 10% of the cellular glutathione became protein bound (2 nmol/mg of protein). There was no increase in glutathione disulfide suggesting that S-thiolation of the proteins did not occur by thiol/disulfide exchange. Approximately 10 mol% of one heavily modified band (29 kDa) was S-thiolated after 30 min. A second major band of 42 kDa was identified as actin. It contained 1/10th of the total protein-bound glutathione and approximately 5 mol% was S-thiolated after 30 min. These experiments identify a subset of S-thiolated neutrophil proteins, including actin, whose modification is related to the phorbol diester stimulation of superoxide anion production in human neutrophils. Ten percent of the total glutathione pool became protein-bound without an appreciable change in non-bound concentration of glutathione or glutathione disulfide. These results suggest that glutathione was synthesized during initial phases of the respiratory burst, compensating for the amount of glutathione that became protein-bound. Since there was no significant increase in glutathione disulfide, it was probably not important in the observed protein S-thiolation.

Actins↗
PubMed1994

Effects of substitution of Thr63 by alanine on the structure and function of Lactobacillus casei dihydrofolate reductase.

A mutant of Lactobacillus casei dihydrofolate reductase has been constructed in which Thr63, a residue which interacts with the 2'-phosphate group of the bound coenzyme, is replaced by alanine. This substitution does not affect kcat, but produces an 800-fold increase in the Km for NADPH, which reflects dissociation of NADPH from the enzyme-NADPH-tetrahydrofolate complex, and a 625-fold increase (corresponding to 3.8 kcal/mol) in the dissociation constant for the enzyme-NADPH complex. The difference in magnitude of these effects indicates a small effect of the substitution on the negative cooperativity between NADPH and tetrahydrofolate. Stopped-flow studies of the kinetics of NADPH binding show that the weaker binding arises predominantly from a decrease in the association rate constant. NMR spectroscopy was used to compare the structures of the mutant and wild-type enzymes in solution, in their complexes with methotrexate and with methotrexate and NADPH. This showed that only minimal structural changes result from the mutation; a total of 47 residues were monitored from their resolved 1H resonances, and of these nine in the binary complex and six in the ternary differed in chemical shift between mutant and wild-type enzyme. These affected residues are confined to the immediate vicinity of residue 63. There is a substantial difference in the 31P chemical shift of the 2'-phosphate of the bound coenzyme, reflecting the loss of the interaction with the side chain of Thr63. The only changes in nuclear Overhauser effects (NOEs) observed were decreases in the intensity of NOEs between protons of the adenine ring of the bound coenzyme and the nearby residues Leu62 and Ile102, showing that the substitution of Thr63 does cause a change in the position or orientation of the adenine ring in its binding site.

Alanine↗
PubMed1994

Are incentive spirometry, intermittent positive pressure breathing, and deep breathing exercises effective in the prevention of postoperative pulmonary complications after upper abdominal surgery? A systematic overview and meta-analysis.

BACKGROUND AND PURPOSE: The purpose of this meta-analysis was to quantitatively assess the conflicting body of literature concerning the efficacy of incentive spirometry (IS), intermittent positive pressure breathing (IPPB), and deep breathing exercises (DBEX) in the prevention of postoperative pulmonary complications in patients undergoing upper abdominal surgery. METHODS: Computerized searches of MEDLINE and the Cumulative Index to Nursing and Allied Health databases were performed for the years 1966 through 1992. Citations were selected based on the following relevance criteria: (1) patients undergoing any type of upper abdominal surgery; (2) any combination of IS, IPPB, and DBEX; (3) an outcome of pulmonary complications; and (4) randomized trials. Review of 116 citations yielded 55 potential trials and 10 review articles. Of these, 14 citations were included in the overview based on relevance criteria requirements. Study validity was assessed by two independent observers, and data were extracted. RESULTS: The common odds ratio (COR) for the occurrence of pulmonary complications for IS versus no physical therapy was 0.44 in favor of IS. The COR for DBEX versus no physical therapy was 0.43 in favor of DBEX. Both findings were statistically significant. The CORs for IS versus IPPB, IS versus DBEX, and IPPB versus DBEX were 0.76 (95% confidence interval [CI] = 0.39-1.4), 0.91 (95% CI = 0.57-1.4), and 0.94 (95% CI = 0.28-3.17), respectively. None of these comparisons reached statistical significance. CONCLUSION AND DISCUSSION: Incentive spirometry and DBEX appear to be more effective than no physical therapy intervention in the prevention of postoperative pulmonary complications. There is no evidence to support a significant difference between any of the three modalities. [Thomas JA, McIntosh JM. Are incentive spirometry, intermittent positive pressure breathing, and deep breathing exercises effective in the prevention of postoperative pulmonary complications after upper abdominal surgery? a systematic overview and meta-analysis.

Abdomen↗
PubMed1994

A murine cytomegalovirus-neutralizing monoclonal antibody exhibits autoreactivity and induces tissue damage in vivo.

The autoreactivity of murine cytomegalovirus (MCMV)-neutralizing monoclonal antibody (mAb) AC1 was examined in vitro and in vivo. Both mAb AC1 and a human antiserum reactive with U1-small nuclear ribonucleoprotein (U1-snRNP) stained uninfected mouse embryo fibroblasts (MEF) in a speckled nuclear pattern and reacted with 70,000 molecular weight (MW) MEF nuclear antigens by immunoblotting, suggesting that mAb AC1 cross-reacted with the 70,000 MW component of U1-snRNP. However, only mAb AC1 cross-reacted with an additional epithelial cytoplasmic autoantigen present in cultured HEp2 cells. On tissue sections from uninfected mice, mAb AC1 predominantly reacted with a component of central and peripheral nervous systems, although cross-reactivity with the stratum spinosum of the skin and the outer sheath of hair follicles was also observed. Immunoblotting revealed that mAb AC1 reacted with phosphorylated epitopes present on a 98,000 MW MCMV structural protein and the 200,000 MW mouse neurofilament protein (NFP). Treatment of uninfected mice with mAb AC1 resulted in a severe interstitial pneumonia with greatly thickened and congested alveolar septa. Severe oedema of the hypodermis and a mild mesangial proliferative glomerulonephritis were also observed. These results demonstrate that a mAb reacting with a MCMV structural phosphoprotein which can protect mice against the dissemination of MCMV, can also promote the development of autoimmune disease. Therefore, the production of such cross-reactive antibodies may be an important mechanism in the development of autoimmunity following viral infection.

Animals↗