Biomedical subjects
C Bowman
Publications and source records attributed to C Bowman.
Specific tryptophan substitution in catalytic sites of Escherichia coli F1-ATPase allows differentiation between bound substrate ATP and product ADP in steady-state catalysis.
Tryptophan was specifically inserted as the residue immediately preceding the P-loop sequence in F1-ATPase catalytic sites. The mutant enzyme (betaF148W) showed normal enzymatic characteristics. The fluorescence responses of beta-tryptophan 148 enabled us to differentiate between nucleoside di- and triphosphate bound in catalytic sites; MgADP quenched at 350 nm, whereas MgAMPPNP and MgADP.BeFx complex enhanced the fluorescence at 325 nm. With MgATP, both effects were seen simultaneously. This allowed analysis of bound catalytic site nucleotides directly under steady-state MgATP hydrolysis conditions. At mM concentration of MgATP (Vmax conditions) one of the three catalytic sites was filled with substrate MgATP and the other two sites were filled with product MgADP. A model for F1-ATPase steady-state turnover is presented that encompasses these findings. Given the structural similarity of the P-loop in nucleotide-binding proteins, this approach may prove widely useful.
alpha-Aspartate 261 is a key residue in noncatalytic sites of Escherichia coli F1-ATPase.
X-ray structure analysis of the noncatalytic sites of F1-ATPase revealed that residue alpha-Asp261 lies close to the Mg of bound Mg-5'-adenylyl-beta,gamma-imidodiphosphate. Here, the mutation alpha D261N was generated in Escherichia coli and combined with the alpha R365W mutation, allowing nucleotide binding at F1 noncatalytic sites to be specifically monitored by tryptophan fluorescence spectroscopy. Purified alpha D261N/alpha R365W F1-ATPase showed catalytic activity similar to wild-type. An important feature was that, without any resort to nucleotide-depletion procedures, the noncatalytic sites in purified native enzyme were already empty. Binding studies with MgATP, MgADP, and the corresponding free nucleotides led to the following conclusions. Residue alpha-Asp261 interacts with the Mg of Mg-nucleotide in noncatalytic sites and provides a large component of the binding energy (approximately 3 kcal/mol). It is the primary determinant of the preference of noncatalytic sites for Mg-nucleotide. The natural ligands at these sites in wild-type enzyme are the Mg-nucleotides and free nucleotides bind poorly. Under conditions where noncatalytic sites were empty, alpha D261N/alpha R365W F1 showed significant hydrolysis of MgATP. This established unequivocally that occupancy of noncatalytic sites by nucleotide is not required for catalysis.
Methicillin-resistant Staphylococcus aureus (MRSA) in the community.
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Health system reform. Physicians' views on the critical choices.
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Position of The American Dietetic Association: health implications of dietary fiber.
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Community and medical staff collaborate in needs assessment.
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Community-based case management of HIV disease.
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The effects of immersion and exercise on prolactin during pregnancy.
Prolactin is an important hormone during pregnancy, affecting mother, fetus, and amniotic fluid volume. Immersion is known to affect prolactin levels significantly. To determine the effect of immersion and exercise on the prolactin response during pregnancy, we examined serum prolactin levels at 15, 25, and 35 weeks' gestation and 10 weeks post partum. Twelve women completed 20 min land rest, 20 min immersion in 30 degrees C water to the xiphoid, and 20 min exercise in the water at 60% VO2max. Resting prolactin levels were 1.91 +/- 0.32, 4.55 +/- 0.5, and 5.85 +/- 0.27 nmol.l-1 +/- standard error of the mean at 15, 25, and 35 weeks' gestation, respectively. Postpartum lactating women had a resting mean prolactin level of 3.95 +/- 1.6 versus 0.22 +/- 0.4 nmol.l-1 in non-lactating women. Prolactin levels declined significantly during immersion even after correction for dilution by plasma volume shifts. The immersion response was inversely related to the duration of pregnancy with 29%, 22%, and 12% drops during 15-, 25- and 35-week trials, respectively. Compared to rest, exercise prolactin levels remained depressed during the 15th and 25th week trials. We hypothesize that immersion in water caused prolactin levels to decline.
Renal responses to immersion and exercise in pregnancy.
Twelve healthy pregnant women were studied at 15, 25, and 35 weeks' gestation and at 8 to 12 weeks postpartum. Women were immersed for 20 minutes at 30 degrees C. They then exercised at 60% maximum oxygen capacity on a modified ergometer. Substantial diuresis and natriuresis occurred without changes in osmolarity or serum sodium. The diuresis was significantly greater during pregnancy than postpartum. The natriuresis was similar. Diuresis and natriuresis were greater than would be expected from investigations in nonpregnant subjects. This study suggests that immersion may be a beneficial therapy for edema without decreasing plasma volume.
Kinetics and pathogenicity of autoantibodies induced by mercuric chloride in the brown Norway rat.
Repeated low-dose injections of mercuric chloride (HgCl2) in the brown Norway (BN) rat result in polyclonal activation which includes the induction of anti-glomerular basement membrane (GBM) autoantibodies. We examined the kinetics of various autoantibodies produced in vivo, general features of polyclonal activation such as total IgG levels and immune complex formation, and the relationship between organ specific autoimmunity and tissue injury in the kidney and thyroid. The production of immune complexes and autoantibodies to GBM and thyroglobulin was short lived, and the increase in levels of total IgG and antibodies to ssDNA and dsDNA was prolonged; the antibody response to collagen types I and II was intermediate in duration. Autoantibodies induced by HgCl2 caused only mild and variable tissue injury in the kidneys and did not induce abnormalities in the thyroid. These studies demonstrate that immunostimulation by mercury may result in the formation of a range of autoantibodies, with variable kinetics and pathogenicity.
Transfacial resection of intracranial tumor.
Tumors of the skull base with intracranial and extracranial components pose special access problems. Traditional craniotomy and extensive external incisions produce morbidity that can be avoided in properly selected cases. Current surgical techniques extend previously described operations to allow good visualization and safe resection through the face for certain intracranial tumors. Meticulous attention to surgical techniques and wide exposure for complete tumor visualization are essential to ensure the safety of these procedures.
The effect of endotoxin on peripheral blood lymphocytes in the rat.
We have studied the effects on peripheral blood lymphocytes of the administration to rats of a single dose of endotoxin. Lymphopenia occurred during the first hour, and persisted during 24 hours. Lymphocyte subpopulations were examined by flow cytometry. T-lymphocytes were relatively resistant to the initial effects of endotoxin, but there were no differences in the responses of cells identified as T-helper or T-suppressor/cytotoxic lymphocytes. The effects of endotoxin on peripheral blood lymphocytes is less extreme but appears more persistent than the effects on granulocytes.
Autoimmunity and glomerulonephritis.
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Circulating T-cell populations during mercuric chloride-induced nephritis in the Brown Norway rat.
Fluorescence-activated cell sorter analysis was used to study the peripheral lymphocyte populations during mercuric chloride (HgCl2)-induced autoimmune nephritis in the Brown Norway (BN) rat. Sequential studies showed a transient loss of T cells from peripheral blood attributable to decreases in the percentage of T-helper cells. In addition, there was a decrease in the percentage of T-cytotoxic/suppressor cells prior to the appearance of circulating anti-GBM antibodies, followed by elevated levels of T-suppressor cells during down-regulation of the response. This method may allow closer inspection of the events linking changes in T-cell populations and induction and termination of an autoimmune response.
Restriction of human IgG subclass expression in the population of auto-antibodies to glomerular basement membrane.
To study the possible relationship between individual subclass expression and pathogenesis of antibody-mediated disease, we examined the immunoglobulin-G subclass of antiglomerular basement membrane (GBM) antibodies, in the sera of 20 patients with auto-antibody mediated nephritis, as well as in a limited number of kidney eluates, using a solid phase radioimmunoassay and monoclonal antibodies specific for human IgG subclasses. Only anti-GBM antibodies of the IgG1 and/or IgG4 subclass were detectable, both in the circulation and in renal eluates. Recurrence of circulating anti-GBM antibody during convalescence occurred in two patients out of 49 studied sequentially for more than 12 months. In both cases only antibodies of a single subclass were involved. The recurrence of IgG1 antibody (which can fix complement and bind macrophages) was associated with clinical manifestations of disease, whereas the reappearance of IgG4 antibody (which cannot engage amplifiers of the inflammatory response) did not appear to be harmful. Thus, in anti-GBM auto-antibody mediated nephritis, clear restriction in the subclass of IgG auto-antibody occurs, and this may be important in disease expression.
Antiglomerular basement membrane antibody mediated disease in the British Isles 1980-4.
Clinical and pathological data on 71 patients from throughout the British Isles who developed antiglomerular basement membrane antibody mediated nephritis in the period 1980-4 were studied. Two principle patterns of disease were recognised: young men presenting in their 20s with Goodpasture's syndrome (glomerulonephritis and lung haemorrhage) and women presenting in their 60s with glomerulonephritis alone. The effect of treatment on prognosis of a total of 108 patients was also reviewed (the 71 patients plus patients seen before 1980 at Hammersmith Hospital). Treatment with prednisolone, cytotoxic drugs, and plasma exchange hastened the time to clearance of autoantibody and improved the outlook of patients who were not dependent on dialysis and those with lung haemorrhage.