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Biomedical subjects

S Marshall

Publications and source records attributed to S Marshall.

At least 19 recordsLinked to original sources
PubMed1992

Molecular cloning, cDNA sequence, and bacterial expression of human glutamine:fructose-6-phosphate amidotransferase.

Glutamine:fructose-6-phosphate amidotransferase (GFAT) has recently been shown to be an insulin-regulated enzyme that plays a key role in the induction of insulin resistance in cultured cells. As a first step in understanding the molecular regulation of this enzyme the human form of this enzyme has been cloned and the functional protein has been expressed in Escherichia coli. A 3.1-kilobase cDNA was isolated which contains the complete coding region of 681 amino acids. Expression of the cDNA in E. coli produced a protein of approximately 77 kDa and increased GFAT activity 4.5-fold over endogenous bacterial levels. Recombinant GFAT activity was inhibited 51% by UDP-GlcNAc whereas bacterial GFAT activity was insensitive to inhibition by UDP-GlcNAc. On the basis of these results we conclude that: 1) functional human GFAT protein was expressed, and 2) the cloned human cDNA encodes both the catalytic and regulatory domains of GFAT since the recombinant GFAT was sensitive to UDP-GlcNAc. Overall, the development of cloned GFAT molecular probes should provide new insights into the development of insulin resistance by allowing quantitation of GFAT mRNA levels in pathophysiological states such as non-insulin-dependent diabetes mellitus and obesity.

Amino Acid Sequence↗
PubMed1992

Impairment of left ventricular diastolic function in systemic lupus erythematosus.

Left ventricular (LV) diastolic performance was evaluated with pulsed-wave Doppler echocardiography in a cross-sectional population of patients with systemic lupus erythematosus (SLE) in search of subclinical myocardial involvement. Such involvement is reported to occur infrequently, despite pathohistologic evidence of myocarditis in up to 70% of patients with SLE. Thirty-five consecutive patients with SLE were evaluated, 14 with active and 21 with inactive disease, and were compared with 30 age-matched healthy control subjects. Twenty-six patients were restudied at 7 months. All had normal LV systolic function, normal pericardial and valvular structures, and no significant valvular regurgitation on Doppler echocardiography. In SLE patients with active disease, indexes of LV diastolic function differed significantly from the inactive group and from control subjects, with marked prolongation of isovolumic relaxation time (104 +/- 18 vs 74 +/- 13 ms, p = 0.0001), as well as reduced peak early diastolic filling velocity (E) (0.69 +/- 0.19 vs 0.83 +/- 0.17 ms, p = 0.01), reduced ratio of early to late diastolic flow velocity (E/A) (1.15 +/- 0.53 vs 1.47 +/- 0.35, p = 0.02), and prolonged mitral pressure halftime (74 +/- 14 vs 65 +/- 8 ms p = 0.01). Similar significant differences were found between the active and inactive SLE patient groups. SLE patients with inactive disease differed from control subjects in only mild prolongation of mitral pressure halftime. Abnormal prolongation of isovolumic relaxation (greater than 100 ms) was found to be the most useful marker of diastolic impairment, being present in 64% of SLE patients with active disease and in 14% of patients with inactive disease.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1992

Insulin regulation of pyruvate kinase activity in isolated adipocytes. Crucial role of glucose and the hexosamine biosynthesis pathway in the expression of insulin action.

We recently identified glutamine:fructose-6-phosphate amidotransferase (GFAT) as an insulin-regulated enzyme in adipocytes. Moreover, we found that loss of GFAT activity is not due to a direct action of insulin but rather is mediated by enhanced glucose uptake and the subsequent routing of glucose through the hexosamine biosynthesis pathway. To assess whether other cytosolic enzymes are controlled through formation of hexosamine products, we treated adipocytes for 5 h with physiological concentrations of insulin (ED50 = 0.33 ng/ml), glucose (ED50 = 4.5 mM), and glutamine (ED50 = 4.4 mM) and then measured pyruvate kinase (PK) activity. Combined treatment resulted in a progressive (t 1/2 of 2.5 h) and marked (3-fold) increase in PK activity, whereas omission of one or more of these components failed to alter enzyme activity. Several lines of additional evidence implicated the hexosamine biosynthesis pathway in PK regulation; therefore, it appears that the M2 isoform of pyruvate kinase represents another enzyme regulated by insulin through stimulation of glucose uptake and formation of hexosamine products. Related studies revealed that enhancement of PK activity is dependent upon ongoing mRNA synthesis and de novo protein synthesis and is mediated by an increase in enzyme content. Considered together, these findings provide new insights into the cascade of metabolic events triggered by insulin and implicated a novel metabolic pathway in the pretranslational control of enzyme function.

Adipose Tissue↗
PubMed1992

Cardiac function after domino-donor heart transplantation.

A major limitation in cardiac transplantation is donor availability. A possible way to increase the supply of donor hearts is to use explanted hearts from patients undergoing heart-lung transplantation for primary lung disease. One potential advantage of this approach, termed domino-donor transplantation, is the existence of a donor right ventricle already adapted to pulmonary hypertension, which would therefore theoretically decrease the likelihood of acute donor right heart failure in recipients with preexisting elevation of pulmonary vascular resistance. Potential disadvantages include graft failure secondary to chronic effects of pulmonary hypertension on the right ventricle, arrhythmia and infections. Seven domino-donor transplants were performed at Stanford University Hospital; graft and patient survival to date are 100% at a mean follow-up of 20 months (range 1 to 26). Infection and rejection rates have been comparable to those of the current Stanford experience for conventional orthotopic transplantation. Right ventricular function and size have either improved or remained unchanged in all patients after transplantation. Transient early postoperative donor right ventricular dilation, a characteristic adaptive response seen in nondomino transplants, occurred in 4 patients with pulmonary hypertension before surgery. These data indicate that, with adequate assessment before surgery, domino-donor cardiac transplantation is an appropriate means of augmenting the donor pool.

Actuarial Analysis↗
PubMed1992

Prooxidant properties of 5-aminosalicylic acid. Possible mechanism for its adverse side effects.

There is a growing body of experimental and clinical evidence to suggest that oral or rectal administration of 5-ASA or 5-ASA conjugates is associated with significant adverse side effects including pancreatitis, hepatitis, and renal toxicity. The objective of this study was to assess the ability of 5-ASA to interact with low-molecular-weight iron to yield oxygen-derived free radicals and to determine whether these oxidants could damage model biological compounds. We found that 5-ASA was very effective at chelating ferric iron (Fe3+), and it rapidly reduced Fe3+ to the ferrous form (Fe2+). Addition of the 5-ASA/Fe2+ chelate to solutions containing polyunsaturated fatty acids or deoxyribose resulted in lipid peroxidation and oxidative carbohydrate degradation, respectively. These results are consistent with the formation of the highly reactive (and cytotoxic) hydroxyl radical. Formation of this free radical species was confirmed by the ability of hydroxyl radical scavengers (dimethyl sulfoxide, dimethyl thiourea) to inhibit the 5-ASA/Fe-mediated oxidative reactions. Maximum hydroxyl radical formation was achieved at a 5-ASA-to-Fe3+ ratio of 1.0 (20 microM 5-ASA and 20 microM Fe3+). Increasing this ratio significantly inhibited OH. formation with a concomitant reduction in lipid peroxidation and deoxyribose degradation. Finally, we demonstrated that 5-ASA promotes the reductive release of Fe3+ from ferritin. Data obtained in this study suggest that 5-ASA may, under certain conditions, promote the formation of potentially injurious free radical species. These oxidative reactions may contribute to some of the adverse side effects known to be associated with the newer preparations of 5-ASA.

Aminosalicylic Acids↗
PubMed1992

A comparative analysis of two models of colitis in rats.

Two models of colitis produced in rats that have received significant attention over the past few years are the acetic acid and trinitrobenzene sulfonic acid (TNBS) models. The objective of this study was to quantify and compare the temporal relationship among mucosal permeability, epithelial injury, and inflammation induced by acetic acid, ethanol (vehicle), ethanol plus TNBS (unbuffered, pH 1.0), and ethanol plus TNBS (pH 7.4). Data obtained show that the inflammation induced by these four irritants results from caustic injury to the colonic epithelium and interstitium as measured by the rapid and dramatic increases in mucosal permeability and tissue water content as well as by histological analysis. The injurious nature of TNBS was confirmed in a separate series of studies showing that buffered TNBS (pH 7.4), in the absence of ethanol, is toxic to cultured rat intestinal epithelial cell monolayers. Only after 1-2 days of the initial insult, were signs of classical inflammation observed, including increases in colonic myeloperoxidase activity (neutrophil infiltration) and colon weight as well as hyperemia and mucosal ulcerations. Although ethanol plus TNBS (pH 1.0 or 7.4) tended to produce higher mucosal permeabilities (epithelial cell injury) at 1-2 weeks after the enemas than acetic acid or ethanol groups, only the ethanol plus TNBS (pH 7.4) permeabilities were found to be significantly enhanced. In addition, all four groups showed significant elevations in colonic myeloperoxidase activity and colon weight at 1-2 weeks after enema. It is suggested that these models of colitis are useful to study events that occur at the time of inflammation and repair. However, these models may have significant limitations in understanding events that initiate inflammation of the intestine in human inflammatory bowel disease.

Acetates↗
PubMed1992

Nebulized fenoterol causes greater cardiovascular and hypokalaemic effects than equivalent bronchodilator doses of salbutamol in asthmatics.

The pulmonary and extrapulmonary effects of two doses of nebulized fenoterol (5 mg) salbutamol (5 mg) and ipratropium bromide (0.5 mg) at 60 min intervals were compared in nine patients with asthma in a double-blind, randomized study. Measurements of heart rate, blood pressure, electromechanical systole (QS2I), QTc interval, FEV1 and plasma potassium were made at baseline and at 15, 30 and 60 min after each nebulization. Both beta-agonists caused significantly greater inotropic (QS2I), chronotropic (HR), electrocardiographic (QTc) and hypokalaemic effects than ipratropium bromide (IB), with fenoterol being more potent than salbutamol. Fenoterol had no greater effect on FEV1 than salbutamol although both were superior to IB. Only the first four subjects had two doses as originally intended, because the second administration of fenoterol resulted in marked cardiovascular effects and hypokalaemia. The observed differences in extrapulmonary effects between fenoterol and salbutamol provide a plausible group of mechanisms which may explain the increased risk of death associated with fenoterol in severe asthmatics.

Adult↗
PubMed1992

Normal growth with renal insufficiency owing to posterior urethral valves: value of long-term diversion. A twenty-year follow-up.

Twenty-five patients with posterior urethral valves have been monitored for 10-29 years (mean 18 years). Eight underwent diversion because of impaired renal function (creatinine greater than 0.8 mg/dl) accompanied by unimproved upper tract form and function after valve ablation. In the nondiverted group, 6 patients (35%) progressed to renal failure; in the diverted group 5 patients (63%) did so (p = 0.30). Of 13 patients whose lowest serum creatinine in the first year after valve ablation was greater than 0.8 mg/dl, 9 (69%) went on to renal failure (p = 0.02); in the remaining 12 patients, only 2 (17%) did so (p = 0.008). The mean age for renal failure was 13 years for both groups. The Tanner scale rating (mean percent height) for all patients at 10 years was 61% for the diverted group and 35% for those without diversion (p = 0.004). When the patients treated after the age of 1 year (n = 7) were excluded from the calculation, the rating remained different (54% diversion, 26% no diversion; p = 0.012). We conclude that the boy born with urethral valves and serious upper tract damage is at risk for renal failure, especially during the teen years. An undefined relationship appears to exist between urinary obstruction and growth. In our study, the boys who underwent diversion continued to grow along a normal Tanner height curve.

Adolescent↗
PubMed1992

Detection of inspiratory resistive loads by heart-lung transplant recipients.

Human heart-lung transplantation recipients (HL) are models for the effects of selective lower respiratory tract denervation. We used a series resistor to examine thresholds of inspiratory flow resistance perception among 16 HL in comparison with 15 normal control subjects (C) and 12 heart transplant recipients (H). HL and C were closely age matched. H subjects were older and had mildly reduced lung volumes compared with the other groups. The perception threshold was defined as the added resistance (delta R) detected by subjects at 50% of random, blinded presentations. These delta R were subsequently expressed as a fraction of the airway resistance of the subject and baseline resistance of the apparatus (delta R/Raw + Rapparatus), otherwise known as the Weber fraction. The mean +/- SEM Weber fractions were nearly identical for HL and C (0.32 +/- 0.05 versus 0.34 +/- 0.05, respectively). The Weber fraction of H (0.74 +/- 0.17) was significantly greater than those of both other groups (p less than 0.05 by Kruskal-Wallis test). We conclude that lower respiratory tract afferents do not play a significant role in the perception of inspiratory resistive loads. The finding of an abnormally high Weber fraction in the heart transplant population remains unexplained but may be a function of age or restrictive pulmonary defects.

Adult↗
PubMed1991

No evidence for social class inequalities in intervention for coronary heart disease in Otago 1987-9.

OBJECTS: to investigate whether social class differences in access to cardiac surgical interventions (angioplasties and coronary artery bypass grafting) could explain social class inequalities in mortality from coronary heart disease. METHODS: rates of therapeutic interventions to treat coronary heart disease were calculated for male patients aged less than 65 years admitted for the first time to Dunedin Hospital with a principal diagnosis of ischaemic heart disease (ICD codes 410-414) during the three year period from 1 January 1987 to 31 December 1989. Patients were categorised into different socioeconomic groups using the Elley-Irving social class scale. RESULTS: no statistically significant trend across social class was observed in the cumulative incidence of cardiac surgical interventions (angioplasties, coronary artery bypass grafts). Similarly there was no statistically significant trend across social class in the incidence rates of cardiac surgical interventions, even after adjustment for age. CONCLUSIONS: social class inequalities in access to cardiac surgical intervention do not appear to explain the observed inequalities in mortality from coronary heart disease.

Angioplasty, Balloon, Coronary↗
PubMed1991

Coordinated regulation of glutamine:fructose-6-phosphate amidotransferase activity by insulin, glucose, and glutamine. Role of hexosamine biosynthesis in enzyme regulation.

We reported previously that glutamine:F-6-P amidotransferase (GFAT) plays an integral role in the development of insulin resistance by directing the flow of incoming glucose into the hexosamine biosynthesis pathway. To determine whether the enzymatic activity of GFAT is altered during desensitization of the glucose transport system, we treated isolated rat adipocytes with various combinations of insulin, glucose, and glutamine. Treatment with insulin or glucose alone (or in combination) failed to reduce cytosolic GFAT activity after 4 h, whereas combined treatment with all three components elicited a progressive loss of GFAT activity that was rapid (t1/2 of 2 h) and extensive (70% loss). A pronounced loss of GFAT activity was also seen in cells exposed to glucosamine, an agent known to directly enter the hexosamine pathway (55% loss at 4 h, ED50 of 360 microM). Moreover, a close correlation was observed between the induction of desensitization and the loss of GFAT activity as a function of glucose, insulin, glutamine, and glucosamine concentrations. When total intracellular hexosamine products were measured, we found that hexosamine formation was unaltered by insulin or glucose (or a combination) but was elevated by greater than 4-fold in the presence of insulin, glucose, and glutamine (t1/2 of 22 min), a condition known to cause both desensitization and loss of GFAT activity. Additional studies indicated that the loss of GFAT activity under desensitizing conditions is not due to allosteric regulation since removal of potential allosteric factors from the cytosol of desensitized cells by G-25 column chromatography failed to restore enzyme activity. Overall, these studies indicate that 1) GFAT is an insulin-regulated enzyme; however, control of enzyme activity is not due to a direct action of insulin, but rather is mediated by insulin-induced enhancement of glucose uptake; 2) the routing of incoming glucose through the hexosamine pathway and the formation of hexosamine products appears to regulate GFAT activity; and 3) the progressive loss of GFAT activity over several hours is probably not due to allosteric regulation.

Adipose Tissue↗
PubMed1991

Complete inhibition of glucose-induced desensitization of the glucose transport system by inhibitors of mRNA synthesis. Evidence for rapid turnover of glutamine:fructose-6-phosphate amidotransferase.

Glutamine:fructose-6-phosphate amidotransferase (GFAT) plays a key role in desensitizing the insulin-responsive glucose transport system (GTS), and recent studies have revealed that loss of GFAT activity accompanies desensitization. To gain insights into the mechanisms underlying loss of enzyme activity, we have used primary cultured adipocytes and two well established inhibitors of mRNA synthesis to estimate GFAT turnover. Both actinomycin D and 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB) caused a rapid and extensive loss in GFAT activity (greater than 70% loss, t1/2 of 45 min) indicating that GFAT has a relatively short half-life. Since induction of insulin resistance requires GFAT, we next examined the ability of mRNA inhibitors to block glucose-induced desensitization. When adipocytes were cultured for 18 h with 20 mM glucose, amino acids, and 25 ng/ml insulin, maximal insulin responsiveness of the GTS was reduced by greater than 70%. Both actinomycin D and DRB rapidly and completely prevented desensitization in a dose-dependent manner (ED50 of 16 nM and 15 microM, respectively). These findings are the predicted functional consequence of diminished GFAT activity. Evidence that actinomycin D acts selectively on GFAT without influencing other steps within the desensitization pathway was obtained using glucosamine, an agent that enters the hexosamine biosynthesis pathway at a point distal to the action of GFAT. Actinomycin D inhibited glucose-induced desensitization but failed to block glucosamine-induced desensitization. From these studies we conclude that 1) glucose-induced desensitization of the GTS can be completely prevented by actinomycin D and DRB, two potent and diverse inhibitors of mRNA synthesis; 2) the functional integrity of the desensitization pathway is maintained by a short-lived protein; and 3) the identity of this short-lived protein is most likely GFAT, the first and rate-limiting enzyme of the hexosamine biosynthesis pathway.

Animals↗
PubMed1991

Undiminished social class mortality differences in New Zealand men.

Social class mortality differences in New Zealand men aged 15-64 years have previously been examined for the period 1975-7. It was found that the lower social classes had mortality rates higher than the upper social classes with the mortality rate of the lowest class being approximately twice that of the highest class on a six category scale. The greatest relative social class differences were in men aged less than 35 years. The analysis has now been repeated for the period 1985-7. Mortality declined by 15% between 1975-7 and 1985-7, but the social class mortality differences were undiminished, and the mortality slope was actually slightly greater in the more recent time period. The primary significance of social class analyses is that they identify groups in the community which have an excess mortality that is potentially preventable. The findings of this study indicate that this potential has not been fully realised in New Zealand, since social class differences are undiminished despite the continuing decline in overall mortality.

Adolescent↗
PubMed1991

Discovery of a metabolic pathway mediating glucose-induced desensitization of the glucose transport system. Role of hexosamine biosynthesis in the induction of insulin resistance.

Based on our previous finding that desensitization of the insulin-responsive glucose transport system (GTS) requires three components, glucose, insulin, and glutamine, we postulated that the routing of incoming glucose through the hexosamine biosynthesis pathway plays a key role in the development of insulin resistance in primary cultured adipocytes. Two approaches were used to test this hypothesis. First, we assessed whether glucose-induced desensitization of the GTS could be prevented by glutamine analogs that irreversibly inactivate glutamine-requiring enzymes, such as glutamine:fructose-6-phosphate amidotransferase (GFAT) the first and the rate-limiting enzyme in hexosamine biosynthesis. Both O-diazoacetyl-L-serine (azaserine) and 6-diazo-5-oxonorleucine inhibited desensitization in 18-h treated cells without affecting maximal insulin responsiveness in control cells. Moreover, close agreement was seen between the ability of azaserine to prevent desensitization of the GTS in intact adipocytes (70% inhibition, ED50 = 1.1 microM), its ability to inactivate GFAT in intact adipocytes (64% inhibition, ED50 = 1.0 microM) and its ability to inactivate GFAT activity in a cytosolic adipocyte preparation (ED50 = 1.3 microM). From these results we concluded that a glutamine amidotransferase is involved in the induction of insulin resistance. As a second approach, we determined whether glucosamine, an agent known to preferentially enter the hexosamine pathway at a point distal to enzymatic amidation by GFAT, could induce cellular insulin resistance. When adipocytes were exposed to various concentrations of glucosamine for 5 h, progressive desensitization of the GTS was observed (ED50 = 0.36 mM) that culminated in a 40-50% loss of insulin responsiveness. Moreover, we estimated that glucosamine is at least 40 times more potent than glucose in mediating desensitization, since glucosamine entered adipocytes at only one-quarter of the glucose uptake rate, yet induced desensitization at an extra-cellular dose 10 times lower than glucose. In addition, we found that glucosamine-induced desensitization did not require glutamine and was unaffected by azaserine treatment. Thus, we conclude that glucosamine enters the hexosamine-desensitization pathway at a point distal to GFAT amidation. Overall, these studies indicate that a unique metabolic pathway exists in adipocytes that mediates desensitization of the insulin-responsive GTS, and reveal that an early step in this pathway involves the conversion of fructose 6-phosphate to glucosamine 6-phosphate by the first and rate-limiting enzyme of the hexosamine pathway, glutamine:fructose-6-phosphate amidotransferase.

Animals↗