Glucose-induced oxidative stress in mesangial cells.

Background: Hyperglycaemia is a well recognized pathogenic factor of long term complications in diabetes mellitus. Hyperglycaemia not only generates reactive oxygen species but also attenuates antioxidant mechanisms creating a state of oxidative stress. Methods: Porcine mesangial cells were cultured in high glucose (HG) for ten days to investigate the effects on the antioxidant defences of the cell. Results: Mesangial cells cultured in HG conditions had significantly reduced levels of glutathione (GSH) compared with those grown in normal glucose (NG). The reduced GSH levels were accompanied by decreased gene expression of both subunits of gamma-glutamylcysteine synthetase (gamma-GCS), the rate-limiting enzyme in de novo synthesis of GSH. Elevated levels of intracellular malondialdehyde (MDA) were found in cells exposed to HG conditions. HG also caused elevated mRNA levels of the antioxidant enzymes CuZn superoxide dismutase (SOD) and MnSOD. These changes were accompanied by increased mRNA levels of extracellular matrix proteins (ECM), fibronectin (FN) and collagen IV (CIV). Addition of antioxidants to high glucose caused a significant reversal of FN and CIV gene expression; alpha-lipoic acid also upregulated gamma-GCS gene expression and restored intracellular GSH and MDA levels. Conclusions: We have demonstrated the existence of glucose induced-oxidative stress in mesangial cells as evidenced by elevated MDA and decreased GSH levels. The decreased levels of GSH are as a result of decreased mRNA expression of gamma-GCS within the cell. Antioxidants caused a significant reversal of FN and CIV gene expression suggesting an aetiological link between oxidative stress and increased ECM protein synthesis.

High glucose decreases intracellular glutathione concentrations and upregulates inducible nitric oxide synthase gene expression in intestinal epithelial cells

Diabetes is associated with oxidative stress and increased levels of inflammatory cytokines. The aim of the study was to assess the effects of inflammatory cytokines and oxidative stress associated with raised glucose levels on inducible nitric oxide synthase (iNOS) promoter activity in intestinal epithelial cells. High glucose (25 mmol/l) conditions reduced glutathione (GSH) levels in the human intestinal epithelial cell line, DLD-1. Addition of the antioxidant alpha-lipoic acid resulted in the restoration of GSH levels to normal. Upregulation of basal iNOS promoter activity was observed when cells were incubated in high glucose alone. This effect was significantly reduced by the addition of the antioxidant, alpha-lipoic acid and completely blocked with inhibition of NFkappa B activity. Cytokine stimulation [interleukin-1 beta, tumor necrosis factor-alpha, interferon-gamma] induced iNOS promoter activity in all conditions and this was accompanied by an increase in nitric oxide (NO) production. Inhibition of NFkappa-B activity decreased but did not completely inhibit cytokine-induced iNOS promoter activity and subsequent NO production. In conclusion, high glucose-induced iNOS promoter activity is mediated in part through intracellular GSH and NFkappa-B.

Differences in mitochondrial and nuclear DNA status of high-density and low-density sperm fractions after density centrifugation preparation

Background: In order to isolate the â??bestâ?? sperm for assisted conception a discontinuous two-step density gradient centrifugation is usually employed. This technique is known to isolate a subpopulation with good motility, morphology and nuclear DNA (nDNA) integrity. As yet its ability to isolate sperm with unfragmented mitochondrial DNA (mtDNA) is unknown. Methods: Semen was obtained from men (n=28) attending our Regional Fertility Centre for infertility investigations. We employed a modified long polymerase chain reaction to study mtDNA and a modified alkaline Comet assay to determine nDNA fragmentation. Results: The high- density fraction displayed significantly more wild type mtDNA (75% of samples) than that of the low- density fraction (25% of samples). In the high-density fraction, there was a higher incidence of single, rather than double or multiple deletions and the deletions were predominantly small scale (0.1-4.0kb). There was a strong correlation between nDNA fragmentation, the number of mtDNA deletions (r=0.7, p

Modelling the flow of congestive heart failure patients through a hospital system

The number of hospital admissions in England due to heart failure is projected to increase by over 50% during the next 25 years. This will incur greater pressures on hospital managers to allocate resources in an effective manner. A reliable indicator for measuring the quantity of resources consumed by hospital patients is their length of stay (LOS) in care. This paper proposes modelling the length of time heart failure patients spend in hospital using a special type of Markov model, where the flow of patients through hospital can be thought of as consisting of three stages of care—short-, medium- and longer-term care. If it is assumed that new admissions into the ward are replacements for discharges, such a model may be used to investigate the case-mix of patients in hospital and the expected patient turnover during some specified period of time. An example is illustrated by considering hospital admissions to a Belfast hospital in Northern Ireland, between 2000 and 2004.

Interaction of glucose and long chain fatty acids (C18) on antioxidant defences and free radical damage in porcine vascular smooth muscle cells in vitro.

Aims/hypothesis: Abnormalities of glucose and fatty acid metabolism in diabetes are believed to contribute to the development of oxidative stress and the long term vascular complications of the disease therefore the interactions of glucose and long chain fatty acids on free radical damage and endogenous antioxidant defences were investigated in vascular smooth muscle cells. Methods: Porcine vascular smooth muscle cells were cultured in 5 mmol/l or 25 mmol/l glucose for ten days. Fatty acids, stearic acid (18:0), oleic acid (18:1), linoleic acid (18:2) and gamma-linolenic acid (18:3) were added with defatted bovine serum albumin as a carrier for the final three days. Results. Glucose (25 mmol/l) alone caused oxidative stress in the cells as evidenced by free radical-mediated damage to DNA, lipids, and proteins. The addition of fatty acids (0.2 mmol/l) altered the profile of free radical damage; the response was J-shaped with respect to the degree of unsaturation of each acid, and oleic acid was associated with least damage. The more physiological concentration (0.01 mmol/l) of gamma-linolenic acids was markedly different in that, when added to 25 mmol/l glucose it resulted in a decrease in free radical damage to DNA, lipids and proteins. This was due to a marked increase in levels of the antioxidant, glutathione, and increased gene expression of the rate-limiting enzyme in glutathione synthesis, gamma-glutamylcysteine synthetase. Conclusion/Interpretation: The results clearly show that glucose and fatty acids interact in the production of oxidative stress in vascular smooth muscle cells.