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Biomedical subjects

David A Hughes

Publications and source records attributed to David A Hughes.

18 recordsLinked to original sources
PubMed2006

Fission yeast Dss1 associates with the proteasome and is required for efficient ubiquitin-dependent proteolysis.

Human DSS1 associates with BRCA2, a tumour suppressor protein required for efficient recombinational DNA repair, but the biochemical function of DSS1 is not known. Orthologues of DSS1 are found in organisms such as budding yeast and fission yeast that do not have BRCA2-related proteins, indicating that DSS1 has a physiological role independent of BRCA2. The DSS1 orthologue in Saccharomyces cerevisiae has been shown to associate with the 26 S proteasome and, in the present paper, we report that in the distantly related fission yeast Schizosaccharomyces pombe, Dss1 associates with the 19 S RP (regulatory particle) of the 26 S proteasome. A role for S. pombe Dss1 in proteasome function is supported by three lines of evidence. First, overexpression of two components of the 19 S RP, namely Pad1/Rpn11 and Mts3/Rpn12, rescued the temperature-sensitive growth defect of the dss1 mutant. Secondly, the dss1 mutant showed phenotypes indicative of a defect in proteasome function: growth of the dss1 mutant was inhibited by low concentrations of L-canavanine, an amino acid analogue, and cells of the dss1 mutant accumulated high molecular mass poly-ubiquitylated proteins. Thirdly, synthetic growth defects were found when the dss1 mutation was combined with mutations in other proteasome subunit genes. These findings show that DSS1 has an evolutionarily conserved role as a regulator of proteasome function and suggest that DSS1 may provide a link between BRCA2 and ubiquitin-mediated proteolysis in human cells.

Adenosine Triphosphate↗
PubMed2004

Effect of a beta-3 agonist on food intake in two strains of rats that differ in susceptibility to obesity.

CONTEXT: Beta-3 agonists acutely reduce food intake, but the mechanism is not well understood. OBJECTIVE: To evaluate the effect of a beta3 agonist on food intake in two strains of rats that differ in their sensitivity to becoming obese while eating a high-fat (HF) diet. METHODS: Male Osborne-Mendel (OM) and S5B/Pl (S5B) rats were treated with a beta3-adrenergic agonist (CL 316,243) at 8 weeks of age, after an adaptation to either an HF (56% fat energy) or a low-fat (LF; 10% fat energy) diet that was equicaloric for protein (24% energy). Ad-lib-fed rats were injected intraperitoneally with CL 316,243, at doses of 0.03, 0.1, 0.3, 1.0 or 3.0 mg/kg, or with vehicle at the beginning of the dark cycle. Food intake was measured at 1, 3, 6 and 24 h after injections. RESULTS: The beta3 agonist CL 316,243 significantly decreased food intake at all timepoints in both strains of rats eating both diets. However, this inhibition of food intake was significantly greater in the S5B rat. CL 316,243 significantly decreased serum leptin and serum glucose in both the OM and the S5B rats, and again, the inhibition was greater in the S5B rat. Whereas CL 316,243 increased serum insulin levels in the OM rat, it decreased them in the S5B rat on an LF diet. In a second experiment, chow-fed rats were implanted with vascular ports into the jugular vein and allowed to recover. When CL 316,243 was injected into the animals that were fasted overnight, rats of both strains significantly increased their serum insulin at 30 min, but the increase was much more pronounced in the S5B rat. Serum glucose was decreased significantly at both the 30- and 60-min timepoints in the OM rat and at 30 min in the S5B rat. CONCLUSION: These experiments demonstrate that a beta3 agonist (CL 316,243) has a much greater effect in a strain of rats that resist fat-induced obesity.

Adipose Tissue↗
PubMed2004

Plasma matrix metalloproteinases, low density lipoprotein oxidisability and soluble adhesion molecules after a glucose load in Type 2 diabetes.

BACKGROUND: Acute hyperglycaemia is an independent cardiovascular risk factor in Type 2 diabetes which may be mediated through increased oxidative damage to plasma low density lipoprotein, and in vitro, high glucose concentrations promote proatherogenic adhesion molecule expression and matrix metalloproteinase expression. METHODS: We examined these atherogenic risk markers in 21 subjects with Type 2 diabetes and 20 controls during an oral 75 g glucose tolerance test. Plasma soluble adhesion molecule concentrations [E-selectin, VCAM-1 and ICAM-1], plasma matrix metalloproteinases [MMP-3 and 9] and plasma LDL oxidisability were measured at 30 minute intervals. RESULTS: In the diabetes group, the concentrations of all plasma soluble adhesion molecules fell promptly [all p < 0.0001] related principally to glycaemic excursions, but such changes also occurred in the control group. Plasma MMP-3 and -9 concentrations were lower [p < 0.05], and LDL oxidisability greater [p < 0.01] in the diabetes group but did not change in either group. There was a direct relationship between plasma MMP-9 and s ICAM-1 in the controls [r = 0.62; p = 0.006] perhaps suggesting a functional relationship between s ICAM-1 shedding and MMP-9. CONCLUSIONS: A glucose load leads to a rapid fall in plasma soluble adhesion molecule concentrations in Type 2 diabetes and controls, perhaps reflecting reduced generation of soluble from membrane forms during enhanced leukocyte-endothelial adhesion or increased hepatic clearance, without changes in plasma matrix metalloproteinase concentrations or low density lipoprotein oxidisability. These in vivo findings are in contrast with in vitro data.

Journal Article↗
PubMed2004

p120 Ras GTPase-activating protein associates with fibroblast growth factor receptors in Drosophila.

Btl (breathless) and Htl (heartless), the two FGFRs (fibroblast growth factor receptors) in Drosophila melanogaster, control cell migration and differentiation in the developing embryo. These receptors signal through the conserved Ras/mitogen-activated protein kinase pathway, but how they regulate Ras activity is not known. The present study shows that there is a direct interaction between p120 RasGAP (Ras GTPase-activating protein), a negative regulator of Ras, and activated FGFRs in Drosophila. The interaction is dependent on the SH2 (Src homology 2) domains of RasGAP, which have been shown to interact with a phosphotyrosine residue within the consensus sequence (phospho)YXXPXD. A potential binding site that matches this consensus is found in both Btl and Htl, located between the transmembrane and kinase domains of each receptor. A peptide corresponding to this region was capable of binding RasGAP only when the tyrosine residue was phosphorylated. This tyrosine residue appears to be conserved in human FGFR-1 and mediates the association with the adapter protein CrkII, but no association between dCrk (Drosophila homologue of CrkII) and the activated FGFRs was detected. RasGAP was a substrate of the activated FGFR kinase domain, and mutation of the tyrosine residue within the potential binding site on the receptor prevented tyrosine phosphorylation of RasGAP. RasGAP attenuated FGFR signalling in vivo and this ability was dependent on both its SH2 domains and its GAP activity. On the basis of these results, we propose that RasGAP is directly recruited into activated FGFRs in Drosophila and plays a role in regulating the strength of signalling through Ras and the mitogen-activated protein kinase pathway.

Animals↗
PubMed2004

Humans.

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Biological Evolution↗
PubMed2004

Hepatic production of VLDL1 but not VLDL2 is related to insulin resistance in normoglycaemic middle-aged subjects.

Insulin resistance is probably the defining feature of the metabolic syndrome and is an important determinant of plasma triglyceride (TG) concentrations. We sought to investigate whether insulin resistance influenced the metabolism of VLDL1 (Sf 60-400) and VLDL2 (Sf 20-60). Sixteen (eight men, eight women) middle-aged, normoglycaemic subjects participated. VLDL1and VLDL2 apolipoprotein (apo) B metabolism was followed using a deuterated leucine tracer and insulin resistance was estimated using homeostasis model assessment (HOMA). HOMA-estimated insulin resistance (HOMAIR) significantly and strongly correlated with the VLDL1 production rate (r = 0.69, P < 0.01) and VLDL1 apo B pool size (r = 0.59, P = 0.02), but these relationships were not evident for VLDL2. Conversely, HOMAIR was not significantly related to the fractional rate of transfer of VLDL1 to VLDL2 but was significantly related to the fractional rate of transfer from VLDL2 to IDL (r = 0.61, P = 0.01). HOMAIR was not significantly related to the fractional rate of direct catabolism for either VLDL1 or VLDL2. These results suggest a role for insulin resistance in the determination of hepatic VLDL1 production and highlight the independent regulation of VLDL1 and VLDL2 metabolism.

Adult↗
PubMed2004

Significant presence of terminally differentiated T cells and altered NF-kappaB and I-kappaBalpha interactions in healthy ageing.

The risk of infection and cancer increases dramatically beyond middle age, when T-cell function is noticeably altered. Nevertheless, many elderly people remain in apparently good health. To identify immunological adaptations favouring longevity, a pilot study was undertaken to compare peripheral blood T cells from healthy volunteers aged 18-25 years with those >65 years. Instead of preserved immune function in the elderly, there was an emergence of haematopoietic space particularly affecting T- and B-cell numbers, together with early signs of immunoglobulin dysregulation. Age-associated proliferative defects were present irrespective of the stimuli used. A higher constitutive expression of NF-kappaB and I-kappaBalpha in the nuclei of peripheral lymphocytes from the elderly remained unaltered by activation stimuli, despite the presence of exogenous cytokines. Nevertheless, activation resulted in their higher CD95 upregulation and more intracellular bcl-2 (suggesting a survival advantage), but lower CD27, CD28 and CD45Rb expression. The presence of CD45RO(+) CD45Rb(-) populations was unique to the elderly and their lower replicative potential was not due to the presence of CD25(+) regulatory T cells. These data collectively suggest altered gene regulation and the accumulation of terminally differentiated T cells during healthy ageing.

Adolescent↗
PubMed2004

Quantitative PCR for DNA identification based on genome-specific interspersed repetitive elements.

We have designed and evaluated a series of class-specific (Aves), order-specific (Rodentia), and species-specific (equine, canine, feline, rat, hamster, guinea pig, and rabbit) polymerase chain reaction (PCR)-based assays for the identification and quantitation of DNA using amplification of genome-specific short and long interspersed elements. Using SYBR Green-based detection, the minimum effective quantitation levels of the assays ranged from 0.1 ng to 0.1 pg of starting DNA template. Background cross-amplification with DNA templates derived from sixteen other species was negligible prior to 30 cycles of PCR. The species-specificity of the PCR amplicons was further demonstrated by the ability of the assays to accurately detect known quantities of species-specific DNA from mixed (complex) sources. The 10 assays reported here will help facilitate the sensitive detection and quantitation of common domestic animal and bird species DNA from complex biomaterials.

Animals↗
PubMed2004

DNA damage and susceptibility to oxidative damage in lymphocytes: effects of carotenoids in vitro and in vivo.

Reports on the effects of carotenoids are conflicting. The present paper examines similarities and differences from contiguous studies in vitro and in vivo. Single-cell gel electrophoresis was used to measure the frequency of single-strand breaks (SSB) in the cell line MOLT-17 (as a model system) and human peripheral blood lymphocytes (PBL). MOLT-17 cells were supplemented with beta-carotene, lutein or lycopene at a range of concentrations (0.00-8.00 micromol/l) using a liposome delivery method. Uptake was dose-dependent. beta-Carotene concentration in the media had no effect on SSB in control cells, but incubation with lycopene or lutein (>2.00 micromol/l) increased the numbers of SSB in control cells. MOLT-17 DNA was less susceptible to oxidative damage (100 micromol H2O2/l, 5 min, 4 degrees C) following incubation with carotenoids between 0.50 and 1.00 micromol/l; at >1.00 micromol/l the effects were ambiguous. Apparently healthy male volunteers supplemented their habitual diets with lutein, beta-carotene or lycopene (natural isolate capsules, 15 mg/d, 4 weeks) in three independent studies, raising plasma concentrations to different extents. Lycopene and lutein had no effect on SSB in control PBL or following oxidative challenge. However, increased plasma beta-carotene was associated with more SSB in control cells whilst PBL DNA resistance to oxidative damage ex vivo was unaffected. These results suggest that the carotenoids are capable of exerting two overlapping but distinct effects: antioxidant protection by scavenging DNA-damaging free radicals and modulation of DNA repair mechanisms.

Adolescent↗
PubMed2003

Metalloproteinase expression in PMA-stimulated THP-1 cells. Effects of peroxisome proliferator-activated receptor-gamma (PPAR gamma) agonists and 9-cis-retinoic acid.

The PPAR gamma agonists, thiazolidinediones (TZDs), have anti-inflammatory properties as well as increasing insulin sensitivity. This has widened their therapeutic scope to treat inflammatory diseases such as atherosclerosis in addition to Type 2 Diabetes. TZDs are known to reduce monocyte/macrophage expression of Matrix metalloproteinase (MMP)-9, which is implicated in atherosclerotic plaque destabilization. This study aims to identify other metalloproteinase genes of the ADAM (A Disintegin And Metalloproteinase) and ADAMTS families that are regulated by PPAR gamma or RXR agonists, which are potentially important in type 2 diabetes and/or related atherosclerosis. The synthetic PPAR gamma agonist, GW7845, and the natural agonist 15d-PGJ2, suppressed PMA stimulated MMP-9 in human monocyte-like cells (THP-1) only in the presence of 9-cis-retinoic acid. Quantitative Real-Time PCR showed that this reduction was regulated at the mRNA level. Expression of ADAMs 8, 9, and 17 were increased, and ADAM15 was decreased by stimulation of THP-1 with PMA, although these ADAMs were not regulated by PPAR gamma or RXR agonists. PMA-induced ADAM28 expression was further enhanced by the addition of 9-cis-retinoic acid. ADAMTS4, implicated in rheumatoid arthritis, was expressed in THP-1 cells, and significantly increased after 24 h of PMA stimulation. ADAMTS4 expression was suppressed by both PPAR gamma and RXR agonists and was undetectable when the agonists were combined. Pretreatment of THP-1 cells with the PPAR gamma antagonist, GW9662, suggests that PPAR gamma plays subtly different roles in the regulation of MMP-9, ADAMTS4 and ADAM28 gene expression. These results indicate that PPAR gamma and RXR agonists have complex effects on monocyte metalloproteinase expression, which may have implications for therapeutic strategies.

ADAM Proteins↗
PubMed2003

Quantitative intra-short interspersed element PCR for species-specific DNA identification.

We have designed and evaluated four assays based upon PCR amplification of short interspersed elements (SINEs) for species-specific detection and quantitation of bovine, porcine, chicken, and ruminant DNA. The need for these types of approaches has increased drastically in response to the bovine spongiform encephalopathy epidemic. Using SYBR Green-based detection, the minimum effective quantitation levels were 0.1, 0.01, 5, and 1 pg of starting DNA template using our bovine, porcine, chicken, and ruminant species-specific SINE-based PCR assays, respectively. Background cross-amplification with DNA templates derived from 14 other species was negligible. Species specificity of the PCR amplicons was further demonstrated by the ability of the assays to accurately detect trace quantities of species-specific DNA from mixed (complex) sources. Bovine DNA was detected at 0.005% (0.5 pg), porcine DNA was detected at 0.0005% (0.05 pg), and chicken DNA was detected at 0.05% (5 pg) in a 10-ng mixture of bovine, porcine, and chicken DNA templates. We also tested six commercially purchased meat products using these assays. The SINE-based PCR methods we report here are species-specific, are highly sensitive, and will improve the detection limits for DNA sequences derived from these species.

Adult↗
PubMed2003

Monocyte matrix metalloproteinase production in Type 2 diabetes and controls--a cross sectional study.

BACKGROUND: Coronary plaque rupture may result from localised over expression of matrix metalloproteinases (MMPs) within the plaque by infiltrating monocyte-macrophages. As MMP expression can be promoted by the modified lipoproteins, oxidative stress and hyperglycaemia that characterises Type 2 diabetes, we hypothesised that peripheral monocytes in these patients, exposed to these factors in vivo, would demonstrate increased MMP production compared to controls. METHODS: We examined peripheral venous monocyte expression of MMP and tissue inhibitor of metalloproteinase-1 (TIMP-1) in 18 controls and 22 subjects with Type 2 diabetes and no previous cardiovascular complications. RESULTS: No significant difference in MMP-1, 3 or 9 or TIMP-1 production was observed between control and diabetes groups. CONCLUSIONS: Monocyte MMP-1, 3, and 9, and TIMP-1, production are not abnormal in Type 2 diabetes. This data cannot be extrapolated to monocyte-macrophage behaviour in the vessel wall, but it does suggest MMP and TIMP-1 expression prior to monocyte infiltration and transformation are not abnormal in Type 2 diabetes.

Journal Article↗
PubMed2003

Increased expression of a scavenger receptor (CD36) in monocytes from subjects with Type 2 diabetes.

Uptake of modified low density lipoprotein (LDL) by monocyte-macrophages is mediated by the scavenger receptor CD36, which is upregulated in vitro by high glucose concentrations and oxidatively modified LDL. We hypothesised that monocyte CD36 expression would be higher in Type 2 diabetes, and would increase during acute hyperglycaemia. Sixteen subjects with Type 2 diabetes and 11 controls underwent a 75 g oral glucose load. Monocyte CD36 expression (by laser flow cytometry), plasma LDL diene conjugates, plasma LDL hydroxyoctadecadienoic acid-13 (a peroxisome proliferator activator receptor gamma agonist) were measured at 0, 2 and 4 h. Mean monocyte CD36 expression at baseline was 34% higher in the diabetes group (P=0.01), did not change during acute hyperglycaemia and plasma LDL conjugated diene concentration was the only variable directly related to CD36 expression (F=4.53; P=0.05; r=0.51). Higher baseline CD36 expression in Type 2 diabetes could reflect increased post-transcriptional efficiency of CD36 mRNA in response to chronic hyperglycaemia and could be a proatherogenic mechanism in Type 2 diabetes.

Adult↗
PubMed2003

Deregulation of the Egfr/Ras signaling pathway induces age-related brain degeneration in the Drosophila mutant vap.

Ras signaling has been shown to play an important role in promoting cell survival in many different tissues. Here we show that upregulation of Ras activity in adult Drosophila neurons induces neuronal cell death, as evident from the phenotype of vacuolar peduncle (vap) mutants defective in the Drosophila RasGAP gene, which encodes a Ras GTPase-activating protein. These mutants show age-related brain degeneration that is dependent on activation of the EGF receptor signaling pathway in adult neurons, leading to autophagic cell death (cell death type 2). These results provide the first evidence for a requirement of Egf receptor activity in differentiated adult Drosophila neurons and show that a delicate balance of Ras activity is essential for the survival of adult neurons.

Aging↗
PubMed2003

Effects of dietary monounsaturated fatty acids on lipoprotein concentrations, compositions, and subfraction distributions and on VLDL apolipoprotein B kinetics: dose-dependent effects on LDL.

BACKGROUND: Replacing dietary saturated fatty acids (SFAs) with monounsaturated fatty acids (MUFAs) lowers LDL cholesterol, but the underlying mechanisms remain unclear. OBJECTIVE: We assessed the effects of replacing dietary SFAs with MUFAs on concentrations and subclass distributions of VLDL, intermediate-density lipoprotein, LDL, and HDL and on VLDL apolipoprotein B kinetics. DESIGN: Thirty-five moderately hypercholesterolemic, middle-aged volunteers consumed for 6 wk, in random order, diets containing low (L-MUFA; 7.8% of energy from MUFAs), moderate (M-MUFA; 10.3% from MUFAs), or high (H-MUFA; 13.7% from MUFAs) amounts of MUFAs. Fasting blood samples were taken from all subjects after each intervention. VLDL apolipoprotein B kinetic studies were performed in a subgroup after the L-MUFA and H-MUFA diets. RESULTS: Plasma cholesterol concentrations decreased in a dose-dependent manner with increasing intakes of dietary MUFAs. This change was entirely accounted for by reduced LDL cholesterol (-0.20 and -0.49 mmol/L after the M-MUFA and H-MUFA diets, respectively, compared with the concentration after the L-MUFA diet; P for trend < 0.01). Plasma triacylglycerol and HDL cholesterol were not significantly affected by the dietary intervention, nor were the concentrations of VLDL(1) (S(f) 60-400), VLDL(2) (S(f) 20-60), or intermediate-density lipoprotein (S(f) 12-20). Production and catabolic rates for VLDL(1) and VLDL(2) were also unaffected. HDL and LDL subclass distributions were not significantly altered, but as a consequence of the overall LDL lowering, concentrations of atherogenic LDL-III were 25% lower after the H-MUFA diet than after the L-MUFA diet (P = 0.02). CONCLUSION: The effects of replacing dietary SFAs with MUFAs on lipoprotein metabolism appear to be almost exclusively limited to the LDL density class.

Apolipoproteins↗
PubMed2002

Plasma F2 isoprostanes: direct evidence of increased free radical damage during acute hyperglycemia in type 2 diabetes.

OBJECTIVES: Acute hyperglycemia in type 2 diabetes increases the generation of plasma 8-epi-prostaglandin F2 (8-epi-PGF2alpha) isoprostane, a sensitive direct marker of in vivo free radical oxidative damage to membrane phospholipids. RESEARCH DESIGN AND METHODS: A total of 21 patients with type 2 diabetes underwent an oral 75-g glucose tolerance test. Plasma 8-epi-PGF2alpha isoprostane concentrations (by gas chromatography [GC]/mass spectrometry [MS]), intralymphocyte reduced-to-oxidized glutathione ratios, and plasma total antioxidant capacity were measured at baseline and 90 min after glucose loading. RESULTS: Plasma 8-epi-PGF2alpha isoprostane concentrations rose significantly (P=0. 010) from 0.241 +/- 0.1 to 0.326 +/- 0.17 ng/l after 90 min. Intracellular oxidative balance and plasma antioxidant capacity did not change in either group. CONCLUSIONS: Plasma concentrations of 8-epi-PGF2alpha isoprostane increase during acute hyperglycemia in type 2 diabetes, providing direct evidence of free radical-mediated oxidative damage and demonstrating a pathway for an association between acute rather than fasting hyperglycemia and macrovascular risk in type 2 diabetes.

Adult↗
PubMedDate not supplied

Alpha-lipoic acid modulates NF-kappaB activity in human monocytic cells by direct interaction with DNA.

The constitutive activity of the redox-sensitive transcription factor, NF-kappaB, which regulates the production of many inflammatory cytokines and adhesion molecules, appears to be up-regulated in an age-associated manner and it is thought this might contribute to the increased incidence of chronic inflammatory conditions observed with increasing age. As some antioxidants have demonstrated protective effects against rheumatoid arthritis, we are investigating the effects of vitamin E, vitamin C and alpha-lipoic acid (ALA) on NF-kappaB activity and on the expression of intracellular adhesion molecule (ICAM)-1. MonoMac6 cells (a human monocytic cell line) stimulated with tumour necrosis factor-alpha (TNF-alpha) were treated with antioxidants at physiological achievable levels and ICAM-1 mRNA levels investigated. Both vitamin E and vitamin C had no effect on ICAM-1 expression at the doses used, but ALA reduced the TNF-alpha-stimulated ICAM-1 expression in a dose-dependent manner, to levels observed in unstimulated cells. Alpha-lipoic acid also reduced NF-kappaB activity in these cells in a dose-dependent manner. Addition of ALA to the binding reaction of nuclear extract with DNA prior to gel-shift analysis showed that it caused inhibition at this level. These initial results suggest that antioxidant modulation of monocyte activity might have potential benefits in inhibiting the dysregulated activity of redox-sensitive transcription factors that occurs with increasing age.

Antioxidants↗