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Farah T. van Genderen Frans K. Gorus Ilse Vermeulen Evilien M.F. Vekens Pieter E.M. De Pauw Daniel G. Pipeleers Chris Van Schravendijk 《Analytical biochemistry》2010,404(1):8-13
We present a time-resolved fluorescence immunoassay (TR-FIA) for the measurement of rat insulin in cell extracts and culture media. This assay is based on the binding of two monoclonal antibodies to different parts of the insulin molecule in a 96-well microtiter plate. For the detection, europium-labeled streptavidin that interacts with the second biotinylated antibody is used. Samples of 25 μl could be analyzed in less than 2 days with a measuring range between 5 and 1250 pg (0.2-50 μg/L or 34.4-8600 pM). The inter- and intraassay percentage coefficients of variation were less than 8.3 and 5.1, respectively. Recoveries of 0.48 to 40 μg/L rat insulin, added to culture medium, ranged between 94 and 107%. Results were significantly correlated with those of an in-house radioimmunoassay (RIA) for rodent insulin (P < 0.0001, r2 = 0.99). The TR-FIA method had a similar detection limit (0.16 μg/L), but its working range was at least 5-fold larger. Additional advantages include the lower cost, the applicability to measurements in tissue and serum, and the quantification of insulin from other species. 相似文献
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Beta-endorphin and ACTH levels in peripheral blood during and after aerobic and anaerobic exercise 总被引:1,自引:0,他引:1
K de Meirleir N Naaktgeboren A Van Steirteghem F Gorus J Olbrecht P Block 《European journal of applied physiology and occupational physiology》1986,55(1):5-8
Beta-endorphin (beta-End) and adrenocorticotrophic hormone (ACTH) were determined in the peripheral blood of 14 human volunteers exercising on a bicycle ergometer. After 1 h of submaximal work below anaerobic threshold (AT), defined as the 4 mmol X l-1 lactic acid level in arteriolar blood (Kindermann 1979; Mader 1980), beta-End and ACTH levels did not change from control conditions. Eleven of the same 14 subjects performed an uninterrupted graded exercise test on the same bicycle ergometer until exhaustion. This time beta-End and ACTH levels increased concomitantly with exercise of high intensity: at each moment, during and after this maximal test, a highly significant correlation (P less than 0.0001) was noted between the levels of beta-End and ACTH. The peak values of these hormones were reached within 10 min after stopping maximal exercise, and coincided with lactic acid peak levels. A rise in lactic acid levels above the anaerobic threshold always preceded the exercise-induced rise in beta-End and ACTH. Within the population tested, two subgroups could be distinguished: one comprising individuals whose hormonal response nearly coincided with the rise in lactic acid (rapid responders) and a second group composed of subjects whose normal response appeared delayed with respect to the lactic acid rise (slow responders). These results support the view that beta-End and ACTH are secreted in equimolar quantities into the blood circulation in response to exercise, and suggest that metabolic changes of anaerobiosis play a key role in the regulation of stress-hormone release.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
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Differences in glucose handling by pancreatic A- and B-cells 总被引:10,自引:0,他引:10
Glucose exerts opposite effects upon glucagon and insulin release from the endocrine pancreas. Glucose uptake and oxidation were therefore compared in purified A- and B-cells. In purified B-cells, the intracellular concentration of glucose or 3-O-methyl-D-glucose equilibrates within 2 min with the extracellular levels, and, like in intact islets, the rate of glucose oxidation displays a sigmoidal dose-response curve for glucose. In contrast, even after 5 min of incubation, the apparent distribution space of D-glucose or 3-O-methyl-D-glucose in A-cells remains much lower than the intracellular volume. In A-cells, both the rate of 3-O-methyl-D-glucose uptake and glucose oxidation proceed proportional to the hexose concentration up to 10 mM and reach saturation at higher concentrations. Addition of insulin failed to affect 3-O-methyl-D-glucose or D-glucose uptake and glucose oxidation by purified A-cells. Glucose releases 30-fold more insulin from islets than from single B-cells, but this marked difference is not associated with differences in glucose handling. The rate of glucose oxidation is virtually identical in single and reaggregated B-cells and is not altered after addition of glucagon or somatostatin. It is concluded that the dependency of glucose-induced insulin release upon the functional coordination between islet cells is not mediated through changes in glucose metabolism. 相似文献
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Multiple isoforms of UDP-GalNAc:polypeptide N-acetylgalactosaminyl-
transferase (ppGaNTase) have been cloned and expressed from a variety of
organisms. In general, these isoforms display different patterns of
tissue-specific expression, but exhibit overlapping substrate
specificities, in vitro . A peptide substrate, derived from the sequence of
the V3 loop of the HIV gp120 protein (HIV peptide), has previously been
shown to be glycosylated in vitro exclusively by the ppGaNTase-T3 (Bennett
et al. , 1996). To determine if this isoform- specificity is maintained in
vivo , we have examined the glycosylation of this substrate when it is
expressed as a reporter peptide (rHIV) in a cell background (COS7 cells)
which lacks detectable levels of the ppGaNTase-T3. Glycosylation of rHIV
was greatly increased by coexpression of a recombinant ppGaNTase-T3.
Overexpression of ppGaNTase- T1 yielded only partial glycosylation of the
reporter. We have also determined that the introduction of a proline
residue at the +3 position flanking the potential glycosylation site
eliminated ppGaNTase- T3 selectivity toward rHIV observed both in vivo and
in vitro .
相似文献
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Alloxan rapidly binds to or accumulates in pancreatic B-cells as distinct from non-B-cells. The selective uptake of this cytotoxic agent by the insulin-producing B-cells might account for its well-known diabetogenic effect. 相似文献
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Martens GA Jiang L Verhaeghen K Connolly JB Geromanos SG Stangé G Van Oudenhove L Devreese B Hellemans KH Ling Z Van Schravendijk C Pipeleers DG Vissers JP Gorus FK 《PloS one》2010,5(12):e14214
Background and Methodology
Pancreatic beta cells show intercellular differences in their metabolic glucose sensitivity and associated activation of insulin production. To identify protein markers for these variations in functional glucose sensitivity, rat beta cell subpopulations were flow-sorted for their level of glucose-induced NAD(P)H and their proteomes were quantified by label-free data independent alternate scanning LC-MS. Beta cell-selective proteins were also identified through comparison with rat brain and liver tissue and with purified islet alpha cells, after geometrical normalization using 6 stably expressed reference proteins.Principal Findings
All tissues combined, 943 proteins were reliably quantified. In beta cells, 93 out of 467 quantifiable proteins were uniquely detected in this cell type; several other proteins presented a high molar abundance in beta cells. The proteome of the beta cell subpopulation with high metabolic and biosynthetic responsiveness to 7.5 mM glucose was characterized by (i) an on average 50% higher expression of protein biosynthesis regulators such as 40S and 60S ribosomal constituents, NADPH-dependent protein folding factors and translation elongation factors; (ii) 50% higher levels of enzymes involved in glycolysis and in the cytosolic arm of the malate/aspartate-NADH-shuttle. No differences were noticed in mitochondrial enzymes of the Krebs cycle, beta-oxidation or respiratory chain.Conclusions
Quantification of subtle variations in the proteome using alternate scanning LC-MS shows that beta cell metabolic glucose responsiveness is mostly associated with higher levels of glycolytic but not of mitochondrial enzymes. 相似文献10.
Czyzyk J Henegariu O Preston-Hurlburt P Baldzizhar R Fedorchuk C Esplugues E Bottomly K Gorus FK Herold K Flavell RA 《Journal of immunology (Baltimore, Md. : 1950)》2012,188(12):6319-6327
Intracellular (clade B) OVA-serpin protease inhibitors play an important role in tissue homeostasis by protecting cells from death in response to hypo-osmotic stress, heat shock, and other stimuli. It is not known whether these serpins influence immunological tolerance and the risk for autoimmune diseases. We found that a fraction of young autoimmune diabetes-prone NOD mice had elevated levels of autoantibodies against a member of clade B family known as serpinB13. High levels of anti-serpinB13 Abs were accompanied by low levels of anti-insulin autoantibodies, reduced numbers of islet-associated T cells, and delayed onset of diabetes. Exposure to anti-serpinB13 mAb alone also decreased islet inflammation, and coadministration of this reagent and a suboptimal dose of anti-CD3 mAb accelerated recovery from diabetes. In a fashion similar to that discovered in the NOD model, a deficiency in humoral activity against serpinB13 was associated with early onset of human type 1 diabetes. These findings suggest that, in addition to limiting exposure to proteases within the cell, clade B serpins help to maintain homeostasis by inducing protective humoral immunity. 相似文献