Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:1.8.1.4 (diaphorase)
2,754 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A nonradioisotope, 96-well-microplate assay to evaluate glucose uptake activity in cultured cells has been developed. 2-Deoxyglucose (2DG) was detected by measuring a potent fluorophore, resorufin, generated after incubation with a single assay solution containing hexokinase, adenosine 5'-triphosphate, glucose 6-phosphate dehydrogenase, beta-nicotineamide adenine dinucleotide phosphate, diaphorase, and resazurin. This amplifying detection system could detect the fluorescence intensity induced by uptake of 2DG into L6 skeletal muscle cells, even at the level of cells cultivated in individual wells in a 96-well microplate. Using this assay system, the effects of insulin, cytochalasin B (hexose uptake inhibitor), LY294002 (inhibitor of glucose transporter translocation), and pioglitazone hydrochloride (insulin-sensitizing agent) on 2DG uptake into L6 myotubes could be assessed clearly. Therefore, our simple method may be useful for in vitro high-throughput screening and for evaluating regulators of glucose uptake.
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PMID:A nonradioisotope, enzymatic assay for 2-deoxyglucose uptake in L6 skeletal muscle cells cultured in a 96-well microplate. 1644 89

The proteins of HL type cytoplasmic male sterility rice anther of YTA (CMS) and YTB (maintenance line) were separated by two-dimensional electrophoresis with immobilized ph (3-10 non-linear) gradients as the first dimension and SDS-PAGE as the second. The silver-stained proteins spots were analyzed using Image Master 2D software, there were about 1800 detectable spots on each 2D-gel, and about 85 spots were differential expressed. With direct MALDI-TOF mass spectrometry analysis and protein database searching, 9 protein spots out of 16 were identified. Among those proteins, there were Putative nucleic acid binding protein, glucose-1-phosphate adenylyltransferase (ADP-glucose pyrophosphorylase, AGPase) (EC: 2.7.7.27) large chain, UDP-glucuronic acid decarboxylase, putative calcium-binding protein annexin, putative acetyl-CoA synthetase and putative lipoamide dehydrogenase etc. They were closely associated with metabolism, protein biosynthesis, transcription, signal transduction and so on, all of which are cell activities that are essential to pollen development. Some of the identified proteins, i.e. AGPase, putative lipoamide dehydrogenase and putative acetyl-CoA synthetase were deeply discussed on the relationship to CMS. AGPase catalyzes a very important step in the biosynthesis of alpha 1,4-glucans (glycogen or starch) in bacteria and plants: synthesis of the activated glucosyl donor, ADP-glucose, from glucose-1-phosphate and ATP. The lack of the AGPase in male sterile line might directly result in the reduction of starch, and the synthesis of starch was the most important processes during the development of pollen. In present research, the descent or reduction of putative lipoamide dehydrogenase and putative acetyl-CoA synthetase seemed involved in pollen sterility in rice. The degeneration and formation of various tissues during pollen development may impose high demands for energy and key biosynthetic intermediates. Under such conditions, the TCA cycle needs to operate fully, because the TCA cycle is an important source for many intermediates required for biosynthetic pathways, in addition to performing an oxidative, energy-producing role. Thus, it seemed reasonable to infer that the decrease of putative lipoamide dehydrogenase and putative acetyl-CoA synthetase in anther might prevent the conversion of pyruvate into acetyl-CoA, and as a result, the TCA cycle could no longer operate at a sufficient rate to meet all requirements in anther cells, leading to pollen sterility. This study gave new insights into the mechanism of CMS in rice and demonstrated the power of the proteomic approach in plant biology studies.
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PMID:[Preliminary proteomics analysis of the total proteins of HL Type cytoplasmic male sterility rice anther]. 1655 98

Alzheimer's disease (AD) is a progressive neurodegenerative disorder that destroys patient memory and cognition, communication ability with the social environment and the ability to carry out daily activities. Despite extensive research into the pathogenesis of AD, a neuroprotective treatment - particularly for the early stages of disease - remains unavailable for clinical use. In this review, we advance the suggestion that lipoic acid (LA) may fulfil this therapeutic need. A naturally occurring precursor of an essential cofactor for mitochondrial enzymes, including pyruvate dehydrogenase (PDH) and alpha-ketoglutarate dehydrogenase (KGDH), LA has been shown to have a variety of properties which can interfere with pathogenic principles of AD. For example, LA increases acetylcholine (ACh) production by activation of choline acetyltransferase and increases glucose uptake, thus supplying more acetyl-CoA for the production of ACh. LA chelates redox-active transition metals, thus inhibiting the formation of hydroxyl radicals and also scavenges reactive oxygen species (ROS), thereby increasing the levels of reduced glutathione. Via the same mechanisms, downregulation redox-sensitive inflammatory processes is also achieved. Furthermore, LA can scavenge lipid peroxidation products such as hydroxynonenal and acrolein. The reduced form of LA, dihydrolipoic acid (DHLA), is the active compound responsible for most of these beneficial effects. R-alpha-LA can be applied instead of DHLA, as it is reduced by mitochondrial lipoamide dehydrogenase, a part of the PDH complex. In this review, the properties of LA are explored with particular emphasis on how this agent, particularly the R-alpha-enantiomer, may be effective to treat AD and related dementias.
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PMID:Lipoic acid as a novel treatment for Alzheimer's disease and related dementias. 1698 5

Prolonged exposure of pancreatic beta cells to the sulfonylureas glibencamide and tolbutamide induces subsequent desensitization to the actions of these drugs. The precise mechanisms underlying this desensitization remain unknown, prompting the present study, which investigated the impact of prolonged sulfonylurea exposure on glucose and energy metabolism using clonal pancreatic BRIN-BD11 beta cells. Following prolonged exposure to tolbutamide, BRIN-BD11 beta cells were incubated in the presence of [U-(13)C]glucose, and isotopomer analysis revealed that there was a change in the ratio of flux through pyruvate carboxylase (EC 6.4.1.1) and pyruvate dehydrogenase (EC 1.2.4.1, EC 2.3.1.12, EC 1.8.1.4). Energy status in intact BRIN-BD11 cells was determined using (31)P-NMR spectroscopy. Exposure to tolbutamide did not alter the nucleotide triphosphate levels. Collectively, data from the present study demonstrate that prolonged exposure of beta cells to tolbutamide results in changes in flux through key enzymes involved in glucose metabolism that, in turn, may impact on glucose-induced insulin secretion.
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PMID:Investigation of the effects of sulfonylurea exposure on pancreatic beta cell metabolism. 1705 12

Replicative life span in Saccharomyces cerevisiae is increased by glucose (Glc) limitation [calorie restriction (CR)] and by augmented NAD+. Increased survival promoted by CR was attributed previously to the NAD+-dependent histone deacetylase activity of sirtuin family protein Sir2p but not to changes in redox state. Here we show that strains defective in NAD+ synthesis and salvage pathways (pnc1delta, npt1delta, and bna6delta) exhibit decreased oxygen consumption and increased mitochondrial H2O2 release, reversed over time by CR. These null mutant strains also present decreased chronological longevity in a manner rescued by CR. Furthermore, we observed that changes in mitochondrial H2O2 release alter cellular redox state, as attested by measurements of total, oxidized, and reduced glutathione. Surprisingly, our results indicate that matrix-soluble dihydrolipoyl-dehydrogenases are an important source of CR-preventable mitochondrial reactive oxygen species (ROS). Indeed, deletion of the LPD1 gene prevented oxidative stress in npt1delta and bna6delta mutants. Furthermore, pyruvate and alpha-ketoglutarate, substrates for dihydrolipoyl dehydrogenase-containing enzymes, promoted pronounced reactive oxygen release in permeabilized wild-type mitochondria. Altogether, these results substantiate the concept that mitochondrial ROS can be limited by caloric restriction and play an important role in S. cerevisiae senescence. Furthermore, these findings uncover dihydrolipoyl dehydrogenase as an important and novel source of ROS leading to life span limitation.
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PMID:Dihydrolipoyl dehydrogenase as a source of reactive oxygen species inhibited by caloric restriction and involved in Saccharomyces cerevisiae aging. 1711 Apr 66

This paper describes a new amperometric biosensor for glucose monitoring. The biosensor is based on the activity of glucose dehydrogenase (GDH) and diaphorase (DI) co-immobilized with NAD(+) into a carbon nanotube paste (CNTP) electrode modified with an osmium functionalized polymer. This mediator was demonstrated to shuttle the electron transfer between the immobilized diaphorase and the CNTP electrode, thus, showing a good electrocatalytic activity towards NADH oxidation at potentials around +0.2V versus Ag|AgCl, where interfering reactions are less prone to occur. The biosensor exhibits a detection limit of 10 micromol L(-1), linearity up to 8 x 10(-4) mol L(-1), a sensitivity of 13.4 microA cm(-2)mmol(-1)L(-1), a good reproducibility (R.S.D. 2.1%, n=6) and a stability of about 1 week when stored dry at 4 degrees C. Finally, the proposed biosensor was applied for the determination of glucose in different samples of sweet wine and validated with a commercial spectrophotometric enzymatic kit.
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PMID:Development of a carbon nanotube paste electrode osmium polymer-mediated biosensor for determination of glucose in alcoholic beverages. 1717 56

Escherichia coli K-12 was cultured under anaerobic conditions to form biofilm on carbon fiber electrodes in glucose-containing medium. The anodic current increased with the development of the biofilm and depended on the glucose concentration. Cyclic voltammetric results support the presence of a redox compound(s) excreted from E. coli cells in the biofilm. The compound remained in the film under conditions of continuous flow and gave a couple of oxidation and reduction waves, which may be assigned to a menaquinone-like compound based on the mid-point potential (-0.22 V vs Ag|AgCl at pH 7.1) and its pH dependence. The catalytic current started to increase around the anodic peak potential of the redox compound and also increased by the permeabilization of the E. coli cell membranes with ethylenediamine tetraacetic acid-treatment. The results indicate that the E. coli-excreted redox compound works as a mediator for the electron transfer from the E. coli cells to the electrode as the final electron acceptor. The activity of the redox compound in the E. coli-biofilm as a mediator with some mobility was also verified for diaphorase-catalyzed electrochemical oxidation of NADH.
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PMID:Self-excreted mediator from Escherichia coli K-12 for electron transfer to carbon electrodes. 1766 90

Under anaerobic growth conditions, an active pyruvate dehydrogenase (PDH) is expected to create a redox imbalance in wild-type Escherichia coli due to increased production of NADH (>2 NADH molecules/glucose molecule) that could lead to growth inhibition. However, the additional NADH produced by PDH can be used for conversion of acetyl coenzyme A into reduced fermentation products, like alcohols, during metabolic engineering of the bacterium. E. coli mutants that produced ethanol as the main fermentation product were recently isolated as derivatives of an ldhA pflB double mutant. In all six mutants tested, the mutation was in the lpd gene encoding dihydrolipoamide dehydrogenase (LPD), a component of PDH. Three of the LPD mutants carried an H322Y mutation (lpd102), while the other mutants carried an E354K mutation (lpd101). Genetic and physiological analysis revealed that the mutation in either allele supported anaerobic growth and homoethanol fermentation in an ldhA pflB double mutant. Enzyme kinetic studies revealed that the LPD(E354K) enzyme was significantly less sensitive to NADH inhibition than the native LPD. This reduced NADH sensitivity of the mutated LPD was translated into lower sensitivity of the appropriate PDH complex to NADH inhibition. The mutated forms of the PDH had a 10-fold-higher K(i) for NADH than the native PDH. The lower sensitivity of PDH to NADH inhibition apparently increased PDH activity in anaerobic E. coli cultures and created the new ethanologenic fermentation pathway in this bacterium. Analogous mutations in the LPD of other bacteria may also significantly influence the growth and physiology of the organisms in a similar fashion.
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PMID:Dihydrolipoamide dehydrogenase mutation alters the NADH sensitivity of pyruvate dehydrogenase complex of Escherichia coli K-12. 1837 66

Current conventional measurement of allantoin levels in human serum uses an HPLC method. However, performing this assay is time-consuming and sample-intensive, and it requires expensive equipment. We have developed a novel enzyme cycling method for measuring allantoin concentrations in human serum. In the first step, serum allantoin is converted to allantoate by the action of allantoinase (EC 3.5.2.5), and endogenous ammonia is simultaneously removed by the action of glutamine synthetase II (EC 6.3.1.2). In the second step, l-methionine sulfoximine is used to inhibit glutamine synthetase II, and ammonia is liberated from allantoate by the activity of allantoate amidohydrolase (EC 3.5.3.9). In the final step, the ammonia is then converted to NAD by NAD synthetase (EC 6.3.1.5). Subsequent action of glucose dehydrogenase (EC 1.1.1.47) and diaphorase (EC 1.6.99.2) in the presence of glucose and 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium (WST-1) acts to cycle the formed NAD between its oxidized and reduced forms, resulting in the production of WST-1 formazan, which is monitored at 450 nm. The assay standard curve is linear from 0 to 70 muM allantoin. The level of allantoin in healthy subjects was measured to be 8.2+/-3.1 microM (n=30).
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PMID:An enzyme cycling method for measurement of allantoin in human serum. 1844 70

Several redox enzymes were examined for enzymatic/electrochemical-recycling systems in order to measure p-aminophenol (PAP) with high sensitivity. Glucose oxidase (GOD) and diaphorase (DI) worked well as catalysts for recycling electrode systems: these enzymes effectively reduced p-iminoquinone (PIQ), the electrochemically-oxidized form of PAP, and caused an enhancement in the electrochemical signals (anodic currents in the voltammogram and amperogram) by approximately 100 fold. The lower detection limits for PAP were estimated to be 50 nM with the GOD system and 2 nM with the DI system. We combined the enzymatic-recycling electrode using DI with an enzyme immunoassay system to measure atrial natriuretic peptide (ANP), an important marker peptide hormone involved in heart diseases. ANPs from serum samples at ppt-levels were determined appropriately using the present assay system.
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PMID:Comparison of enzymatic recycling electrodes for measuring aminophenol: development of a highly sensitive natriuretic peptide assay system. 1846 61


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