Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:1.6.99.3 (diaphorase)
 5,903 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We describe a fully enzymic method for manual and continuous-flow colorimetric assay of triacylglycerols (triglycerides) in serum. Triglycerides are enzymically hydrolyzed in 10 min by lipase and microbial esterase. The resulting free glycerol is measured enzymically by glycerol kinase and glycerol-3-phosphate dehydrogenase. The NADH so formed is oxidized by coupling with a tetrazolium salt/diaphorase system. The test follows Beer's law to 8 g/L, and the final color is stable for at least 1 h for serum, 15 min for aqueous triolein standards. The manual assay requires only 25 microliter of serum and few manipulations. A specific triolein standard was developed for calibrating the manual method. For the continuous-flow method, calibration is made with four concentrations of glycerol standard. The procedure is sensitive, has good precision and accuracy, and gives results that compare well with chemical and enzymic commercial kit methods.
 ...
PMID:Manual and continuous-flow colorimetry of triacylglycerols by a fully enzymic method. 75 21

Up to now, more than 40.000 determinations of urinary estrogens (E1 + E2) have been carried out in routine clinical analysis by the enzymatic method using estradiol dehydrogenase. This method makes use of the transhydrogenating activity of the placental enzyme: this enzyme transfers hydrogen from NADP to NAD with recycling of the specific substrate (E1 + E2). For several years the necessary reagents have been commercially available in the form of a kit. Nonetheless, various improvements have been made to the measurement of reduced NAD, which accumulates in the reaction medium and is directly proportional to the concentration of the two estrogens. Three protocols are available at present: Spectrophotometric measurement at 340 nm (initial technique); Colorimetric measurement at 492 nm. The pink colour measured arises from the reduction of a tetrazolium salt (INT) by reduced NAD in a coupled system using diaphorase; Measurement by bioluminescence of the light energy liberated on the reduction of flavin derivatives by NADH. The reaction is mediated by various enzymes isolated from marine bacteria (FMN oxidoreductase and luciferase) in the presence of an aliphatic aldehyde (decanal). The procedure for each of these protocols is described as well as the means for controlling the linearity of the reaction. The choice of protocol is determined by the biological fluid available, the speed of response desired and the cost of the analysis.
 ...
PMID:[Various protocols for determining estrogens by the enzymatic method using estradiol dehydrogenase. Respective procedures and advantages]. 386 35

This paper describes a detection system for beta-lactams using a commercially prepared carboxypeptidase enzyme (CPase) and a substrate system in which lactic acid is cleaved from a synthetic peptide, N alpha-N epsilon-diacetyl-L-lysyl-d-alanyl-d-lactic acid. The lactate is itself oxidized by lactate dehydrogenase to form NADH. Oxidized NAD+ is regenerated by diaphorase with the simultaneous reduction of the colourless 2-(p-iodophenyl)-3-(p-nitrophenyl)-5-phenyl tetrazolium chloride hydrate (INT) indicator substrate to produce a red-mauve colour that is proportional to CPase activity. The presence of beta-lactams decreases the intensity of colour produced. The lower limit of detection for benzyl penicillin (Pen G) by this system is 20 ng g-1 compared with 50 ng g-1 by the same assay but using a R-d-ala-d-ala substrate from a commercial kit.
 ...
PMID:Improved spectrophotometric assay for beta-lactam residues in kidney tissue. 787 84

3Alpha-hydroxysteroid dehydrogenase (3-HSD) from Pseudomonas testosteroni and diaphorase (lipoyl dehydrogenase) from Clostridium spp. have been immobilized individually onto arylamine glass beads through diazotization. A cost-effective enzymic colorimetric method for determination of bile acid in serum and bile employing a mixture of these immobilized enzymes was developed. The method is based on measurement of reduced nicotinamide adenine dinucleotide generated from bile acid in serum/bile by immobilized 3alpha-HSD with a color reagent consisting of nitro blue tetrazolium chloride salt, oxidized nicotinamide adenine dinucleotide, and immobilized lipoyl dehydrogenase in 0.065 M sodium phosphate buffer, pH 7.0. Analytical recovery of added bile acid (50 and 200 micromol/L) was 95.57 and 85.46% in serum and 97.6 and 91.6% in bile, respectively. Within- and between-batch coefficients of variation (CV) for bile acid determination were <1.2 and <0.2% in serum and >0.1 and <0.1% in bile, respectively. Good correlations for bile acid in serum (r1=0.92) and in bile (r2=0.97) were obtained by use of a standard chemical method and the present method. The mixture of immobilized 3alpha-HSD dehydrogenase and lipoyl dehydrogenase lost 50% of its initial activity after 6 months of regular use. The cost of bile acid determination in 100 serum and bile samples by the present method has been compared with that of the Sigma kit method.
 ...
PMID:Measurement of bile acid in serum and bile with arylamine-glass-bound 3alpha-hydroxysteroid dehydrogenase and diaphorase. 1530 46

In order to elucidate the mechanism of fungal heterokayosis, a wild type strain of Fusarium oxysporum f. sp. vasinfectum was isolated from the cotton field in Anyang, Henan Province. Through single hyphal-tip isolation, a heterokaryon, Ag149, was obtained, and its two different phenotypic segregants, Ag149-I and Ag149-III, were separated from the mutated sectors on colony of the heterokaryon. They have remarkable differences on color of colony, morphology of hypha and pathogenicity. After analyzing by RAPD on their nuclear DNA with 100 random primers, no polymorphic difference was found among them. On going to find the different expressed gene, the mRNA differential display method was performed. Two kinds of reverse transcriptase AMV and MMLV, and a kit which consists of three kinds of 3' terminal anchor primers and eight kinds of 5' terminal arbitrary primers were used in differential display PCR (DD-PCR). Total RNA as template was reverse transcribed into corresponding cDNA by 3' terminal anchor primers, and the cDNA were amplified by polymerase chain reaction with a set of one same 3' anchor primer and one 5' arbitrary primer. The PCR products were then resolved on denaturing polyacylamimide gel, and the cDNA bands were visualized by silver staining. Among the 144 PCR products, 19 differentially expressed cDNA fragments ranged from 300 bp to 700 bp were purified. All of them were ligated to pGEM-T vector respectively for sequencing and Rev-Northern blotting. Two cDNA fragments (G5 and C6) were observed to be positive after Rev-Northern blotting. The C6 was highly expressed in the heterokaryon Ag149 and its segregant Ag149-I. It is 564 bp in length and can be predicted 77 amino acids from the beginning of the 3rd to 233th base, and then searched from GenBank. The amino acid sequence of C6 shared homologies with the 6th subunit of NADH dehydrogenase found in some bacteria, plants and animals at 30% - 70% level. While the G5 was highly expressed in Ag149 and its segregant Ag149-III. It is 432 bp in length and can be predicted 101 amino acids from the beginning of the 2nd to 304th base, and the amino acid sequence is 35% homologies comparing with the tetracycline efflux protein (OtrB) of Streptomyces rimosus. We also checked these two kinds of the pGEM-T vector harboring cDNA fragments (C6 and G5) by Southern blotting with their nuclear DNA and mitochondrial DNA (mtDNA) separately. The positive identification signals only appeared from nuclear DNA,and it addressed that C6 and G5 locate on their nuclear genome. The result indicated that the difference between the heterokaryon and its segregants is distinct on gene transcriptional level. Thus a molecular evidence for the formation of heterokaryon in filamentous fungi was provided.
 ...
PMID:[Transcriptional differences between a heterokaryon and its segregants of Fusarium oxysporum f. sp. vasinfectum]. 1547 7

3alpha-Hydroxysteroid dehydrogenase (3alpha-HSD) from Pseudomonas testosteronei and diaphorase (lipoyl dehydrogenase) from Clostridium spp were immobilized individually onto alkylamine glass beads through glutaraldehyde coupling. A cost-effective enzymic colorimetric method for determination of bile acid in the serum and bile was developed employing mixture of the immobilized enzymes. The method was based upon measurement of NADH generated from NAD+ during oxidation of bile acid by immobilized 3alpha-HSD with a color reagent consisting of nitrobluetetrazolium (NBT) chloride salt and immobilized diaphorase in 0.065 M sodium phosphate buffer (pH 7.0). The minimum detection limit of the method was 4.8 pmol/L in the serum and 19.5 micromol/L in bile. The per cent recovery of added bile acid in the serum and bile was 89.1 and 95.0, respectively. Within and between batch coefficients of variation (CV) for bile acid determination were <1.0% and <0.2% in the serum and <0.2% and <0.6% in bile, respectively. A good correlation for bile acid in the serum (r1= 0.95) and in bile (r2 = 0.93) was obtained by a standard chemical method (a commonly used method in India) and the present method. The mixture of immobilized 3alpha-HSD and diaphorase lost 30% of its initial activity after 4 months of regular use. The cost of bile acid determination for 100 the serum and bile samples by the present method was found to be lower than by a commercially available method (Sigma kit 450-A).
 ...
PMID:Discrete analysis of bile acid in serum and bile with 3 alpha-hydroxysteroid dehydrogenase and diaphorase immobilized onto alkylamine glass beads. 1695 58

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.
 ...
PMID:Development of a carbon nanotube paste electrode osmium polymer-mediated biosensor for determination of glucose in alcoholic beverages. 1717 56

The construction and performance of integrated amperometric biosensors for the determination of glycerol are reported. Two different biosensor configurations have been evaluated: one based on the glycerol dehydrogenase/diaphorase (GDH/DP) bienzyme system, and another using glycerol kinase/glycerol-3-phosphate oxidase/peroxidase (GK/GPOx/HRP). Both enzyme systems were immobilized together with the mediator tetrathiafulvalene (TTF) on a 3-mercaptopropionic acid (MPA) self-assembled monolayer (SAM)-modified gold electrode by using a dialysis membrane. The electrochemical oxidation of TTF at +150mV (vs. Ag/AgCl), and the reduction of TTF(+) at 0mV were used for the monitoring of the enzyme reactions for the bienzyme and trienzyme configurations, respectively. Experimental variables concerning both the biosensors composition and the working conditions were optimized for each configuration. A good repeatability of the measurements with no need of cleaning or pretreatment of the biosensors was obtained in both cases. After 51 days of use, the GDH/DP biosensor still exhibited 87% of the original sensitivity, while the GK/GPOx/HRP biosensor yielded a 46% of the original response after 8 days. Calibration graphs for glycerol with linear ranges of 1.0x10(-6) to 2.0x10(-5) or 1.0x10(-6) to 1.0x10(-5)M glycerol and sensitivities of 1214+/-21 or 1460+/-34microAM(-1) were obtained with GDH/DP and GK/GPOx/HRP biosensors, respectively. The calculated detection limits were 4.0x10(-7) and 3.1x10(-7)M, respectively. The biosensors exhibited a great sensitivity with no significant interferences in the analysis of wines. The biosensors were applied to the determination of glycerol in 12 different wines and the results advantageously compared with those provided by a commercial enzyme kit.
 ...
PMID:Integrated multienzyme electrochemical biosensors for the determination of glycerol in wines. 1826 15

BACKGROUND Osteosarcoma (OS) is a common primary malignant bone tumor for which the molecular mechanisms remain unclear. Studies on coding and non-coding RNAs are needed to determine the molecular mechanism. MATERIAL AND METHODS To explore the potential roles of miRNAs and mRNA in OS, we determined the miRNA and mRNA expression profile of 3 pairs of OS and paracancerous tissues from patients with OS by sequencing and bioinformatics analysis. The expression levels of critical miRNAs and mRNAs were verified in 10 pairs of OS and paracancerous tissues. An miRNA inhibitor and mimics were used to investigate the interactions between miRNAs and target genes. The cell counting kit-8 assay was performed to evaluate OS cell proliferation after miRNA interference. RESULTS A total of 184 miRNAs and 2501 mRNAs were identified (fold-change >2.0 or <2.0, P<0.05), with up-regulation of 82 miRNAs and 1320 mRNAs and down-regulation of 102 miRNAs and 1181 mRNAs in OS tissue. The protein protein interaction network revealed that UQCRC1 (ubiquinol-cytochrome c reductase core protein 1) is a critical gene and a potential target gene of miR-214-3p. Both UQCRC1 and miR-214-3p were significantly differentially expressed in OS tissue and cell lines (down and up-regulated, respectively). Down-regulated miR-214-3p expression increased UQCRC1 expression and suppressed OS cell proliferation. In contrast, overexpression of miR-214-3p decreased UQCRC1 expression and promoted OS cell proliferation. CONCLUSIONS High miR-214-3p expression may promote OS cell proliferation by targeting UQCRC1, providing insight into a potential therapeutic target for preventing and treating OS.
 ...
PMID:RNA Sequencing of Osteosarcoma Gene Expression Profile Revealed that miR-214-3p Facilitates Osteosarcoma Cell Proliferation via Targeting Ubiquinol-Cytochrome c Reductase Core Protein 1 (UQCRC1). 3127 65