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:1.4.1.2 (glutamate dehydrogenase)
4,380 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We describe a bioluminescence method for measuring adenosine deaminase activity in serum. The method involves use of batchwise enzyme reaction containing adenosine, alpha-ketoglutarate, glutamic dehydrogenase and NADH. The resulting solution is injected to the continuous-flow bioluminescence system. In the system, a bacterial luciferase and NAD(P)H:FMN oxidoreductase are covalently co-immobilized on Sepharose 4B. Carrier solution (pH 6.8) for bioluminescence reaction contains FMN and decanal. The continuous-flow light-emitting system, in which the reactor (flow cell packed with immobilized enzyme) is placed in front of a photomultiplier tube inside a photon counter, is versatile and simple. Concentration and response are linearly related from 1.2 to 92.5 pmol per injection of ammonia. The precision of the method is satisfactory (coefficient of variation 3.9-6.8%). We validated the technique by comparing results with conventional assay method (UV method). Normal values for adenosine deaminase activity of serum ranged from 7.0 to 22.0 U/l in agreement with those obtained by other method. The Sepharose 4B-immobilized enzymes are stable for more than one year. This assay system could be used as a routine clinical laboratory test in the diagnosis of liver damage.
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PMID:Bioluminescent assay for serum adenosine deaminase with immobilized bacterial luciferase. 262 Apr 50

We have developed a rapid, simple, specific, and very sensitive bioluminescence method for the measurement of L-glutamate (L-Glu). Oxidation of L-Glu by glutamate dehydrogenase has been coupled with bacterial FMN reductase and luciferase. Light production (i.e., peak height or integral) was linear from less than 0.5 to 500 pmol of L-Glu. Potential interfering substances that may be encountered in brain tissue have been identified. The most potent inhibitors were ascorbate and the biogenic amines. Procedures that conferred long-term stability of the reagent mixture (greater than 8 h) were established. Bioluminescence analysis of L-Glu content in brain tissue extracts, fractions from release experiments, and human CSF corroborated respective results obtained by HPLC analysis. In this study, we have applied the method to monitor changes in the KCl-evoked release of endogenous L-Glu from milligram amounts of brain tissue, i.e., from lateral geniculate nucleus and superior colliculus after visual cortex ablation.
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PMID:A bioluminescence method for the measurement of L-glutamate: applications to the study of changes in the release of L-glutamate from lateral geniculate nucleus and superior colliculus after visual cortex ablation in rats. 287 87

We have developed a gene transfer system for the protozoan parasite Giardia lamblia. This organism is responsible for many cases of diarrhea worldwide and is considered to be one of the most primitive eukaryotes. Expression of a heterologous gene was detected in this parasite after electroporation with appropriate DNA constructs. We constructed a series of transfection plasmids using flanking sequences of the Giardia glutamate dehydrogenase (GDH) gene to drive expression of the firefly luciferase reporter gene. The optimal construct consisted of a GDH/luciferase fusion gene in which the first 18 codons of the GDH gene immediately preceded the luciferase gene; this fusion gene was flanked by the upstream and downstream sequences of the GDH gene. Electroporation of this construct into Giardia yielded luciferase activity that was 3000- to 50,000-fold above background. Removal of either the 5' or 3' GDH flanking sequences from this construct resulted in significantly reduced luciferase activity, and removal of both flanking sequences reduced luciferase activity to background levels. Luciferase activity was proportional to the amount of DNA electroporated and was maximal at 6 hr after electroporation.
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PMID:Transient transfection and expression of firefly luciferase in Giardia lamblia. 777 58

Manipulation of gene expression in Giardia lamblia, one of the most ancient eukaryotes, may provide insights into the evolutionary transition from prokaryotes to eukaryotes. Two recent successes in transient expression of the firefly luciferase (luc) gene in G. lamblia were mediated by a 5'-untranslated region (UTR) of the Giardia glutamate dehydrogenase (gdh) gene and a giardiavirus (GLV) genomic transcript, respectively. We now report a stable coexpression of luc gene with a neomycin phosphotransferase (neo(r)) gene in G. lamblia. An in vitro transcript of the construct pC670-Neo; containing the neo(r) encoding region flanked with the 5'670 nucleotides (nt) and the 3'2022 nt portion of GLV positive strand RNA, was electroporated into G. lamblia trophozoites that were infected with GLV. G418-resistant Giardia trophozoites were cloned, and the neo(r) mRNA in these clones was found to increase with increasing G418 pressure. This drug resistance remained stable upon continuous in vitro cultivation in the absence of G418 for over 15 days. Another plasmid pNeo/GDH/Luc, was constructed by inserting luc gene downstream from the neo(r) gene and the 193 nt 5' portion of gdh gene in pC670-Neo, and its bicistronic in vitro transcript was introduced into GLV-infected G. lamblia by electroporation. The transfectants demonstrated G418-resistance and persistent luciferase activity at levels parallel to the amount of G418 used for selection, peaking at a level of several thousand-fold above the background. Taken together, these data indicate that the neo(r) gene provides an effective selection marker for transformation of Giardia trophozoites, and the bicistronic RNA transfection vector may open the way for functional analysis of other genes in Giardia.
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PMID:Stable coexpression of a drug-resistance gene and a heterologous gene in an ancient parasitic protozoan Giardia lamblia. 901 Aug 44

We studied gene expression in the ancient eukaryote, Giardia lamblia, by taking advantage of assays developed recently in our laboratory, which allow new genetic analyses of this organism. We examined the transcription of a 2.2-kilobase segment of the Giardia genome that contains the glutamate dehydrogenase (GDH) gene and a portion of a second open reading frame encoding an uncharacterized gene. Nuclear run-on analyses showed that the genes are transcribed as two separate units spaced less than 200 base pairs apart, and transcription of the GDH gene initiates just 3-6 nucleotides upstream of its translation start codon. We characterized the GDH promoter by transfecting Giardia with DNA constructs that used the GDH upstream sequence to drive the expression of a luciferase reporter gene. By deletion and mutational analyses, we localized promoter function to three motifs within a 50-base pair region of the GDH upstream sequence. Using band shift assays and UV cross-linking, we demonstrated specific binding of a 68-kDa protein from Giardia nuclear extracts to short poly(T) tracts contained within two of the sequence motifs on single-stranded DNA from the promoter region. This report describes one of the first functional gene promoter and its cognate DNA-binding protein in this primitive eukaryote.
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PMID:Transcriptional analysis of the glutamate dehydrogenase gene in the primitive eukaryote, Giardia lamblia. Identification of a primordial gene promoter. 1075 60

A rat HIRI model was constructed and treated with an intraperitoneal injection of agomir-miR-494 or agomir-NC (negative control) for 7 days after the surgery. The pathophysiological changes in sham-operated rats, HIRI, HIRI + agomir-miR-494, and HIRI + agomir-NC were compared. The effect of miR-494 was also assessed in an H₂O₂-induced apoptosis model. Hepatic AML12 cells were transfected with mimics NC or miR-494 mimics, followed by 6-h H₂O₂ treatment. Cell proliferation and apoptosis were detected by CCK8 assay and flow cytometry, respectively. Further, the miR-494 target gene was identified by luciferase reporter assay, and verified both in vitro and in vivo experiments. The activity of AKT pathway was further analyzed in vivo by Western blot. HIRI + agomir-miR-494 rats exhibited significantly higher miR-494 expression, lower serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), lactate dehydrogenase (LDH), and glutamate dehydrogenase (GLDH) level, lower hepatic MDA, TOA, and OSI, alleviated hepatic necrosis, reduced hepatocyte apoptosis, and decreased expression of apoptosis-related proteins, when compared with HIRI + agomir-NC rats (P<0.05 or 0.01). After H₂O₂ treatment, AML-12 cells transfected with miR-494 mimics had significantly higher proliferation and lower apoptosis rate compared with mimics NC group (P<0.01). PTEN was identified as an miR-494 target gene. PTEN expression was significantly down-regulated in AML12 cells transfected with miR-494 mimics, and was up-regulated by treatment of miR-494 inhibitor (P<0.01). Moreover, HIRI + agomir-miR-494 rats exhibited significantly lower PTEN expression, and higher p-AKT, p-mTOR, and p-p70S6K levels compared with HIRI + agomir-NC rats. Therefore, miR-494 protected rats against hepatic ischemia/reperfusion (I/R) injury through down-regulating its downstream target gene PTEN, leading to the activation of PI3K/AKT signaling pathway.
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PMID:miR-494 up-regulates the PI3K/Akt pathway via targetting PTEN and attenuates hepatic ischemia/reperfusion injury in a rat model. 2884 16

Prevalence studies have demonstrated a global distribution of equine hepacivirus (EqHV), a member of the family Flaviviridae. However, apart from a single case of vertical transmission, natural routes of EqHV transmission remain elusive. Many known flaviviruses are horizontally transmitted between hematophagous arthropods and vertebrate hosts. This study represents the first investigation of potential EqHV transmission by mosquitoes. More than 5000 mosquitoes were collected across Austria and analyzed for EqHV ribonucleic acid (RNA) by reverse transcription quantitative polymerase chain reaction (RT-qPCR). Concurrently, 386 serum samples from horses in eastern Austria were analyzed for EqHV-specific antibodies by luciferase immunoprecipitation system (LIPS) and for EqHV RNA by RT-qPCR. Additionally, liver-specific biochemistry parameters were compared between EqHV RNA-positive horses and EqHV RNA-negative horses. Phylogenetic analysis was conducted in comparison to previously published sequences from various origins. No EqHV RNA was detected in mosquito pools. Serum samples yielded an EqHV antibody prevalence of 45.9% (177/386) and RNA prevalence of 4.15% (16/386). EqHV RNA-positive horses had significantly higher glutamate dehydrogenase (GLDH) levels (p = 0.013) than control horses. Phylogenetic analysis showed high similarity between nucleotide sequences of EqHV in Austrian horses and EqHV circulating in other regions. Despite frequently detected evidence of EqHV infection in Austrian horses, no viral RNA was found in mosquitoes. It is therefore unlikely that mosquitoes are vectors of this flavivirus.
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PMID:No Evidence of Mosquito Involvement in the Transmission of Equine Hepacivirus (Flaviviridae) in an Epidemiological Survey of Austrian Horses. 3168 93

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