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:1.2.1.13 (glyceraldehyde-3-phosphate dehydrogenase)
6,511 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Previous studies have shown that endogenous nitric oxide (NO) potentiates glycolysis in the cytokine-activated murine microvascular endothelial cells (MME). In the present study we investigate the influence of NO on the expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), an enzyme of the glycolytic pathway. Activation of MME with TNF-alpha and IFN-gamma resulted in a strong elevation of GAPDH mRNA levels. This effect was impaired in the presence of L-NMMA, the inhibitor of NO synthesis. We discuss the possibility that NO-mediated elevation of GAPDH mRNA levels may compensate for NO-mediated inhibition of GAPDH enzymatic activity, representing another adaptive mechanism which protects cells producing large amounts of NO against its cytotoxic effects.
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PMID:Stimulation of glyceraldehyde-3-phosphate dehydrogenase mRNA levels by endogenous nitric oxide in cytokine-activated endothelium. 852 35

A specific tumour necrosis factor alpha ribozyme (TNF-alpha-Rz) binding activity has been purified and identified by N-terminal microsequencing as the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The purified protein as well as commercial GAPDH binds tightly to TNF-alpha ribozyme compared to a variety of other ribozymes and RNAs. Binding of GAPDH to the TNF-alpha-Rz and its derivatives was inhibited by NAD+ and ATP, suggesting that the GAPDH Rossmann fold structure is a part of the ribozyme binding site. Interestingly, GAPDH increased the in vitro cleavage rates of hammerhead ribozymes by up to 25-fold, while no significant stimulation was observed with the lactate dehydrogenase (LDH). This effect was found to be due to the unfolding activity of GAPDH. In fact, pulse-chase experiments demonstrate directly that GAPDH has the capacity to accelerate the ribozyme/substrate association, especially of ribozymes and/or substrates whose predicted secondary structure might interfere with the association step. Under our conditions, the presumed unfolding activity of GAPDH also enhances the turnover of ribozymes by increasing the rate of product dissociation, although only for short cleavage products. Longer duplexes required more incubation time to dissociate. In vitro non-specific interaction of the GAPDH with hammerhead ribozymes and RNA substrates was found to be adequate for the cleavage enhancement effect to occur. However, an analysis of the ability of various prototypical ribozymes to inhibit the expression of interleukin-2 suggests that the addition of a sequence having a high affinity for GAPDH improves the efficacy of ribozymes in the cells. Thus the characterization of cellular proteins with unfolding activity, which specifically bind to hammerhead ribozyme, should facilitate the design of a more effective ribozyme in vivo.
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PMID:Enhancement of hammerhead ribozyme catalysis by glyceraldehyde-3-phosphate dehydrogenase. 863 81

The mechanism of inhibition of HIVBa-L replication by interleukin 10 (IL-10) in primary monocytes and macrophages at various stages of maturation was investigated using semiquantitative PCR for reverse-transcribed HIV DNA, and Northern hybridization for HIV mRNA expression in comparison with extracellular p24 antigen. Pretreatment of monocytes with IL-10 markedly inhibited expression of both unspliced and spliced HIV RNA, reaching a nadir at 7 days and recovering to normal levels by 10 days after a single application. The optimum inhibitory concentration was 25 ng/ml. Less inhibition of HIV RNA expression was observed when IL-10 was added after HIV infection of monocytes and the inhibitory effect progressively declined to negligible levels as monocytes matured into macrophages over 10 days. IL-10 also downregulated the expression of cellular genes, including the transferrin receptor, 28S rRNA, and GAPDH. The kinetics of the inhibition of cellular mRNAs correlated with the inhibition of HIV RNA and also declined as monocytes matured into macrophages. In contrast, IL-10 did not inhibit cellular mRNA expression in the macrophage cell line THP-1. Neutralizing polyclonal antibody to IL-10 reversed all its inhibitory effects. Interaction of IL-10 and TNF-alpha in combination were generally antagonistic in their effects on HIV transcription. IL-10 prevented stimulation of HIV RNA expression by TNF-alpha after preincubation with monocytes for 48 hr. IL-10 had no effect on the levels of HIV cDNA or the process of initiation and completion of reverse transcription. The inhibitory effect of IL-10 on HIV replication in maturing monocytes was probably mediated mainly by inhibition of cellular gene expression and inhibition of maturation of monocytes into macrophages and their activation, with consequent downregulation of HIV mRNA.
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PMID:Inhibition of human immunodeficiency virus replication in differentiating monocytes by interleukin 10 occurs in parallel with inhibition of cellular RNA expression. 887 Aug 45

Spontaneous tumor regression, which is observed clinically and histologically in some primary melanomas, occurs in the absence of any effective therapy. It is probably immunologically mediated, because regressing melanomas are infiltrated with larger numbers of activated T cells, primarily CD4+, than nonregressing melanomas. To investigate the hypothesis that spontaneous regression of melanomas is caused by T-cell cytokine production, cytokine mRNA expression in 20 primary melanomas was examined using a noncompetitive, quantitative reverse-transcriptase polymerase chain reaction method. DNA standards were used to generate known numbers of molecules in each sample. Results were standardized to the internal control, glyceraldehyde-3-phosphate dehydrogenase. mRNA for CD35, lymphotoxin (TNF-beta), and IL-2 were significantly elevated in the ten regressing melanomas compared to the ten nonregressing melanomas. IFN-gamma mRNA was also elevated in regressing melanomas but failed to reach statistical significance. The Th2 cytokines IL-10 and IL-13 did not show differences in the regressing melanomas compared to nonregressing melanomas; neither did the pro-inflammatory cytokines IL-1alpha, IL-1beta, IL-6, IL-8, and TNF-alpha, nor the growth factors, bFGF and TGF-beta or GM-CSF. This study shows an association between Th1 cytokines and spontaneously regressing melanomas. Although we have not shown that these cytokines cause regression, these findings support our hypothesis that activated CD4+ T cells may mediate melanoma regression by secretion of Th1 cytokines.
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PMID:T helper 1 cytokine mRNA is increased in spontaneously regressing primary melanomas. 918 21

Following decapitation, during the brief time period of postmortem brain tissue collection, significant changes in neuro-metabolite levels can occur. To circumvent such changes, we routinely kill rats using 1 to 2 sec pulses of focused high-energy microwave irradiation (10 kW). The effects of high-energy microwave irradiation on total RNA and mRNA integrity in brain however, are unknown. Total RNA recovery, per gram wet weight, in brain regions of microwaved rats was less than 50% of that in rats killed by decapitation. Formaldehyde agarose gel electrophoresis showed that ribosomal RNA components were highly degraded in all brain regions of microwaved rats. In contrast, poly A+mRNA, as measured by poly A+driven cDNA synthesis and Northern analysis, in brain samples of microwaved or decapitated rats was of equal integrity and quantity when expressed per mg tissue weight. Furthermore, positive RT-PCR products for GAPDH and TNF-alpha were observed in brain regions of both microwaved and decapitated rats. These observations indicated that high-energy focused microwave irradiation does not reduce mRNA abundance and integrity. Thus, this method of animal sacrifice can be used to simultaneously study, accurately and precisely, levels of brain metabolites as well as molecular biological events in discrete brain regions of experimental animals without postmortem interference.
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PMID:Recovery of high-integrity mRNA from brains of rats killed by high-energy focused microwave irradiation. 1059 38

Here we present a novel methodology to quantitate bovine cytokines and growth factors contributing to immunity against bacterial infections of the mammary gland in cattle. Real-time TaqMan PCR systems were developed to overcome limitations of conventional quantitative PCR methods. The TaqMan method is based on the cleavage of fluorescent dye-labeled probes by the 5'-3' exonuclease activity of the Taq DNA polymerase during PCR and measurement of fluorescence intensity by an automated spectrophotometer integrated in a sequence detection system (Applied Biosystems, Foster City, CA). The bovine-specific TaqMan probes were designed to encompass an intron, thus allowing differentiation between complementary DNA (cDNA) and genomic DNA (gDNA) amplification products. Quantitative analysis of cytokine cDNA was performed in comparison to bovine glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Messenger RNA (mRNA) from the universally expressed housekeeping gene GAPDH proved to be useful as an amplification control and allowed for correction of variations in different numbers of cells in the starting material, in the efficiencies of RNA extraction and reverse transcription. With this method, high-throughput analysis of large numbers of samples was possible within a short time. In addition, decreasing the numbers of working steps shortened the time for analysis and increased accuracy. Profiles of cytokines (interleukin (IL)-2, IL-6, IL-8, IL-12 p40, TNF-alpha, IFN-gamma) and granulocyte-macrophage colony stimulating factor (GM-CSF) were established in normal lactating cattle. Differences of cytokine profiles obtained with the real-time TaqMan PCR system and conventional methods are discussed.
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PMID:Quantitation of bovine cytokine mRNA in milk cells of healthy cattle by real-time TaqMan polymerase chain reaction. 1113 25

NF-kappaB/Rel transcription factors have been implicated in the differentiation of monocytes to either dendritic cells (DCs) or macrophages, as well as in the maturation of DCs from antigen-processing to antigen-presenting cells. Recent studies of the expression pattern of Rel proteins and their inhibitors (IkappaBs) suggest that their regulation during this differentiation process is transcriptional. To investigate differential gene expression between macrophages and DCs, we used commercially available gene microarrays (GEArray KIT), which included four of the NF-kappaB/Rel family genes (p50/p105, p52/p100, RelB, and c-rel) and 32 additional genes either in the NF-kappaB signal transduction pathway or under transcriptional control of NF-kappaB/Rel factors. To generate macrophages and DCs, human adherent peripheral blood monocytes were cultured with M-CSF or GM-CSF + IL-4 respectively for up to 8 days. DCs (and in some experiments, macrophages) were treated with lipopolysaccharide (LPS) for the last 48 h of culture to induce maturation. Cells were harvested after 7 days, cDNA was prepared and radiolabeled with alpha-(32)P-dCTP, then hybridized to gene arrays containing specific gene probes. beta-actin and GAPDH or PUC18 oligonucleotides served as positive or negative controls, respectively. The expression of all four NF-kappaB/Rel family genes examined was significantly upregulated in maturing DCs compared to macrophages. The strongest difference was observed for c-rel. RT-PCR determinations of c-rel, RelB, and p105 mRNAs confirmed these observations. Among the 32 NF-kappaB/Rel pathway genes, 14 were upregulated in mature DCs compared to macrophages. These genes were IkappaBalpha, IKK-beta, NIK, ICAM-1, P-selectin, E-selectin, TNF-alpha, TNFR2, TNFAIP3, IL-1alpha, IL-1R1, IL-1R2, IRAK, and TANK. By contrast, only mcp-1 (monocyte chemotactic protein 1) was upregulated in macrophages compared to DCs. NF-kappaB pathway genes upregulated in DCs compared to macrophages were constitutively expressed in monocytes then selectively downregulated during macrophage but not DC differentiation. LPS did not induce expression of most of these genes in macrophages but LPS did induce upregulation of IL-8 in mature macrophages. We conclude that NF-kappaB/Rel family genes, especially c-rel, are selectively expressed during differentiation of monocytes towards DCs. Moreover, this differential expression is associated both with activation of different NF-kappaB signal transduction pathways in DCs and macrophages and with expression of a unique subset of genes in DCs that are transcriptionally targeted by NF-kappaB/Rel factors. The results illustrate the ability of the NF-kappaB pathway to respond to differentiation stimuli by activating in a cell-specific manner unique signalling pathways and subsets of NF-kappaB target genes.
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PMID:Expression of different NF-kappaB pathway genes in dendritic cells (DCs) or macrophages assessed by gene expression profiling. 1157 45

Bacterial heat shock proteins (hsp) are evolutionary conserved immunodominant proteins that manifest amino acid homologies with hsp present in mammalian cells. Preimmunization with mycobacterial hsp65 has been found to protect against various forms of experimental arthritis. As these protective effects have previously been attributed to induction of self homologue cross-reactive T cell responses, the question was raised as to whether this protective effect could be extended to other highly conserved and immunodominant microbial Ags with mammalian homologues. Therefore, we immunized Lewis rats with conserved bacterial Ags (superoxide dismutase, aldolase, GAPDH, and hsp70). Although all Ags appeared highly immunogenic, we only found a protective effect in experimental arthritis after immunization with bacterial hsp70. The protective effect of hsp70 was accompanied with a switch in the subclasses of hsp70-specific Abs, suggesting the induction of Th2-like response. The most striking difference between immunization with hsp70 and all other immunodominant Ags was the expression of IL-10 found after immunization with hsp70. Even more, while immunization with hsp70 led to Ag-induced production of IL-10 and IL-4, immunization with aldolase led to increased production of IFN-gamma and TNF-alpha. Thus, the protective effect of conserved immunodominant proteins in experimental arthritis seems to be a specific feature of hsp. Therefore, hsp may offer unique possibilities for immunological intervention in inflammatory diseases.
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PMID:Induction of IL-10 and inhibition of experimental arthritis are specific features of microbial heat shock proteins that are absent for other evolutionarily conserved immunodominant proteins. 1159 34

Cytokines play an important role in the regulation of the immune system, but low circulating levels in plasma make routine measurement a difficult task. A new methodology based on single tube RT-PCR has been developed to determine the expression of multiple canine cytokines (TNF-alpha, IL-2, IFN-gamma, IL-18, IL-4, IL-6 and IL-10) using primers and protocols designed allow specific amplification of the mRNAs. The technique is performed in one tube in two consecutive steps, a specific transcription of the mRNA of a given cytokine and amplification of the corresponding gene by PCR. The technique was used to analyse the mRNA cytokine profile of peripheral blood mononuclear cells (PBMCs) from healthy dogs using two approaches: (i) analysis of PBMC isolated ex vivo; (ii) analysis of PBMC after in vitro cultures with or without the mitogen ConA. The samples were separated in agarose gels and the intensity of ethidium bromide signals quantified using standard video imaging equipment. Results were interpreted as the ratio of cytokine to GAPDH expression. The results obtained show that the method is easy to use and reproducible. Therefore, this method of monitoring the mRNA cytokine expression might be an useful tool for understanding the immune response in dogs.
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PMID:Semi-quantitative analysis of multiple cytokines in canine peripheral blood mononuclear cells by [correction of zby] a single tube RT-PCR. 1173 Sep 29

To investigate the pathogenesis of early lesions in canine distemper virus (CDV) leukoencephalomyelitis, the expressions of pro- and anti-inflammatory cytokines such as interleukin (IL)-1beta, IL-2, IL-6, IL-8, IL-10, IL-12, tumor necrosis factor (TNF)-alpha, interferon (IFN)-gamma and transforming growth factor (TGF)-beta and the housekeeping genes beta-actin and GAPDH were studied using semi-quantitative RT-PCR. Relative cytokine values were related to the degree of CDV infection, MHC class II expression and infiltration of CD4-, CD8- and CD3epsilon-positive lymphocytes. Actin up-regulation, in contrast to GAPDH, was influenced by CDV infection and therefore could not be used as an internal standard to study cytokine expression. In early CDV infection of the cerebellum, either no detectable lesions or mild infiltration of CD8 positive cells or demyelination and up-regulation of MHC class II antigen were observed. IL-6, -8, -12 and TNF-alpha transcripts were found in 94%, 94%, 78% and 56% of distemper dogs, respectively, compared to 17%, 33%, 0% and 0% in controls, whereas IL-1beta, -2 and IFN-gamma were not detectable in any of the studied cerebella. Conversely, IL-10 and TGF-beta transcripts were present in 83% and 100% of the investigated cerebella of distemper dogs and controls. Relative RT-PCR results, expressed as %GAPDH, revealed a significant up-regulation of IL-6, -8, -12 and TNF-alpha mRNA in distemper dogs; whereas IL-10 and TGF-beta showed only a weak and not significantly increased expression following infection. Relative pro-inflammatory cytokine expression values were highest following CDV infection, indicating that the virus itself directly triggered the up-regulation of the pro-inflammatory cytokines. Succeeding changes, such as lymphocyte infiltration, MHC class II up-regulation and demyelination resulted only in a minor additional increase in cytokine expression, implying a secondary or by-stander mechanism of cytokine activation by these changes. Disease initiation and progression in early distemper leukoencephalomyelitis seemed to be due to a lacking or inappropriate response of the anti-inflammatory cytokines in the presence of a vigorous up-regulation of pro-inflammatory cytokines.
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PMID:Increased expression of pro-inflammatory cytokines and lack of up-regulation of anti-inflammatory cytokines in early distemper CNS lesions. 1196 Jun 38


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