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)

The steady-state level of the neuromodulin transcript in the neuron-like N1E-115 cell line was measured with a method combining reverse transcription and the polymerase chain reaction (RT/PCR). Total RNA was isolated from N1E-115 cells and treated with DNAse to remove residual DNA; cDNA was synthesized from this RNA by priming with random hexamers. For PCR amplification, primers for neuromodulin were designed for regions of the coding sequence that were identical in mouse, rat, and human. In one approach (the 'ratio method'), variations in RNA yield and cDNA synthesis efficiency were controlled for by amplifying a reference (housekeeping) gene (glyceraldehyde phosphate dehydrogenase; GAPDH). To control for inter-experimental variations in PCR amplification efficiencies the data were analyzed on semi-logarithmic plots, with which the relative levels of the starting templates could be determined by extrapolating the plots to cycle number zero (0). In another approach with RT/PCR (the 'spiking method'), the absolute level of N1E-115 neuromodulin cDNA was assessed by adding known amounts of cloned human neuromodulin template to the RT/PCR assay of N1E-115 nucleic acid and comparing the increased yield of product across cycles. When the spike was added at either the cDNA level (in the form of double-stranded DNA) or at the total RNA level (as sense RNA), the levels of N1E-115 calculated were virtually the same: 509 fg and 495 fg of coding region per ug total RNA in confluent N1E-115 cells, respectively. Treatment of N1E-115 cells with 2% dimethylsulfoxide for three days elevated neuromodulin mRNA levels 5.6-fold. Conversely, treatment of N1E-115 cells with 100 nM phorbol myristate acetate for 24 h decreased the level of neuromodulin mRNA by 70%. Under carefully controlled conditions and within certain limits of precision, the RT/PCR method appears to be suitable for assessing the level of low abundance mRNA under various pharmacologically-induced conditions.
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PMID:Transcriptional regulation of neuromodulin (GAP-43) in mouse neuroblastoma clone N1E-115 as evaluated by the RT/PCR method. 137 7

Housekeeping genes, particularly actin, tubulin, and the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH), are widely used to estimate the amount and integrity of RNA in Northern blotting. In this work, the most reliable housekeeping gene for gene expression analysis of Hodgkin's disease (HD) lymph nodes was determined by comparing the conventional housekeeping genes, beta-actin, beta-tubulin, GAPDH, and the mouse gene LLRep3, that had been used previously in gene expression studies. It was found that the amounts of mRNA in these genes are very heterogeneous in HD lymph nodes. In contrast, their expression was relatively constant in tonsils undergoing a chronic inflammatory process. It is concluded that none of the housekeeping genes tested is suitable for the fine quantitative analysis of gene expression in HD lymph nodes.
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PMID:Expression of housekeeping genes in Hodgkin's disease lymph nodes. 177 60

The action of 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) on transcription and steady-state mRNA levels, protein expression, and enzymatic activity of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in cultured human keratinocytes was examined. GAPDH transcription increased as a function of time of exposure to TCDD, reaching a maximum of 23-fold after 8 days. Northern blot analysis showed a 4-fold increase in mRNA after 8 days exposure. Protein levels were increased 4-fold, and dehydrogenase activity by 5-fold, under the same exposure conditions. GAPDH is commonly thought of as a constitutive housekeeping gene and is often used as a loading control for northern blot analysis. Our data suggest that caution should be used when using GAPDH as an RNA control, especially in studies examining cell proliferation and carcinogenesis.
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PMID:Transcriptional regulation of glyceraldehyde-3-phosphate dehydrogenase by 2,3,7,8-tetrachlorodibenzo-p-dioxin. 761 2

Matrix Gla (gamma-carboxyglutamic acid) protein (MGP) is a vitamin-K-dependent extracellular matrix protein. A method was developed to quantitate MGP mRNA based on competitive polymerase chain reaction following reverse transcription (competitive RT-PCR). The MGP cDNA was coamplified with a mutant MGP cDNA (competitor). The ratio of MGP to competitor after the PCR reaction was compared to standards to determine the amount of MGP mRNA in RT samples. MGP mRNA in as little as 3.125 ng total RNA was accurately quantitated and was far more sensitive than RNA hybridization methods. To control for variations due to sample preparation, a second competitive RT-PCR was developed to measure the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA from the same sample as an internal control. Thus, the amount of MGP is normalized to the amount of the housekeeping gene GAPDH. The accuracy, sensitivity and ease of this new method enables rapid mRNA quantitation without blotting, hybridization or autoradiography. The method is particularly advantageous for MGP mRNA measurement from a small amount of sample. Using this assay, we established that MGP mRNA increases approx. fivefold with co-treatment of retinoic and ascorbic acids.
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PMID:Quantitation of matrix Gla protein mRNA by competitive polymerase chain reaction using glyceraldehyde-3-phosphate dehydrogenase as an internal control. 772 Oct 85

Acute myocardial ischemia leads to a rapid increase of cardiac beta-adrenergic receptors in plasma membranes despite the release of large and desensitizing amounts of endogenous catecholamines. Part of this increase has been shown to occur at the expense of intracellular receptors. To investigate whether an additional expressional regulation of beta-adrenergic receptors due to an increase of mRNA levels is involved, the mRNA levels specific for beta 1- and beta 2-adrenergic receptors were determined after various periods of global ischemia in isolated perfused rat hearts. The subtype-specific quantification of mRNA for beta 1- and beta 2-adrenergic receptors was determined using reverse-transcription followed by PCR (RT-PCR) and RNA protection assays. RT-PCR resulted in single amplification products of the expected sizes (159 bp for beta 1-adrenergic receptors and 240 bp for beta 2-adrenergic receptors). The specificity of these amplification products was confirmed by specific restriction digests. Southern blot hybridizations with internal oligonucleotides and sequencing using the dideoxy chain termination method. For quantification purposes, the mRNAs of housekeeping gene GAPDH and of cardiac alpha-actin were determined as internal standards. Additionally, cRNAs specific for beta 1- and beta 2-adrenergic receptors were used as external standards. Brief periods of global ischemia induced a rapid increase in the steady state level of mRNA for beta 1-adrenergic receptors. There was a statistically significant rise already after 15 min by 57% compared to controls. After 30 min of ischemia the mRNA levels had almost doubled. After 60 min of ischemia, the mRNA levels specific for beta 1-adrenergic receptors tended to decrease, but remained significantly above normoxic controls. In contrast, the mRNA levels specific for beta 2-adrenergic receptors remained constant up to 60 min of global myocardial ischemia. To investigate, whether agonist occupancy of the receptors may contribute to this regulation, the effect of preperfusion with the beta-blocker alprenolol was determined. Contrary to expectation, beta-blockade did not influence the ischemia-induced increase of mRNA levels specific for beta 1-adrenergic receptors. These data demonstrate for the first time, that acute myocardial ischemia induces a rapid, and subtype-selective regulation of mRNA levels for beta 1-adrenergic receptors. However, occupation or activation of beta-adrenergic receptors by an agonist is not involved in this newly characterized regulation of mRNA for beta 1-adrenergic receptors in acute myocardial ischemia.
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PMID:Regulation of beta-adrenergic receptors in acute myocardial ischemia: subtype-selective increase of mRNA specific for beta 1-adrenergic receptors. 776 Mar 63

A number of reports have indicated that RNA recovered from paraffin-embedded tissue can be used as a substrate in the polymerase chain reaction (PCR). Although it is established that RNA in paraffin-embedded tissue undergoes significant degradation, the specific contributions of different fixatives and fixation times to this degradation are not known. Mouse splenic tissue was harvested and fixed immediately for 2, 8, or 24 h in either formalin, Omnifix II, or Carnoy's fixative and then processed and embedded in paraffin. RNA was extracted from deparaffinized cubes of tissue using an adaptation of the technique described by Chomczynski and Sacchi. RNA was reverse transcribed using a random hexamer primed reaction. PCR amplification for cDNAs of the housekeeping genes glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and hypoxanthine phosphoribosyltransferase (HPRT) mRNAs was then performed. Although GAPDH amplification is used routinely on fresh and frozen tissues, we show that the presence of DNA contamination in the RNA preparations limits its usefulness in paraffin-embedded tissue. Amplifiable HPRT mRNA sequences were detected in nine of 12 samples fixed in Omnifix II, in four of 12 samples fixed in Carnoy's fixative, and in none of 12 formalin-fixed samples. Because of primer selection to preclude amplification of genomic HPRT, DNA contamination is not an issue when HPRT is amplified. Thus, HPRT represents the control system of choice for the evaluation of RNA in PET. The techniques described provide a rapid, uniform, and reproducible method of obtaining RNA from PET for molecular analysis, but they indicate limited utility for retrospective analysis of archival tissues.
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PMID:Effects of fixative and fixation time on the extraction and polymerase chain reaction amplification of RNA from paraffin-embedded tissue. Comparison of two housekeeping gene mRNA controls. 752 44

Studies of quantitative changes in gene expression in malignant cells have often used housekeeping genes as controls against which the level of expression of a gene under study could be compared. We have now examined whether the expression of the most commonly used of these housekeeping genes can be regarded as reliable controls for gene expression studies in non-Hodgkin's lymphoma (NHL). We have used Northern blot analysis to compare the levels of expression of beta-actin, alpha-tubulin, beta 2-microglobulin and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) to that of ribosomal RNA. These studies demonstrated that whereas there was a reasonable correlation between the relative levels of rRNA and housekeeping gene expression in reactive hyperplastic nodes, there were major differences in the relative levels of expression of the housekeeping genes in both low and high grade lymphomas; only GAPDH showed any degree of consistency. These observations indicated that housekeeping gene expression was not a reliable control for estimating changes in the level of expression of other genes in NHL, and instead suggested that 18S or 28S rRNA expression offered a more accurate method of RNA quantitation.
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PMID:Investigation of the expression of housekeeping genes in non-Hodgkin's lymphoma. 822 Jan 38

We tested the hypothesis that early alterations in calcium influx induced by an imposed 60 Hz magnetic field are propagated down the signal transduction (ST) cascade to alter c-MYC mRNa induction. To test this we measured both ST parameters in the same cells following 60 Hz magnetic field exposures in a specialized annular ring device (220 G (22 mT), 1.7 mV/cm maximal E(induced), 37 degrees C, 60 min). Ca2+ influx is a very early ST marker that precedes the specific induction of mRNA transcripts for the proto-oncogene c-MYC, an immediate early response gene. In three experiments influx of 45Ca2+ in the absence of mitogen was similar to that in cells treated with suboptimal levels of Con-A (1 micrograms/ml). However, calcium influx was elevated 1.5-fold when lymphocytes were exposed to Con-A plus magnetic fields; this co-stimulatory effect is consistent with previous reports from our laboratory [FEBS Lett. 301 (1992) 53-59; FEBS Lett. 271 (1990) 157-160; Ann. N.Y. Acad. Sci. 649 (1992) 74-95]. The level of c-MYC mRNA transcript copies in non-activated cells and in suboptimally-activated cells was also similar, which is consistent with the above calcium influx findings. Significantly, lymphocytes exposed to the combination of magnetic fields plus suboptimal Con-A responded with an approximate 3.0-fold increase in band intensity of c-MYC mRNA transcripts. Importantly, transcripts for the housekeeping gene GAPDH were not influenced by mitogen or magnetic fields. We also observed that lymphocytes that failed to exhibit increased calcium influx in response to magnetic fields plus Con-A, also failed to exhibit an increase in total copies of c-MYC mRNA. Thus, calcium influx and c-MYC mRNA expression, which are sequentially linked via the signal transduction cascade in contrast to GAPDH, were both increased by magnetic fields. These findings support the above ST hypothesis and provide experimental evidence for a general biological framework for understanding magnetic field interactions with the cell through signal transduction. In addition, these findings indicate that magnetic fields can act as a co-stimulus at suboptimal levels of mitogen; pronounced physiological changes in lymphocytes such as calcium influx and c-MYC mRNA induction were not triggered by a weak mitogenic signal unless accompanied by a magnetic field. Magnetic fields, thus, have the ability to potentiate or amplify cell signaling.
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PMID:Experimental evidence for 60 Hz magnetic fields operating through the signal transduction cascade. Effects on calcium influx and c-MYC mRNA induction. 824 37

A 28-base phosphodiester triple helix-forming oligonucleotide, mostly G and A containing, targeted to a polypurine tract interrupted by a purine-pyrimidine inversion, situated upstream from the TATA box of the promoter of the human HER2 gene, was conceived by computer modeling. The "energetically best choice" was oligo 28(C), which formed the triple helix in vitro, as proved by gel retardation and Fourier transform infrared spectroscopy. When administered as a complex with lipofectin, fluorescence confocal microscopy and electrophoresis confirmed the delivery and persistence of this unprotected oligonucleotide inside MCF7 (breast cancer) cells. At a concentration of 2 microM, the oligonucleotide reduced within 6 h the HER2 mRNA level to 42% (Northern blot) but did not interfere with the transcription of a housekeeping gene, glyceraldehyde-3-phosphate dehydrogenase. During the first day of administration at 0.22 microM, it lowered to 59% the HER2 protein in treated, as compared to nontreated, cells (ELISA). The effect was sequence specific when compared to that of five different negative controls, and it was target selective when compared to the expression of a related, nontargeted protein, the epidermal growth factor receptor. By day 2, the inhibitory effect was overcome by replenishment reactions.
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PMID:Temporary ex vivo inhibition of the expression of the human oncogene HER2 (NEU) by a triple helix-forming oligonucleotide. 856 64

During the shift from a proliferative to a secretory endometrium in the rhesus menstrual cycle, progesterone action causes massive metabolic and structural remodelling. In order to identify genes whose expression is potentially important for the change from estrogen (E) to progesterone (P) dominance we have initiated a study of specific gene regulation using semiquantitative, reverse transcription polymerase chain reaction (RT-PCR). PolyA+ RNA was isolated from both E-dominant (days 9-13 of artificial menstrual cycles [AMCs]) and P-dominant (days 21-23) rhesus monkey endometria. The two pools of mRNA were converted to cDNA, end-ligated to double-stranded oligonucleotide adaptors and amplified by PCR using an adaptor-complementary primer. This procedure resulted in the production of E- and PcDNA template populations for cDNA-specific screening and comparative quantitation by PCR. Initial analysis showed that placental protein 14 (PP14) was P-dependent and human complement 3 (HC3) was up-regulated in E-dominant tissue, whereas the housekeeping genes B-actin and glyceraldehyde-3-phosphate dehydrogenase (G-3-PDH) were expressed at equivalent levels under E and P dominance. Expression of the E receptor (ER), P receptor (PR), epidermal growth factor receptor (EGFR) and insulin-like growth factor (IGF-I) was equivalent under E or P dominance. Expression of epidermal growth factor (EGF) and retinoblastoma (RB) was down-regulated in P-dominant tissue. Conversely IGF-1 receptor (IGF-1-R), transforming growth factor-beta 2 (TGFB-2), TGFB-2 receptor (TGFB-2-R), 17 beta-hydroxysteroid dehydrogenase (17-B-HSD) and leukemia inhibitory factor (LIF) levels were up-regulated in PcDNA. Among these factors, PP14, LIF, IGF-1-R TGFB-2 and 17-B-HSD were also detectable in PCR in a P-dependent cDNA library isolated by subtractive hybridization. These data provide evidence for hormonal regulation of specific gene products that may play important roles in the normal maturation of the primate endometrium in preparation for implantation.
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PMID:Differential gene regulation by estrogen and progesterone in the primate endometrium. 867 69


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