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 appropriate choice of an internal standard is critical for quantitative RNA analyses. As housekeeping genes, GAPDH, beta-actin, cyclophilin, and 28S rRNA are commonly employed as RNA internal standards with the assumption that their expression levels remain relatively constant in different experimental conditions. We tested this assumption under hypoxia (1% O2, 24 hours) compared to normoxia (20% O2, 24 hours) and compared RNA levels of these 4 housekeeping genes head to head using ribonuclease protection assays. Four biologically diverse cell lines with respect to clonal origin, neoplastic transformation, and growth rates were used in the comparison. Expression levels of 28S rRNA were constant, independent of O2 tension, but levels of GAPDH, beta-actin, and cyclophilin varied widely with hypoxia. In particular, GAPDH mRNA expression was increased by 21.2-75.1% under hypoxic conditions. Increased GAPDH transcription in hypoxia was correlated in the cancer cell lines with upregulation of the transcription factor Hypoxia Inducible Factor-1alpha protein levels in identical experimental conditions. These results suggest that 28S rRNA is a reliable internal control for comparative analyses of transcription under hypoxia; GAPDH appears particularly unfavorable for this purpose either in hypoxia or other experimental conditions that upregulate HIF-1alpha.
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PMID:Direct comparison of GAPDH, beta-actin, cyclophilin, and 28S rRNA as internal standards for quantifying RNA levels under hypoxia. 1036 51

Administration of supramaximal doses of cerulein results in acute interstitial pancreatitis. To understand the pathogenesis of this disease, it would be of great importance to elucidate the changes during the early phase of the process. We report changes of gene expression in the pancreas during the first 6 h of cerulein supramaximal stimulation. The expression of genes, including the secretory enzyme amylase, the lysosomal enzyme cathepsin B, as well as the housekeeping genes beta-actin and glyceraldehyde-3-phosphate dehydrogenase (GAPD), was investigated in this study. The most prominent alteration in gene expression is beta-actin messenger RNA (mRNA), which increased continuously after cerulein infusion. Immunostaining for beta-actin was observed along the membrane of large cytoplasmic vacuoles in pancreatic acinar cells. The level of amylase mRNA decreased during the first 30 min of cerulein infusion, recovered to the control level at 1 h and increased twofold at 2 h. An obvious increase in cathepsin B mRNA was observed after 3 h of cerulein infusion and reached sixfold of the control at 6 h. A significant increase of GAPD mRNA level was observed at 6 h of cerulein stimulation. In conclusion, this study provides direct evidence that the changes in gene expression, such as cathepsin B and amylase, after supramaximal cerulein stimulation, are regulated at the transcriptional level. It also suggests that beta-actin is involved in the formation of cytoplasmic vacuoles during supramaximal cerulein administration. Finally, this study indicates that beta-actin and GAPD may not be appropriate as RNA-loading controls for Northern blot analysis of pancreatic tissue.
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PMID:Early changes of gene expression during cerulein supramaximal stimulation. 1189 43

The objective of the present study was to examine the impact of preeclampsia on the relation of leptin and neuropeptide Y (NPY) gene expression in human placenta. A second goal was to monitor the change of leptin messenger RNA (mRNA) with increasing gestational age. Placental tissue was obtained from 17 premature deliveries, 18 term deliveries, and 10 mothers with preeclampsia. Gene expression of leptin, NPY, and two housekeeping genes (beta-actin and glyceraldehyde-3-phosphate dehydrogenase was quantified using real-time PCR. The leptin/beta-actin mRNA ratio was significantly higher in specimens of patients with preeclampsia than in those of gestational age-matched controls (0.63+/-0.23 vs. 0.09+/-0.04 relative U (RU); P = 0.03). NPY/beta-actin mRNA was significantly reduced in the preeclampsia group (0.003+/-0.001 vs. 0.026+/-0.008 RU in controls; P = 0.01). The NPY/leptin ratio was 0.11+/-0.09 for preeclamptic placenta samples and 1.7+/-0.6 RU for the controls (P = 0.02). The leptin/beta-actin ratio was significantly lower in placenta from premature deliveries than in term deliveries (0.02+/-0.004 vs. 0.12+/-0.05 RU; P = 0.01). Similar results were obtained for normalization to glyceraldehyde-3-phosphate dehydrogenase mRNA. Our data suggest an increase of placental leptin production with gestational age. In patients with preeclampsia, elevated leptin expression goes along with suppressed NPY expression. This resembles hypothalamic regulation.
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PMID:Leptin and neuropeptide Y gene expression in human placenta: ontogeny and evidence for similarities to hypothalamic regulation. 1044 74

We investigated the effect of the histone deacetylase inhibitors (HDIs), trichostatin A and trapoxin A on leukemia cells and cell lines from the viewpoint of differentiation induction. TSA induced differentiation in erythroid cell lines by itself, whereas it synergistically enhanced the differentiation that was directed by all-trans retinoic acid (ATRA) or vitamin D3 in U937, HL60 and NB4 cells. The combined treatment of HDI with ATRA induced differentiation in ATRA-resistant HL60 and NB4 cells. The transcriptional expression during the treatment with HDI was examined in HL60, U937 and MEG-O1. Cell cycle-regulator genes (p21waf1 and p16INK4A) were upregulated or constantly expressed, erythroid-specific genes (GATA-1, beta-globin) were silent or downregulated, and housekeeping genes (beta-actin and GAPDH) were constantly expressed. Twelve of 35 (34%) clinical samples from AML patients ranging from M0 to M7 also displayed both phenotypical and morphological changes by the treatment with TSA alone. HDIs are thus the potent inducer or enhancer of differentiation in acute myeloid leukemia and regulate transcription in an ordered manner.
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PMID:Histone deacetylase inhibitors are the potent inducer/enhancer of differentiation in acute myeloid leukemia: a new approach to anti-leukemia therapy. 1048 80

Further characterization of the canine immune system will greatly benefit from the availability of tools to detect canine cytokines. Our interest concerns the study on the role of cytokines in canine visceral leishmaniasis. For this purpose, we have designed specific primers using previously published sequences for the detection of canine IL-2, IFN-gamma and IL10 mRNA by reverse transcription-polymerase chain reaction (RT-PCR). For IL-4, we have cloned and sequenced this cytokine gene, and developed canine-specific primers. To control for sample-to-sample variation in the quantity of mRNA and variation in the RT and PCR reactions, the mRNA levels of glyceraldehyde-3-phosphate dehydrogenase (G3PDH), a housekeeping gene, were determined in parallel. Primers to amplify G3PDH were designed from consensus sequences obtained from the Genbank database. The mRNA levels of the cytokines mentioned here were detected from ConA-stimulated peripheral mononuclear cells derived from Leishmania-infected dogs. A different pattern of cytokine production among infected animals was found.
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PMID:Detection of canine cytokine gene expression by reverse transcription-polymerase chain reaction. 1050 99

AML1/MTG8 was quantified relative to the expression of the GAPDH housekeeping gene by real-time RT-PCR in 22 patients with t(8;21)-positive acute myeloblastic leukaemia (AML) at initial diagnosis and in seven of these patients also during/after chemotherapy and allogeneic bone marrow transplantation. Real-time PCR was able to specifically detect and quantify AML1/MTG8 over a 5 log range. The detection limit for t(8;21)-positive cells was a dilution of 1:105. The AML1/MTG8 expression varied considerably among the 22 AML patients at intial diagnosis with a ratio AML1/MTG8:GAPDH of 0.5135+/-0.536 (range 0.1-2.14, median 0.318). In six patients with t(8;21)-positive AML a marked decline of AML1/MTG8 could be induced by chemotherapy. These patients are in ongoing complete haematological remission (CR) with a constant low-level AML1/MTG8 expression. In another patient a rapid rise of AML1/MTG8 transcripts could be detected in CR after allogeneic bone marrow transplantation and the patient relapsed 10 weeks later. In conclusion, real-time RT-PCR is a suitable approach for the quantification of AML1/MTG8 transcripts in the monitoring of AML patients with t(8;21) during/after chemotherapy and can provide data of prognostic relevance.
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PMID:Real-time RT-PCR for the detection and quantification of AML1/MTG8 fusion transcripts in t(8;21)-positive AML patients. 1052 27

Megencephaly, enlarged brain, is a major sign in several human neurological diseases. The mouse model for megencephaly, mceph/mceph, has an enlarged brain and a lowered body weight. In addition, it displays several neurological and motoric disturbances. Previous studies suggest that the brain enlargement results from hypertrophy of the brain cells, rather than hyperplasia. No structural abnormalities, edema or increased myelination have been found. In this study, a major imbalance in the mRNA expression of molecules in the insulin-like growth factor (IGF) system was found in brains of 9-10 weeks old mceph/mceph mice compared to +/+ wild-type mice. In mceph/mceph brains, we found upregulation of IGF binding proteins (BP)-2, -4, -5, and -6 mRNA, the regulating hormone transforming growth factor (TGF)beta1 mRNA and also a local downregulation of IGFBP-5 mRNA compared to wild-type brains by in situ hybridization. The altered expression of these mRNA species is colocalized in cerebral cortex, hippocampus, amygdala and piriform/entorhinal cortex. The mceph/mceph mice express less of the myelin component proteolipid protein (PLP) mRNA in corpus callosum. No expression difference of the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in brain or IGF system components in liver was found between mceph/mceph and wild-type mice. These data suggest that the IGF system has an important role in the excessive growth of the mceph/mceph brains.
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PMID:The megencephaly mouse has disturbances in the insulin-like growth factor (IGF) system. 1052 1

We describe an improved highly sensitive method for generating cDNA libraries containing a high proportion of cDNAs enriched with 5'-coding sequences from single human preimplantation embryos and a 10 week old whole foetus. The embryonic mRNA was isolated using oligo-(dT) linked to magnetic beads. First-strand cDNA synthesis was carried out directly on the bound mRNA, followed by PCR designed to amplify the cDNA molecules synthesized in their entirety. The complexities of the libraries are between 10(5) and 10(6) independent clones. The average cDNA size is 1.0 kb, and the size range is 0.5-3.0 kb. PCR analysis of the embryonic libraries for specific genes has revealed transcripts for genes known to be transcribed in preimplantation stages, such as the imprinted gene SNRPN, developmental genes WNT11, HOX, OCT-1 and the embryonic OCT-4, cytoskeletal genes keratin-18 and beta-actin, the cell cycle gene C-MOS, and housekeeping genes GAPDH and HPRT. Sequencing of random clones showed the presence of a variety of sequences, such as human chorionic gonadotrophin, ubiquitin, TFIIA, guanine nucleotide-binding protein (beta-subunit), annexin I, a gene encoding a kinesin-like protein, and TWIST, which encodes a basic helix-loop-helix (bHLH) transcription factor implicated in Saethre-Chotzen syndrome (characterized by craniofacial and limb anomalies). Approximately 40% of these randomly analysed clones were full length. In addition to cDNAs matching known ESTs (Expressed Sequence Tags) in the GenBank and dbEST databases, novel sequences were detected at a frequency of 16% of randomly picked clones. The libraries are a valuable resource, providing longer cDNAs representing genes expressed during human preimplantation development.
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PMID:Developmental expression of specific genes detected in high-quality cDNA libraries from single human preimplantation embryos. 1052 61

Because previous PCR-based methodologies for detection of minimal residual disease (MRD) in leukemia patients have been too cumbersome to allow for widespread clinical usefulness, we have employed a real-time quantitative PCR (RQ-PCR) system to develop an MRD assay for t(12;21). We initially determined the expression of the different alternatively spliced TEL-AML1 mRNAs found in t(12;21) breakpoint variants I and II. We then optimized PCR primers for the RQ-PCR system and, using the t(12;21)+ REH cell line in spiking experiments, found a linear detection of TEL-AML1 over at least five logs. Moreover, 1 malignant cell in a background of 1,000,000 normal cells could be detected. The expression of the GAPDH, ABL, and beta(2)-microglobulin (beta2M) housekeeping genes were then compared in normal donors and in leukemic patients, and the very stably expressed beta2M was selected as an internal reference gene, allowing us to compensate for variation in RNA quality and day-to-day variation. In 12 samples from t(12;21)-positive patients at diagnosis, the levels of the TEL-AML1 fusion transcripts were found to vary up to 14-fold after normalization to beta2M. Interestingly, in samples obtained from seven patients at diagnosis, during induction chemotherapy, or relapse, the level of TEL-AML1 in peripheral blood (PB) and bone marrow (BM) was found to differ only by threefold, suggesting that MRD may be evaluated in PB samples in most patients. We conclude that this assay could set new standards for t(12;21) MRD detection with its accuracy, its high throughput, and its short turnover time for samples. Genes Chromosomes Cancer 26:355-365, 1999.
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PMID:Rapid and sensitive minimal residual disease detection in acute leukemia by quantitative real-time RT-PCR exemplified by t(12;21) TEL-AML1 fusion transcript. 1053 71

The polymerase chain reaction (PCR) has proved to be a sensitive and versatile method for the analysis of human and murine cytokine mRNA expression. This paper describes for the first time a reverse transcription-polymerase chain reaction (RT-PCR) at end-point for the quantification of five porcine cytokines: interferon (IFN)-gamma, interleukin (IL)-2, IL-4, IL-10 and IL-18. The main features of the methodology are: (1) a unique RT for all quantifications, (2) the addition of homologous DNA internal controls (IC) of equal length to the corresponding cytokine and consequently co-amplification of the target cytokine and the IC with equivalent efficacy, (3) PCR and detection of amplicons for all cytokines simultaneously, (4) cytokine quantification in relation to a housekeeping gene control (glyceraldehyde-3-phosphate dehydrogenase, GAPDH), (5) detection of the amplicons by enzyme linked immunosorbent assay (ELISA) using a chemiluminescent substrate with high sensitivity and wide dynamic range, (6) automation of the detection system for analysis of a large number of samples. This highly sensitive quantitative RT-PCR assay (able to detect 100-200 cytokines mRNA copies/75x10(3) cells) was validated on peripheral blood mononuclear cells (PBMC) from pigs infected or not with pseudorabies virus (PRV), re-stimulated in vitro by a mitogen or antigens.
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PMID:Quantification of porcine cytokine gene expression using RT-PCR, a homologous internal control and chemiluminescence for microplate detection. 1055 90


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