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:2.7.7.49 (reverse transcriptase)
31,746 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We evaluated the usefulness of a recently developed real-time reverse transcriptase polymerase chain reaction (RT-PCR) system to detect minimal residual diseases (MRD) in patients with acute myelogenous leukaemia (AML) with chromosomal translocation t(8:21). The method was simple, rapid and reproducible for the quantity of chimeric AML1-ETO (MTG8) transcripts. The ratio of the absolute copy number of a target gene (AML1-ETO) to a control gene (glyceraldehyde-3-phosphate dehydrogenase, GAPDH) was calculated by using a fluorescence curve prepared from amplicons of serially diluted standard RNA. The relative points of MRD in bone marrow (BM) of 8 patients in the acute phase of the disease was from 0.85 to 3.0, whereas those of MRD in complete remission (CR) decreased to below 6.4 x 10(-3). This method was also applied to evaluate chimeric transcripts in peripheral blood (PB) samples. The values in patients with t(8;21) AML were from 0.97 to 2.0 in the acute phase, whereas those in CR showed less than 2.2 x 10(-4). There was 10(-5)-fold difference in AML1-ETO mRNA expression between PB samples in the acute phase and those in CR. The results suggest that we may easily monitor MRD in patients with t(8;21) AML through quantitative analysis of AML1-ETO transcripts in blood samples.
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PMID:A quantitative reverse transcriptase polymerase chain reaction method for the detection of leukaemic cells with t(8;21) in peripheral blood. 1077 97

The influence of the gene expression of critical components of the cytoplasmic and lysosomal proteolytic pathways on the rate of protein degradation was evaluated in the leg skeletal muscle of 8 severely traumatized patients. Muscle proteolysis was determined as the intramuscular phenylalanine rate of appearance by L-[ring-2H5]phenylalanine infusion and the leg arteriovenous catheterization technique combined with muscle biopsy. Muscle mRNA levels of UbB polyubiquitin and cathepsin B were determined by reverse transcriptase-competitive polymerase chain reaction and expressed as a percent of the mRNA level of the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH). In the patients, individual values for UbB polyubiquitin mRNA levels directly correlated with the rate of muscle proteolysis (r = .76, P < .05), whereas no correlation (r = .10) was found between cathepsin B mRNA levels and proteolysis. Thus, after trauma, the rate of muscle proteolysis appears to be largely regulated by the ubiquitin-proteasome system at the level of gene transcription.
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PMID:Contribution of the ubiquitin-proteasome pathway to overall muscle proteolysis in hypercatabolic patients. 1087 90

Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer among men in the developed world affecting the oral cavity, salivary glands, larynx and pharynx. Utilizing tissue from patients with HNSCC, we sought to systematically identify and catalog genes expressed in HNSCC progression. Here, we demonstrate the successful use of laser capture microdissection for procuring pure populations of cells from patient tissue sets comprised of oral squamous cell carcinomas (OSCCs) and matching normal tissue. From the estimated 5000 cells procured for each sample, we were able to extract total RNA (14.7-18.6 ng) of sufficient quality to transcribe GAPDH by reverse transcriptase-polymerase chain reaction (RT-PCR). The RNA was used for the synthesis of blunt-ended, double-strand complementary DNAs (cDNAs) by oligo (dT)-mediated reverse transcription, followed by addition of linkers. Primers specific for these linkers with uracil deglycosylase-compatible ends were used to amplify these cDNAs by PCR and the product was subcloned into the pAMP10 cloning vector. Ninety-six clones from each of six libraries were randomly sequenced and results indicated that 76-96% of the inserts represent either anonymous expressed sequence tags (ESTs) (25-48%), known genes (9-29%) or novel sequences (27-51%), respectively, with very little redundancy. These results demonstrate that high quality, representative cDNA libraries can be generated from microdissected OSCC tissue. Furthermore, these finding suggest the existence of at least 132 novel genes expressed in our cDNA libraries, which may have a role in the pathogenesis of HNSCC, and may represent novel markers for early detection as well as targets for pharmacological intervention in this disease.
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PMID:Gene expression profiles in squamous cell carcinomas of the oral cavity: use of laser capture microdissection for the construction and analysis of stage-specific cDNA libraries. 1096 57

We developed a real-time one-step reverse transcriptase-polymerase chain reaction (RT-PCR) method for the routine quantification of c-erbB-2 oncogene expression in breast cancer, using a 7700 ABI PRISM Sequence Detector System (Perkin Elmer-Applied Biosystems, Courtaboeuf, France). The real-time quantification of the polymerase chain reaction products is based on the TaqMan 5' nuclease assay. The optimal experimental conditions we determined were as follows: 6 mM MgCl2, 200 nM of fluorogenic probe, 200 nM of each primer, and 12.5 units MuLV reverse transcriptase. The GAPDH housekeeping gene was used for normalization of c-erbB-2 expression. In human breast cancer cell lines, the normalized expression of c-erbB-2 ranged from 8 x 10(-6) to 2,600 x 10(-6), the two highest values corresponding to the c-erbB-2 overexpressing cells MDA-MB-453 and SK-BR-3. In a series of 100 breast cancer samples, c-erbB-2 normalized expression was found to range from 0.4 x 10(-6) to 350 x 10(-6). A close correlation was observed between this real-time one-step quantitative RT-PCR method and both semiquantitative conventional RT-PCR (N = 22; r = 0.8543; P < .0001) and c-erbB-2 protein expression (p185) quantified by an enzyme immunoassay (EIA) (N = 27; r = 0.71; P < .0001). The current realtime RT-PCR assay is rapid, sensitive, and reproducible and appears particularly suitable to quantify gene expression in large series of samples.
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PMID:A real-time one-step reverse transcriptase-polymerase chain reaction method to quantify c-erbB-2 expression in human breast cancer. 1097 82

Amplification of cytokeratin 19 (CK19) transcripts by reverse transcriptase-polymerase chain reaction (RT-PCR) has been shown to be a highly sensitive assay for the detection of bone marrow micrometastases (BMM) of breast cancer, but recent studies have demonstrated the occurrence of false-positive results due to low-level, illegitimately transcribed CK19 in normal bone marrow tissue. One approach to solve this problem is to develop a quantitative CK19 RT-PCR assay and to introduce a cut-off value which can distinguish between illegitimate expression and cancer-specific expression levels. In the present paper, we describe a quantitative CK19 RT-PCR assay using a real-time automated PCR system. The number of CK19 transcripts was normalized to that of GAPDH transcripts as an internal control for quality and quantity of cDNA. The cut-off value for the ratio of CK19 to GAPDH transcripts was set at 10(-4) since the ratio never exceeded this value in the control bone marrow samples (n = 12). In total, 117 bone marrow aspirates from stage I - III patients with invasive breast cancers were subjected to CK19 RT-PCR assay and immunocytological examination. Forty (34.2%) were found to be BMM-positive by CK19 RT-PCR assay whereas only three (2.6%) were found to be BMM-positive by immunocytology. Multivariate analysis has shown that occult BMM detected by CK19 RT-PCR is a significant risk factor for relapse, being independent of axillary lymph node metastases.
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PMID:Prognostic significance of occult bone marrow micrometastases of breast cancer detected by quantitative polymerase chain reaction for cytokeratin 19 mRNA. 1101 Nov 20

The study of drug metabolism in cultured rat hepatocytes is hampered by the rapid loss of the expression of cytochrome P450 enzymes. Nevertheless, the activity of cytochrome P450 3A (CYP3A), one of the most important isoenzymes for drug metabolism, can be elevated by chemical inducers. In the present study, we investigated in cultured rat hepatocytes the induction of all four currently identified CYP3A isoforms by dexamethasone, and compared the results obtained in vitro with the induction profile of dexamethasone in vivo. To this end, CYP3A mRNA levels were quantified with a novel, radioactive reverse transcriptase-polymerase chain reaction (RT-PCR) assay, and CYP3A enzymatic activity was measured by a testosterone hydroxylation assay. In the RT-PCR assay, CYP3A isoforms were co-amplified with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in the presence of radioactively labeled nucleotides. This resulted in an extremely sensitive and accurate determination of CYP3A expression levels, relative to those of GAPDH. Using this RT-PCR assay, it was found that the expression of all CYP3A isoforms in rat hepatocytes, cultured on a collagen matrix, was decreased by 80-90% within one day of cultivation. After addition of dexamethasone, at one day after isolation, CYP3A1 mRNA levels were elevated to levels comparable to those in freshly isolated hepatocytes within two days. In contrast, CYP3A2, CYP3A9, and CYP3A18 mRNA levels were not affected by dexamethasone treatment, and were hardly detectable after three days of cultivation. CYP3A enzymatic activity was also induced in cultured hepatocytes (approximately 6-fold) after addition of dexamethasone. In vivo, CYP3A1 mRNA levels increased 45-fold after dexamethasone administration. However, in contrast to the situation in cultured hepatocytes, CYP3A2 and CYP3A18 were also induced, albeit to a lesser extent (4- and 7-fold elevated mRNA levels, respectively). We conclude that the selective induction of CYP3A1 in dexamethasone-treated rat hepatocytes allows the study of biotransformation reactions by CYP3A1, without interference by any of the other CYP3A isoenzymes.
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PMID:Selective induction of cytochrome P450 3A1 by dexamethasone in cultured rat hepatocytes: analysis with a novel reverse transcriptase-polymerase chain reaction assay section sign. 1102 Apr 54

Surgical and orthodontic treatment of retrognathia aims to improve orofacial function by adaptation and training of muscle capacity, which is connected with a change in muscle fibre-type proportions. The aim here was to analyse the proportion of myosin-heavy chain (MyHC) gene expression in type I (slow twitch/ST) and type IIb (fast twitch/FT) fibres during sagittal advancement of the mandible by reverse transcriptase-polymerase chain reaction (RT-PCR). The experiments were carried out on 10-week-old pigs (six test animals, six controls) over a 28-day period. Six pigs were fitted with acrylic bite blocks for sagittal advancement of the mandible. Tissue was taken from seven different regions of the masseter, temporal, medial pterygoid, and geniohyoid muscles. The 84 samples were used for histological fibre differentiation with ATPase staining and for isolation of total RNA. To measure the two MyHC isoforms, RT-PCR (in a single tube reaction with MyHC I, MyHC IIb, and GAPDH primers) was used. A significant increase was registered in the percentage of ST fibres and in mRNA from MyHC I in the anterior region of the masseter and in the posterior region of the temporal muscle of the treated animals. The proportion of ST fibres to FT fibres was increased by up to 12% after functional advancement of the mandible. The histological findings corresponded with the data for fibre mRNA generated by RT-PCR.
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PMID:Differential expression of myosin heavy-chain mRNA in muscles of mastication during functional advancement of the mandible in pigs. 1116 67

The plasma membrane Ca(2+) pump is a key regulator of cytosolic free Ca(2+). Recent studies have demonstrated the dynamic expression of the plasma membrane Ca(2+) pump in a variety of cell types. Furthermore, alterations in plasma membrane calcium pump activity have now been implicated in human disease. In this study, the development of a technique to quantitatively assess mRNA expression of the human plasma membrane Ca(2+) ATPase (PMCA1) isoform of the plasma membrane Ca(2+) pump, using a real-time reverse transcriptase-polymerase chain reaction (real-time RT-PCR) assay in a human breast epithelial cell line (MCF-7) is described. The sequences of the PMCA1 primers and probe for real-time RT-PCR are presented. The results also indicate that PMCA1 mRNA can be normalized to both 18S ribosomal RNA (18S rRNA) and human glyceraldehyde-3-phosphate dehydrogenase (hGAPDH) in MCF-7 cells. Real-time RT-PCR will be most useful in assessing PMCA1 mRNA expression in cases where only low amounts of RNA are available and/or when numerous samples must be assessed simultaneously.
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PMID:Development of a real-time RT-PCR assay for plasma membrane calcium ATPase isoform 1 (PMCA1) mRNA levels in a human breast epithelial cell line. 1139 29

The aim of this study was to assess human intracranial tumours for their gene expression pattern of the vasoactive peptide adrenomedullin (AM), its receptor (AM-R) and leptin, which exerts multiple biological effects including proliferation and angiogenesis via the leptin receptor (OB-Rb). Gene activity of neuropeptide Y (NPY) was monitored additionally. We investigated whether there was a characteristic gene expression pattern of AM and leptin in different intracranial tumours, depending on their proliferation activity and biological behaviour. We investigated 35 non-functioning pituitary adenomas (including eight null cell, four silent plurihormonal, 23 silent gonadotroph adenomas), seven somatotropinomas, seven prolactinomas, eight meningiomas, five astrocytomas, two glioblastoma multiformes and unaffected temporal lobe (n = 8). Quantitative reverse transcriptase-polymerase chain reaction (TaqMan RT-PCR) was performed. AM mRNA was detectable in all tumour specimens. AM/GAPDH (glyceraldehyde-3-phosphate dehydrogenase) ratio was significantly higher in somatotropinomas, as was AM/CD31 ratio in prolactinomas, compared with inactive adenomas (P < 0.05). AM-R mRNA was found in all tumour subgroups in small quantities but, in general, higher in tumours than in temporal lobe tissue, respectively. AM-R/CD31 ratio was significantly higher in prolactinomas than in inactive adenomas (P < 0.05). Leptin was detectable in very low quantities in each subgroup. OB-Rb gene expression was found in all tumour subgroups, OB-Rb/GAPDH ratio was highest for meningiomas (P < 0.0001, compared with temporal lobe). NPY mRNA was detectable in temporal lobe in higher quantities than in tumours (P < 0.0001), and almost undetectable in prolactinomas and astrocytomas. Our data demonstrate that AM and AM-R, NPY, as well as leptin and OB-Rb, are expressed in various intracranial tumours in humans but their particular function has to be elucidated further. At present, there is no evidence for a cross-talk on transcriptional level between the peptidergic vasodilative system AM and the putative angiogenic and proliferation affecting factor leptin.
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PMID:Gene expression of adrenomedullin, leptin, their receptors and neuropeptide Y in hormone-secreting and non-functioning pituitary adenomas, meningiomas and malignant intracranial tumours in humans. 1148 41

Conventional hormone replacement therapy acts primarily by preserving bone, but cannot restore lost bone in women with established osteoporosis. Studies in rodents have shown that high doses of estrogens have anabolic skeletal effects, and recent observations in a group of women treated long term with high doses of estrogen indicated that similar effects occur in humans. This study examines the hypothesis that locally produced growth factors, including transforming growth factor-beta (TGF-beta) and platelet-derived growth factors (PDGFs), are involved in mediating the anabolic effects of high-dose estrogen. Transiliac-crest bone biopsies were taken from ten women, aged 52-67 years (mean 58 years), who had been treated with high-dose estrogen for 15 years. Control samples were obtained from four age-matched postmenopausal women not receiving estrogen therapy. TGF-betas and PDGFs were analyzed for mRNA and protein expression by reverse transcriptase-polymerase chain reaction (RT-PCR) and immunohistochemistry. Results showed both TGF-beta1 and -beta2 mRNA, expressed as a ratio to GAPDH, were increased in the estrogen-treated group with an eightfold increase for TGF-beta1 (0.258 +/- 0.246 [mean +/- SD] vs. 0.032 +/- 0.053 in the control group, p = 0.02) and a twofold increase for TGF-beta2 (p = n.s.). TGF-beta3 analysis showed only negligible amounts in both groups. Protein expression levels for TGF-beta1, -beta2, -betaRI and -RII were higher in the estrogen-treated group than in controls, the most marked effects being seen for TGF-beta1. PDGF-A protein expression was also significantly higher in osteoblasts and osteocytes in women treated with estrogen, whereas PDGF-B showed only modest differences. The percentage of bone surface occupied by osteoclasts, as determined by tartrate-resistant acid phosphatase (TRAP) staining, was significantly reduced in the estrogen-treated group (p = 0.001). These results demonstrate that high-dose estrogen therapy is associated with increased TGF-beta, TGF-betaR, and PDGF synthesis and decreased osteoclast activity, consistent with the hypothesis that these growth factors may mediate the actions of estrogen in bone.
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PMID:Mechanisms by which high-dose estrogen therapy produces anabolic skeletal effects in postmenopausal women: role of locally produced growth factors. 1155 64


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