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)

In this study, we describe the presence of Na(+)-dependent high-affinity L-glutamate transport activity in the human U373 astrocytoma cell line. U373 cells exhibited a robust accumulation of L-glutamate which was predominantly (85%) extracellular Na(+)-dependent. Kinetic analysis of this transport activity revealed that the uptake followed first-order Michaelis-Menten kinetics and was high-affinity in nature. The kinetic parameters estimated by Eadie-Hofstee transformation of the saturable uptake were 37.3 +/- 5.1 microM for K(m) and 0.13 +/- 0.02 nmol min-1 mg-1 protein for Vmax. A total of 14 known inhibitors of high-affinity L-glutamate transport were examined for their abilities to inhibit L-glutamate uptake by U373 cells. Three compounds, kainate (KA), dihydrokainate (DHK) and alpha-aminoadipic acid produced less than 30% inhibition at 1 mM. The lack of effect of both KA and DHK indicates that the predominant astroglial L-glutamate transporter EAAT2 (excitatory amino acid transporter 2) does not contribute to the uptake activity present in these cells. The rank order of inhibitory potency for the remaining 11 compounds tested was L-cysteine sulphinate = L-CCG-III = L-cysteate = L-aspartate = threo-beta-hydroxyaspartate > trans-PDC > D-aspartate = MPDC > beta-glutamate > L-CCG-IV = L-aspartate-beta-hydroxamate. Pre-treatment of U373 cells with phorbol ester for 30 min resulted in a 56% decrease in L-glutamate uptake and this effect was blocked in a concentration-dependent manner by the PKC inhibitor bisindolylmaleimide I. Expression of L-glutamate transporters by U373 cells was examined by reverse transcriptase polymerase chain reaction (RT-PCR) and Western analysis. Transcripts for both the EAAT1 and EAAT3 transporter subtypes were detected but not for EAATs 2, 4, and 5. Immunoblot analysis confirmed the presence of EAAT3 protein, however, we were unable to detect EAAT1 protein. In conclusion, the Na(+)-dependent high-affinity L-glutamate transport into human U373 astrocytoma cells appears to be mediated predominantly by the EAAT3 subtype.
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PMID:Properties of excitatory amino acid transport in the human U373 astrocytoma cell line. 1051 46

Type 1B astrocytes of the human optic nerve head (ONH) constitutively express neural cell adhesion molecule (NCAM) in vivo and in vitro. Increased synthesis of NCAM has been detected in reactive astrocytes in the glaucomatous ONH of human donor eyes. Several NCAM isoforms are generated through alternate RNA splicing in tissue- and disease-specific patterns. In this study, we analyzed expression of NCAM isoforms in ONH of normal donors at different ages and in glaucoma. Total RNA was extracted from ONH of fetal, normal adult and glaucomatous eyes, and cultured human ONH astrocytes, fetal brain astrocytes and an astrocytoma cell line, for reverse transcriptase-polymerase chain reaction (RT-PCR) analysis. To distinguish between NCAM 180 and 140 isoforms, exon-specific primer sets covering exons 13-19 were used. Isoform-specific riboprobes were used for in situ hybridization (ISH) in glaucomatous and in age-matched ONH. By RT-PCR, NCAM 140 was the predominant isoform in adult ONH as well as in all cultured cells. NCAM 180 mRNA was strongly expressed in glaucoma, whereas in normal adult tissues it was not detectable. ISH confirmed expression of NCAM in normal adult ONH and localized NCAM 140 mRNA to astrocytes. ISH demonstrated expression of NCAM 180 mRNA in reactive astrocytes in glaucomatous ONH. Our results demonstrate that the NCAM 180 isoform is induced in glaucoma. NCAM 180 may play a role in astrocyte interaction with extracellular matrix (ECM), vessels, axons and other astrocytes and, through its expanded cytoplasmic domain, serve as a signaling molecule for reactive astrocytes during remodeling of the ONH in glaucoma.
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PMID:Differential expression of neural cell adhesion molecule isoforms in normal and glaucomatous human optic nerve heads. 1064 Jun 77

We describe a reverse transcriptase-polymerase chain reaction method for the semiquantitative detection of mRNAs encoding the human heat shock proteins alphaB-crystallin, Hsp27, and Hsp60. The method involves the coamplification of cellular mRNA-derived cDNA with a dilution series of a competitor fragment (internal standard), using 1 primer pair common to both templates. Internal standards were based on cellular-derived cDNA engineered to be slightly smaller to differentiate between the target and the standard on electrophoretic separation. Initial cDNA quantitations can be corrected for possible variations during cDNA synthesis by standardizing to the levels of beta-actin-encoding cDNA. We show that the coamplified templates accumulate in a parallel manner with the cellular-derived cDNA throughout both the exponential and the nonexponential phase of amplification. Furthermore, we illustrate the utility of this technique by quantifying increased expression of alphaB-crystallin, Hsp27, and Hsp60 mRNA in astroglioma cells on heat shock.
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PMID:The stress kit: a new method based on competitive reverse transcriptase-polymerase chain reaction to quantify the expression of human alphaB-crystallin, Hsp27, and Hsp60. 1070 37

Estramustine is a chemotherapeutic drug, used in the treatment of prostatic carcinoma. In the prostate, it binds specifically to a 46 kDa glycoprotein called estramustine-binding protein (EMBP), which consists of three polypeptide components; C1, C2, and C3, each coded for by a specific gene. Expression of EMBP and binding of estramustine has also been detected in malignant glioma in both rats and humans. Elevated levels of this protein in astrocytoma have proved to correlate with poor prognosis. In the present work, expression of all three polypeptide components of EMBP was confirmed in an orthotopic rat glioma model with nested reverse transcriptase PCR and Western blot (molecular weights of 8, 10, and 12 kDa). Specific binding of estramustine with a Kd of 40 for male and 50 for female rats, and a total number of binding sites of 0.7 and 0.4 pmol/mg proteins for male and female rats respectively, was demonstrated with Scatchard plot analysis. These binding characteristics are similar to those of prostatic EMBP. Further studies to elucidate how EMBP expression affects the effect of estramustine treatment, and its putative prognostic value is of special clinical interest. The confirmation of BMBP expression in BT4C rat glioma demonstrates its suitability as a model system for such studies.
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PMID:Estramustine-binding protein in malignant glioma in rat. 1113 83

Tumor necrosis factor receptor type 1 (TNFR1) and c-Myc are important in signal transduction in tumor necrosis factor-alpha (TNF-alpha)-induced cytotoxicity, whereas activation of nuclear factor-kappa B (NF-kappa B) protects against TNF-alpha-induced apoptosis. This study investigated the expression of NF-kappa B, TNFR1, and c-Myc in human astrocytoma tissues by reverse transcriptase-polymerase chain reaction (PCR) and immunohistochemical analysis. TNFR1 messenger ribonucleic acid (mRNA) and c-Myc mRNA were frequently expressed in malignant astrocytomas, especially in glioblastomas, compared with low-grade astrocytomas by PCR analysis. TNFR1 and c-Myc mRNAs were barely detectable in normal brain tissues. NF-kappa B p50 and p65 subunit mRNAs were detected in various grades of astrocytomas, with frequent expression in malignant astrocytomas. The presence of activated NF-kappa B was confirmed by nuclear localization in neoplastic astrocytes as determined by immunohistochemistry. Both p50 and p65 subunits were inhomogeneously expressed in neoplastic astrocytes of glioblastoma, but only in a few scattered tumor cells in low-grade astrocytoma, and almost undetectable in normal brain tissues. These results indicate that TNFR1 and c-Myc are overexpressed in malignant astrocytomas, and this may increase the cellular sensitivity to the cytotoxic action of TNF-alpha. NF-kappa B p50 and p65 were simultaneously induced and activated in malignant astrocytomas. Our results suggest that the constitutive activation of NF-kappa B subunits in malignant astrocytoma, especially in glioblastoma, could be associated with the resistance to TNF-alpha immunotherapy, and indicates new therapeutic strategies for malignant astrocytomas.
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PMID:Expression of nuclear factor-kappa B, tumor necrosis factor receptor type 1, and c-Myc in human astrocytomas. 1138 77

Recent molecular studies indicate two different genetic pathways leading to the development of glioblastoma; final progression of astrocytoma and de novo formation. To define the mutual relationships of cytogenetic changes in the pathogenesis of glioblastoma, molecular histopathologic alterations of p53 and epidermal growth factor receptor (EGFR) were evaluated by single stranded conformational polymorphion, reverse transcriptase-polymerase chain reaction and immunohistochemical stains in 15 primary and 21 secondary glioblastomas. Mutations in p53 gene and positive immunoreactivity to p53 protein (DO1) were more prevalent in secondary glioblastomas than in primary glioblastomas. A correlation between p53 mutations and p53 immunopositivities in glioblastomas was observed in 83.3% of the cases. All cases with positive p53 immunoreactivities showed p53 mutations; however, 13.9% of glioblastomas with p53 immuno-positivities lacked the relevant mutations. EGFR amplifications were detected in 73.3% of primary glioblastomas and 9.5% of secondary glioblastomas (p<0.001). The concurrence of p53 mutation and EGFR amplification was revealed in only 2 out of 15 primary glioblastomas and none among the secondary glioblastomas. Immunoreactivities for EGFR were noted in 66.7% of primary glioblastomas and in 9.5% of secondary glioblastomas (p<0.001). A correlation between EGFR amplification and EGFR immunopositivity in glioblastomas was observed in 91.7% of the cases. These data indicate that EGFR amplification and p53 mutations are two independent genetic events in the development of glioblastomas.
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PMID:p53 mutation and epidermal growth factor receptor overexpression in glioblastoma. 1151 95

Wnt regulates developmental and oncogenic processes through its downstream effector, beta-catenin, and a set of other intracellular regulators that are largely conserved among species. E-cadherin was discovered as a protein associated with beta-catenin which plays a crucial role in cell-cell adhesion. To further understand the molecular basis of Wnt signaling pathway and E-cadherin in brain tumorigenesis, the expression of four Wnt genes (Wnt1, Wnt5a, Wnt10b and Wnt13) and E-cadherin were analyzed by reverse transcriptase-polymerase chain reaction. In addition, their downstream effector, beta-catenin, was also investigated. The results showed that the expression of Wnt5a (41/45), Wnt10b (37/45), and Wnt13 (35/45) were found in brain tumors, whereas Wnt1 (6/45) was shown to be less related. Interestingly, E-cadherin was only expressed in a few cases of astrocytoma (2/16), whereas it was expressed in most meningioma (14/15) and pituitary adenoma tumors (12/14). There was no apparent difference of beta-catenin expression profile in brain tumors; however, the sequencing data of beta-catenin showed two mutations on speculative phosphorylation sites, S73F and S23G in astrocytoma. Furthermore, an in vitro functional assay showed that S73F and S23G mutants of beta-catenin did not affect transcriptional activity in TCF-4-leuciferase reporter construct, suggesting that they may need more complex factors to participate in astrocytoma. Taken together, our data suggest that the mutations of beta-catenin together with E-cadherin and Wnt signaling might be involved in brain tumorigenesis.
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PMID:Differential expression of Wnt genes, beta-catenin and E-cadherin in human brain tumors. 1204 19

The properties of a transport system specific for gamma-aminobutyric acid (GABA) expressed in human U373 MG astrocytoma cells were examined. The uptake of [(3)H]GABA was dependent on both extracellular Na(+) and Cl(-) ions and was inhibited by (+/-)-nipecotic acid, guvacine, and beta-alanine, with a pharmacological profile corresponding to that reported for the human homologue of the GABA/betaine transporter (BGT-1). Accordingly, [(3)H]GABA uptake was also inhibited by betaine, and reverse transcriptase-polymerase chain reaction (RT-PCR) analysis of total RNA from U373 MG cells with specific BGT-1 primers resulted in the amplification of a 440 bp fragment that was further characterized by restriction analysis and sequencing. In addition, Western blot analysis with anti-BGT-1 antiserum revealed the presence of a characteristic 60 kDa band. The primary structure of the human BGT-1 protein predicts two putative phosphorylation sites for the Ca(2+)/diacylglicerol-dependent protein kinase (PKC), and treatment of U373 MG cells with the PKC activator phorbol 12-myristate-13-acetate (TPA) led to a concentration- and time-dependent decrease in [(3)H]GABA uptake. The maximal effect was detected at 2 hr of incubation, to disappear after 4 hr. TPA-induced reduction in [(3)H]GABA uptake was reversed by preincubation with staurosporine. Taken together, these results indicate that U373 MG cells express a GABA transporter of the BGT-1 subtype whose function is regulated by phosphorylation events through PKC.
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PMID:Gamma-aminobutyric acid transporter (BGT-1) expressed in human astrocytoma U373 MG cells: pharmacological and molecular characterization and phorbol ester-induced inhibition. 1211 24

A role for neuropeptide receptors in glial tumorigenesis has recently been proposed. Although angiotensin receptors are known to mediate proliferative effects in many cell types, including brain astrocytes, the possible participation of these receptors in glial tumorigenesis remains unknown. In the present study, we have examined the expression of the molecularly defined angiotensin receptor subtypes AT(1a), AT(1b), and AT(2) in normal perinatal rat astrocytes and in a panel of tumor adult astrocytoma cells, using the reverse transcriptase-polymerase chain reaction (RT-PCR). Subsequently, we compared the mitogenic effect of the angiotensins A(1-8), A(2-8), A(3-8) and the heptapeptide "metabolite" A(1-7), on both normal and tumor astrocytes, measured in terms of the incorporation of tritiated thymidine. Our results indicate that AT(1a), AT(1b), and AT(2) angiotensin receptor mRNA is commonly expressed by many of these cells. Of notable exception is the astrocytoma U373 which was not found to express AT(1) or AT(2) mRNA. Chronic (24-h) incubation of cells with A(1-8) and A(1-7) lead to the induction of mitogenesis, even in the AT(1) and AT(2) mRNA negative astrocytoma cell line U373. Moreover, pharmacological analysis indicated that the observed mitogenic effects are not mediated by the AT(1) or AT(2) type receptors, but rather by a novel, specific A((1-7)) angiotensin receptor, since mitogenesis was shown to be partially blocked by the A(1-7) analogue D-Ala(7)A(1-7) and by the protease inhibitor orthophenanthroline (100 microM). Using Fura-2 spectrophotometry, we found that activation of this receptor does not alter intracellular calcium levels; however, preincubation with the protein kinase kinase inhibitor U0126 (10 microM) was found to inhibit these mitogenic effects partially. Overall, these results which demonstrate that normal and tumor astrocytes express a greater variety of angiotensin receptor subtypes than previously thought, support the idea that A(1-7) and its receptor signaling system may play an important role in shaping the astrocyte population during development. Moreover, the untimely expression of this A((1-7)) receptor may represent an important etiological component in the development of brain astrocytomas.
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PMID:Multiple angiotensin receptor subtypes in normal and tumor astrocytes in vitro. 1220 96

The present work aims to determine the relevance of an astrocytoma cell line U373 MG, for assessing the role of some astroglial cytochrome P450 in neurotoxicity and neuroprotection. CYP1B1, CYP2C8, CYP2C9, CYP2D6, CYP2J2, CYP2E1 and CYP4A11 mRNA were detected by reverse transcriptase-polymerase chain reaction in control U373 MG cell cultures. Among them we focused on CYP1B1 expression. After 48 h treatment with a range of concentrations of interleukin-1beta (1, 5, 10 ng/ml) used to simulate stress conditions, CYP1B1 mRNA expression was enhanced in a dose-dependent way. This increased expression was followed 24 h later by an increase in protein level, determined by Western-blot. N-acetylcysteine (NAC) partially inhibited this effect both on the mRNA and protein levels. As CYP1B1 activates procarcinogenic compounds to reactive metabolites, an increase in this P450 isoform will participate to toxic consequences of an inflammatory/oxidative stress. NAC will prevent this deleterious effect.
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PMID:Astroglial CYP1B1 up-regulation in inflammatory/oxidative toxic conditions: IL-1beta effect and protection by N-acetylcysteine. 1256 1


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