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)

CPT-11, a recently developed topoisomerase I (Topo I) inhibitor, attracts the attention not only of basic researchers but also of clinicians because of its high antitumor activity. The CPT-11 resistant human lung cancer cell line, PC-7/CPT, showed 10-fold resistance compared to parental cell line, PC-7. The total activity of Topo I in the resistant cell line was one fourth that of the parental sensitive cell line. The Topo I from the resistant cells was also 5-fold more resistant to the inhibitory effect of CPT-11 than that of the parental cells. We speculated that the alteration of the Topo I gene may be responsible for the change in topoisomerase activity of the CPT-11 resistant cell line. Therefore, we analyzed the mutation of Topo I gene using the method of single strand conformation polymorphism of polymerase chain reaction and the reverse transcriptase. We divided Topo I cDNA into ten fragments which overlapped each other and covered whole coding sequences of the Topo I cDNA. We observed mobility shift of two fragments in the PC-7/CPT, suggesting the presence of some mutations in these fragments. We performed the direct-sequencing of these portions by the dideoxy chain termination method and observed an altered sequence having a G to A base change in PC-7/CPT. This base substitution results in replacement of the conserved threonine at 729 position with alanine. These results suggest that the point mutation of Topo I gene is related to the decreases of Topo I activity and the sensitivity to Topo I inhibitor in PC-7/CPT cells.
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PMID:Detection of topoisomerase I gene point mutation in CPT-11 resistant lung cancer cell line. 133 3

Previously we demonstrated that the inner medullary collecting duct cell line mIMCD-K2 secretes Cl- by an electrogenic mechanism [N. L. Kizer, B. Lewis, and B. A. Stanton. Am. J. Physiol. 268 (Renal Fluid Electrolyte Physiol. 37): F347-F355, 1995; N. L. Kizer, D. Vandorpe, B. Lewis, B. Bunting, J. Russell, and B. A. Stanton. Am. J. Physiol. 268 (Renal Fluid Electrolyte Physiol. 37): F854-F861, 1995]. The goal of the present study was to characterize the Cl- channel responsible for adenosine 3',5'-cyclic monophosphate (cAMP)-stimulated Cl- secretion. To this end, using the patch-clamp technique, we measured Cl- currents. In whole cell patch-clamp experiments, 8-(4-chlorophenylthio)adenosine 3',5'-cyclic monophosphate (CPT-cAMP) activated Cl- currents that were time and voltage independent, inhibited by diphenylamine 2-carboxylate (DPC), and had a linear current-voltage (I-V) relation. In cell-attached patches of the apical membrane, we identified 7-pS Cl- channels that were stimulated by CPT-cAMP. In inside-out patches with Cl- in the pipette and bath solutions, Cl- currents had a linear I-V relation. The halide permeability sequence was PCl = PBr > PI. The Cl- channel inhibitors DPC, 5-nitro-2-(3-phenylpropylamino)-benzoic acid, and glibenclamide blocked the 7-pS Cl- channel, whereas 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid was ineffective. By reverse transcriptase polymerase chain reaction, we isolated a partial cDNA clone encoding the cystic fibrosis transmembrane conductance regulator in mIMCD-K2 cells. We conclude that cAMP stimulates electrogenic Cl- secretion in inner medullary collecting duct cells by activating cystic fibrosis transmembrane conductance regulator Cl- channels.
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PMID:CFTR mediates electrogenic chloride secretion in mouse inner medullary collecting duct (mIMCD-K2) cells. 757 98

Long-term, serum supplemented cultures of rat adult ventriculocytes were utilized to study the tropic effects of the alpha-agonist phenylephrine and of the carnitine palmitoyltransferase I inhibitor etomoxir. Cell protein and the rate of incorporation of phenylalanine were measured, corrected for cellular DNA content and utilized as an index for hypertrophy and of anabolic activity of the cells, respectively. The mRNA level of ANF was utilized as an index for the pathological phenotypic change (i.e., switch to fetal gene program), and that of the Na-channel--a constantly expressed gene in normal and hypertrophic cardiomyocytes--served as an internal control. Both mRNAs were quantified at various stages in culture by competitive reverse transcriptase PCR. The size of control myocytes steadily increased for over 3 weeks. The cells were completely redifferentiated and reached a maximum of anabolic activity 2 weeks after plating. Secretion and mRNA levels of ANF were increased severalfold after 7-8 days. Addition of 10 microM phenylephrine considerably speeded up cell growth. Maximum anabolic activity and complete redifferentiation were reached already after 1 week. Levels of mRNA and of ANF release increased 30-40 fold. Interestingly, induction of ANF gene transcription lagged behind the redifferentiation of the cells. Ten microM etomoxir inhibited the oxidation of palmitic acid and stimulated that of exogenous glucose by adult cardiomyocytes. In spite of its clear effect on fuel utilization, etomoxir had no direct hypertrophic effect on the myocytes in culture and did not inhibit the stimulatory action of alpha-agonists. Reactivation of the fetal gene program, as visualized by ANF production, was not reversed by etomoxir.
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PMID:Effect of alpha adrenergic stimulation and carnitine palmitoyl transferase I inhibition on hypertrophying adult rat cardiomyocytes in culture. 775 39

A camptothecin analog, ((4s)-4,11-diethyl-4-hydroxy-9-[(4- piperidinopiperidino)carbonyloxy]dione hydrochloride trihydrate), (CPT-11), is a recently developed topoisomerase I (Topo I) inhibitor which attracts the attention of clinicians because of its high antitumor activity. We established a CPT-11-resistant human ovarian cell line, HAC2/CPT, from the parental HAC2 cell line. An in vitro drug sensitivity assay revealed that HAC2/CPT was 9.7 and 4.7 times as resistant as HAC2 to CPT-11 and 7-ethyl-10-hydroxy-CPT-11 (SN-38), an active metabolite of CPT-11, respectively. HAC2/CPT showed no cross-resistance to other drugs tested (adriamycin, etoposide, cisplatin and Taxol), suggesting that HAC2/CPT acquired a phenotype specifically resistant to the Topo I inhibitor. In order to elucidate the mechanism of the resistance, we measured Topo I activity of HAC2 and HAC2/CPT. The activity of Topo I of HAC2/CPT was reduced to half of that of the parental HAC2 cells. To determine the cause of the decreased activity of Topo I, the mutation of the Topo I gene was searched for by the polymerase chain reaction and the reverse transcriptase analysis. Topo I gene mutation was not detected. The amount of Topo I protein was measured by immunoblotting and a marked decrease of Topo I protein was observed in HAC2/CPT. These results suggest that the decreased protein content of Topo I causes the decreased activity of Topo I and the decreased sensitivity of HAC2/CPT to Topo I inhibitors.
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PMID:Establishment of a CPT-11-resistant human ovarian cancer cell line. 807 81

Carnitine palmitoyltransferase I (CPT-I) catalyses the rate-determining step in mitochondrial fatty acid beta-oxidation. The enzyme has two cognate structural genes that are preferentially expressed in liver (alpha) or fat and muscle (beta). We hypothesized the existence of additional isoforms in heart to account for unique kinetic characteristics of enzyme activity in this tissue. Hybridization and PCR screening of a human cardiac cDNA library revealed the expression of two novel CPT-I isoforms generated by alternative splicing of the CPT-Ibeta transcript, in addition to the beta and alpha cDNA species previously described. Ribonuclease protection and reverse transcriptase-mediated PCR assays confirmed the presence of mRNA species of each splicing variant in heart, skeletal muscle and liver, with differing relative concentrations in the tissues. The novel splicing variants omit exons or utilize a cryptic splice donor site within an exon. Deduced polypeptide sequences of the novel enzymes include omissions in the region of putative membrane-spanning and malonyl-CoA regulatory domains compared with the previously described CPT-Is, implying that the encoded enzymes will exhibit unique features with respect to outer mitochondrial membrane topology and response to physiological and pharmacological inhibitors.
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PMID:Expression of novel isoforms of carnitine palmitoyltransferase I (CPT-1) generated by alternative splicing of the CPT-ibeta gene. 969 24

Gene expression, protein expression, and function of amiloride-sensitive sodium channels were examined in human lymphocytes from normal individuals and individuals with Liddle's disease. Using reverse transcriptase polymerase chain reactions, expression of all three cloned epithelial sodium channel (ENaC) subunits was detected in lymphocytes. Polyclonal antibodies to bovine alpha-ENaC bound to the plasma membrane of normal and Liddle's lymphocytes. A quantitative analysis of fluorescence-tagged ENaC antibodies indicated a 2.5-fold greater surface binding of the antibodies to Liddle's lymphocytes compared with normal lymphocytes. The relative binding intensity increased significantly (25%; p < 0.001) for both normal and Liddle's cells after treatment with 40 microM 8-CPT-cAMP. Amiloride-sensitive whole cell currents were recorded under basal and cAMP-treated conditions for both cell types. Liddle's cells had a 4.5-fold larger inward sodium conductance compared with normal cells. A specific 25% increase in the inward sodium current was observed in normal cells in response to cAMP treatment. Outside-out patches from both cell types under both treatment conditions revealed no obvious differences in the single channel conductance. The P(open) was 4.2 +/- 3.9% for patches from non-Liddle's cells, and 27.7 +/- 5.4% in patches from Liddle's lymphocytes. Biochemical purification of a protein complex, using the same antibodies used for the immunohistochemistry, yielded a functional sodium channel complex that was inhibited by amiloride when reconstituted into lipid vesicles and incorporated into planar lipid bilayers. These four independent methodologies yielded findings consistent with the hypotheses that human lymphocytes express functional, regulatable ENaC and that the mutation responsible for Liddle's disease induces excessive channel expression.
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PMID:Expression and regulation of normal and polymorphic epithelial sodium channel by human lymphocytes. 1111 30

The granulocyte-derived hemoregulatory peptide pyroGlu-Glu-Asp-Cys-Lys = pEEDCK is known to keep hematopoietic cells quiescent. When oxidized to its dimeric form (pEEDCK)2, it activates growth of hematopoietic progenitors in association with stroma-derived cytokines. (pEEDCK)2 has a Cys-Cys motif which is also a typical feature of the macrophage inflammatory protein (MIP-1alpha). The present study was designed to analyze differences between the response of normal and leukemic progenitor cells to (pEEDCK)2 or MIP-1alpha. When long-term bone marrow cultures (LTBMCs) were incubated with (pEEDCK)2 or MIP-1alpha and/or cytokines, the stimulatory effect on colony-forming units-granulocyte/erythroid/macrophage/megakaryocyte of LTBMC from chronic myeloid leukemia (CML) patients was less than 50% compared to LTBMC from healthy humans. No difference in oncogene expression could be observed in LTBMC from CML patients regarding reduction of Philadelphia chromosome-associated transcription of the BCR-ABL gene. With respect to the expression of growth and differentiation-associated genes (Galpha16, 5-lipoxygenase, phospholipaseA2, c-kit, and CD34), which were analyzed from LTBMC by semiquantitative reverse transcriptase-polymerase chain reaction, the same transcription rate was observed in CML patients and healthy donors. However, two isoforms of a key enzyme of oxidative metabolism, carnitine palmitoyltransferase (CPT1A and CPT1B), showed 50-fold higher expression rates in LTBMC cells of healthy donors compared to CML patients. It is known that a decrease in oxidative metabolism is associated with an increase in redox equivalents in malignancy. This might result in a reduction of disulphide bonds in (pEEDCK)2 or MIP-1alpha, thus inducing a downregulation of these factors in bone marrow from CML patients.
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PMID:Effect of the hemoregulatory peptide (pEEDCK)2 (pyroGlu-Glu-Asp-Cys-Lys)2 and MIP-1alpha is reduced in bone marrow cultures from patients with chronic myeloid leukemia (CML). 1146 52

Changes in key enzymes of oxidative metabolism at the mitochondrial level are known to be associated with the aging process, apoptosis, and many diseases. Considering the risk of acquiring a myelodysplastic syndrome (MDS) with age, the aim of this study was to quantify mRNA synthesis of the carnitine palmitoyltransferases (CPT1 and CPT2), carnitine acetyltransferase (CRAT), human specific microsomal CPT, and OCTN2 (organic cation transporter) in mononuclear cells of healthy humans of different age groups and MDS patients. Using quantitative reverse transcriptase real-time PCR we compared mRNA synthesis of the above mentioned enzymes in mononuclear cells from peripheral blood of 23 healthy persons (mean age 45 years), 9 blood and 22 bone marrow samples of 31 MDS patients with varying proportions of apoptotic cells (mean age 78 years), and blood samples of 30 age-matched controls. In addition, plasma carnitine levels were determined. Compared to younger adults, there was a 50% downregulation of CPT1 in elderly persons and in MDS patients. Reduction in CRAT, CPT 2, and OCTN2 was more than 85%. Reduction in microsomal CPT was more pronounced in MDS patients than in age-matched controls (96% vs. 43%). In MDS bone marrow cells there was a negative correlation of CPT1 and CRAT with the relative proportion of apoptotic cells. Plasma carnitine values were similar in all groups. The described reduction in transcription of different genes in blood cells which is well known in different tissues may reflect a systemic signaling process, associated with aging, apoptosis, and MDS.
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PMID:Downregulation of carnitine acyltransferases and organic cation transporter OCTN2 in mononuclear cells in healthy elderly and patients with myelodysplastic syndromes. 1280 1

Although treatments with nucleoside reverse transcriptase inhibitors (NRTIs) can modify fat metabolism and fat distribution in humans, the mechanisms of these modifications and the roles of diverse NRTIs are unknown. We studied the mitochondrial and metabolic effects of stavudine (d4T), zidovudine (AZT), didanosine (ddI), lamivudine (3TC), zalcitabine (ddC), and three combinations (AZT-3TC, d4T-3TC, and d4T-ddI) in mice treated for 2 weeks with daily doses equivalent to the human dose per body area. Concentrations of AZT and d4T in plasma were lower when these drugs were administered with 3TC or ddI. Whatever the treatment, mitochondrial DNA was not significantly decreased in muscle, heart, brain, or white adipose tissue but was moderately decreased in liver tissue after the administration of AZT, 3TC, or d4T alone. Blood lactate was unchanged, even when NRTIs were administered at supratherapeutic doses. In contrast, the level of plasma ketone bodies increased with the administration of AZT or high doses of d4T but not with ddC, 3TC, or ddI, suggesting that the thymine moiety could be involved. Indeed, the levels of plasma ketone bodies increased in mice treated with beta-aminoisobutyric acid, a thymine catabolite. Treatment with AZT, d4T, or beta-aminoisobutyric acid increased hepatic carnitine palmitoyltransferase I (CPT-I) mRNA expression and the mitochondrial generation of ketone bodies from palmitate. In conclusion, therapeutic doses of NRTIs have no or moderate effects on mitochondrial DNA and no effects on plasma lactate in mice. However, AZT and high doses of d4T increase the levels of hepatic CPT-I, mitochondrial fatty acid beta-oxidation, and ketone bodies, and these catabolic effects are reproduced by beta-aminoisobutyric acid, a thymine metabolite.
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PMID:Mitochondrial and metabolic effects of nucleoside reverse transcriptase inhibitors (NRTIs) in mice receiving one of five single- and three dual-NRTI treatments. 1457 92

A sensitive and accurate liquid chromatography-tandem mass spectrometric (LC-MS/MS) method for the intracellular determination of nine antiretroviral drugs in human peripheral blood mononuclear cells (PBMCs) is proposed. PBMCs are isolated by density gradient centrifugation using Vacutainer CPT tubes and cell count is performed with a Coulter instrument. Single-step extraction of drugs from PBMCs pellets is performed with MeOH 50% (with clozapine added as internal standard, I.S.) and the supernatant is injected onto a 2.1 mm x 30 mm SymmetryShield 3.5 microm-RP18 column equipped with a 2.1 x 10 mm guard column. Chromatographic separations are performed using a gradient program with a mixture of 2 mM ammonium acetate containing 0.1% formic acid and acetonitrile with 0.1% formic acid. Analytes quantification is performed by electro-spray ionisation-triple quadrupole mass spectrometry using the selected reaction monitoring (SRM) detection mode. The positive mode is used for the HIV protease inhibitors (PIs) indinavir, amprenavir, saquinavir, ritonavir, nelfinavir, lopinavir, atazanavir and the non-nucleoside reverse transcriptase inhibitors (NNRTIs) nevirapine, and the negative mode is applied for efavirenz. The calibration curves are prepared using blank PBMCs spiked with antiretroviral drugs at concentrations ranging from 0.5 to 100 ng/ml of cell extracts and fitted to a quadratic regression model weighted by 1/(concentration)(2). The lower limit of quantification is less than 0.5 ng/ml. The mean extraction recovery for all PIs/NNRTIs is always above 88%. The method is precise, with mean inter-day CV% within 0.6-10.2%, and accurate (range of inter-day deviation from nominal values -7.2 to +8.3%). This analytical method can be conveniently used in clinical research for the assessment of intracellular levels of all PIs/NNRTIs commercially available at present using a simple one-step cell extraction of PBMCs followed by liquid chromatography coupled with tandem triple quadripole mass detection.
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PMID:Intracellular measurements of anti-HIV drugs indinavir, amprenavir, saquinavir, ritonavir, nelfinavir, lopinavir, atazanavir, efavirenz and nevirapine in peripheral blood mononuclear cells by liquid chromatography coupled to tandem mass spectrometry. 1583 90


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