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)

Recent reports have demonstrated that prostaglandin F2alpha-induced generation of reactive oxygen species or their intermediates inhibits progesterone synthesis and may also serve as a trigger for apoptosis in the corpus luteum (CL). BCL-2, an inhibitor of apoptosis in a wide variety of cell types, has been reported to prevent oxidative stress-induced cell death. Thus, the present studies were conducted to determine whether levels of mRNA encoding BCL-2 and related members of this gene family (BAX and BCL-Xshort, which induce apoptosis; BCL-Xlong, a BCL-2 homologue that prevents apoptosis) differed in functional (Day 21 of pregnancy) versus regressed (Day 21 of the estrous cycle) CL in the bovine ovary. Levels of mRNAs encoding p53, a transcriptional regulator of the bcl-2 and bax genes, and interleukin-1beta-converting enzyme (ICE), a protein recently implicated in the induction of apoptosis whose expression may be enhanced by oxidative stress, were also assessed. Partial cDNA clones encoding bovine bax, bcl-x, p53, and Ice were isolated using the reverse transcriptase-polymerase chain reaction (RT-PCR) technique with total RNA prepared from functional or regressed CL. A bovine bcl-2 cDNA could not be isolated from luteal tissue RNA despite the use of several primer pairs for amplification. Total RNA was then extracted from functional or regressed CL and analyzed by Northern blot analysis. The occurrence of apoptosis in regressed CL, as evidenced by the presence of internucleosomal DNA cleavage, was associated with a significant increase in both bax and Ice mRNA levels as compared with levels of bax and Ice expression in functional CL (p < 0.05, n = 3). There were no significant differences in bcl-x or p53 mRNA levels in functional versus regressed CL. Analysis of bcl-x mRNA by RT-PCR revealed that the long form was the primary, if not only, mRNA expressed in functional and regressed bovine luteal tissue. On the basis of data that increased expression of bax is associated with, and may be required for, apoptosis in ovarian granulosa cells and germ cells, we propose that BAX may play a similar role in apoptosis induction during luteal regression. Moreover, the increased Ice mRNA levels in regressed CL provides the first evidence that the ICE family of death proteases may be involved in luteolysis.
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PMID:Increased bax and interleukin-1beta-converting enzyme messenger ribonucleic acid levels coincide with apoptosis in the bovine corpus luteum during structural regression. 900 48

The effect of the transmethylation inhibitor 3-deazaadenosine on transcription levels of genes associated with apoptosis was investigated in HL-60 cells. After incubation of HL-60 cells with 100 microM 3-deazaadenosine for 45 min., a schedule known to perturb transmethylation metabolites and initiate apoptosis in these cells, a 50% decrease in c-myc and a 50% increase in bcl-2 RNA steady-state levels compared to control cells were observed. Transcription levels of c-myc continued to decrease after extended exposure to 3-deazaadenosine, while bcl-2 mRNA levels dropped to 25% and 30% below those in control cells after 1.5 hr and 3 hr, respectively. The expression levels of the bcl-2 related bax gene, showed a similar pattern as bcl-2; a 60% increase was initially measured, but after 1.5 and 3 hr, bax transcripts were 80% and 70% respectively, of those found in untreated cells. Another bcl-2 related gene, bcl-x, was previously reported to generate two transcripts in human cells. The long variant bcl-x1 acts as bcl-2, while the short form bcl-xs induces apoptosis. We were unable to detect bcl-xs transcripts in untreated and 3-deazaadenosine treated cells by the highly sensitive reverse transcriptase polymerase chain reaction method. This suggests that this gene product may not be involved in 3-deazaadenosine induced apoptosis in HL-60 cells. Bcl-x1 mRNA levels, however, slowly decreased with about 50% after 1.5 or 3 hr 3-deazaadenosine treatment. It is concluded that 3-deazaadenosine initiated apoptosis affects c-myc, bcl-2, bax and bcl-x1 mRNA levels.
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PMID:Effects of 3-deazaadenosine on apoptosis-related gene transcripts in HL-60 cells. 939 83

The bcl-2 family of proteins includes some important regulators of apoptosis. Among these, bcl-2 and bcl-xL prevent cells from entering apoptosis, whereas bax and bcl-xS can induce cell death. Alterations in the control of this process can lead to a decrease in cell death, thus contributing to neoplastic growth. Diminished susceptibility to chemotherapy has also been attributed, in in vitro systems, to alterations in the levels of bcl-2, bax, or bcl-x. We analyzed the expression of bcl-2, bax, bcl-xL, and bcl-xS in normal and neoplastic ovarian tissues by reverse transcriptase-PCR and Western blotting. The RNA and protein levels were significantly correlated for all genes. Interestingly, the levels of these genes in normal and neoplastic tissues were significantly different: bcl-2 was higher in normal tissue (P < 0.002), whereas bax and bcl-xL were higher in carcinoma (P < 0.018 and P < 0.030, respectively). bcl-xS was present at low levels in 83% of neoplastic samples and was undetectable in normal tissue. Reverse transcriptase-PCR analysis of 74 tumors showed no major correlation with clinicopathological parameters or with response to chemotherapy. Only bax and bcl-xL were correlated with progesterone receptor levels (n = 29, r = +0.44, P < 0.0189, and r = -0.40, P < 0.035, respectively). No correlation was found with estrogen receptor levels or with p53 immunostaining. Our data indicate that the regulation of the bcl-2 family of proteins differs between normal and neoplastic ovarian tissues. Moreover, the modulation of these genes in ovarian carcinoma is different compared to other tissues; therefore, tissue specificity is very important in regulation of the bcl-2 family of proteins.
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PMID:bcl-2, bax, bcl-XL, and bcl-XS expression in normal and neoplastic ovarian tissues. 951 44

We performed balloon injury in the rat carotid artery and identified intimal thickening after injury. Balloon-injured carotid arteries showed maximum thickness of the neointima on the 14th day before complete endothelial cell regeneration. In this lesion we identified apoptosis of vascular smooth muscle cells (VSMCs) by in situ DNA labelling and electron microscopy in the neointima on the 14th day after injury. mRNA expression levels of bcl-2, bax, bcl-x, p53 and caspase-1 were determined by the reverse transcriptase-polymerase chain reaction method both in injured and uninjured carotid arteries. Neither bcl-2 nor bcl-xl mRNA expression was detected in either injured or uninjured arteries, whereas bax and p53 mRNA expression was identified and their mRNA levels were not altered after balloon injury. In contrast, both bcl-xs and caspase-1 mRNA was detected and was markedly induced only in the injured carotid artery. Positive staining for immunoreactive Bcl-x was observed specifically in the injured arterial wall and co-localized with positive staining of nuclei identified by in situ DNA labelling. We conclude that two opposite cellular responses, VSMC proliferation and apoptosis, exist together in the neointima of the rat carotid artery after balloon injury, and selective induction of Bcl-xs expression is a key regulator of VSMC apoptosis in the process of vascular remodelling.
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PMID:Apoptosis and Bcl-xs in the intimal thickening of balloon-injured carotid arteries. 1033 66

Calpain, a Ca2+-activated cysteine protease, has been implicated in apoptosis of immune cells. Since central nervous system (CNS) is abundant in calpain, the possible involvement of calpain in apoptosis of CNS cells needs to be investigated. We studied calpain expression in rat C6 glioma cells exposed to reactive hydroxyl radical (.OH) [formed via the Fenton reaction (Fe2++H2O2+H+-->Fe3++H2O+.OH)], interferon-gamma (IFN-gamma), and calcium ionophore (A23187). Cell death, cell cycle, calpain expression, and calpain activity were examined. Diverse stimuli induced apoptosis in C6 cells morphologically (chromatin condensation as detected by light microscopy) and biochemically [DNA fragmentation as detected by TdT-mediated dUTP Nick-End Labeling (TUNEL) assay]. Oxidative stress arrested a population of C6 cells at the G2/M phase of cell cycle. The levels of mRNA expression of six genes were analyzed by the reverse transcriptase-polymerase chain reaction (RT-PCR). Diverse stimuli did not alter beta-actin (internal control) expression, but increased calpain expression, and the upregulated bax (pro-apoptotic)/bcl-2 (anti-apoptotic) ratio. There was no significant increase in expression of calpastatin (endogenous calpain inhibitor). Western blot analysis showed an increase in calpain content and degradation of myelin-associated glycoprotein (MAG), a calpain substrate. Pretreatment of C6 cells with calpeptin (a cell-permeable calpain inhibitor) blocked calpain overexpression, MAG degradation, and DNA fragmentation. We conclude that calpain overexpression due to.OH stress, IFN-gamma stimulation, or Ca2+ influx is involved in C6 cell death, which is attenuated by a calpain-specific inhibitor.
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PMID:Diverse stimuli induce calpain overexpression and apoptosis in C6 glioma cells. 1035 May 26

Neutrophils possess a very short lifespan, dying by apoptosis. HL-60 cells undergo apoptosis after neutrophil differentiation with dimethyl sulfoxide (DMSO). We have found that the onset of apoptosis in neutrophil-differentiating HL-60 cells correlates with the achievement of an apoptosis-related gene expression pattern similar to that of peripheral blood mature neutrophils. Using reverse transcriptase-polymerase chain reaction, cloning, and sequencing techniques, we have found that HL-60 cells express bak, bik, bax, bad, bcl-2, bcl-xL, bcl-w, bfl-1, fas, and caspases 1-4 and 7-10. After DMSO treatment, bak, bcl-w, bfl-1, fas, and caspases 1 and 9 were up-regulated, whereas bik, bcl-2, and caspases 2, 3, and 10 were down-regulated at different degrees, achieving mRNA expression levels that correlated with those detected in peripheral blood neutrophils. Caspase-2 mRNA and protein expression was drastically reduced after HL-60 cell differentiation, being absent in both HL-60-differentiated neutrophils and mature neutrophils, whereas caspase-3 and -10 mRNA and protein expression were diminished upon HL-60 cell differentiation until achieving the respective levels found in mature neutrophils. Bak and bfl-1 mRNA levels were largely increased during DMSO-induced differentiation of HL-60 cells, and these genes were the bcl-2 family members that were expressed most abundantly in mature neutrophils. Bcl-2 overexpression or caspase inhibition prevented differentiation-induced apoptosis in HL-60 cells, but not their differentiation capability. Neutrophil spontaneous apoptosis was also blocked by the caspase inhibitor z-Asp-2,6-dichlorobenzoyloxymethylketone. Peripheral blood neutrophils expressed bak, bad, bcl-w, bfl-1, fas, and caspases 1, 3, 4, and 7-10, but hardly expressed bcl-2, bcl-xL, bik, bax, and caspase-2. These results suggest that the above gene expression changes in neutrophil-differentiating HL-60 cells may play a role in the acquisition of the neutrophil apoptotic features.
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PMID:Expression of genes involved in initiation, regulation, and execution of apoptosis in human neutrophils and during neutrophil differentiation of HL-60 cells. 1081 Oct 13

Calpain, a Ca(2+)-dependent cysteine protease, has been implicated in cytoskeletal protein degradation and neurodegeneration in the lesion and adjacent areas following spinal cord injury (SCI). To attenuate apoptosis or programmed cell death (PCD) in SCI, we treated injured rats with E-64-d, a cell permeable and selective inhibitor of calpain. SCI was induced on T12 by the weight-drop (40 g-cm force) method. Within 15 min, E-64-d (1 mg/kg) in 1.5% DMSO was administered i.v. to the SCI rats. Following 24 h treatment, a 5-cm long spinal cord section with the lesion in the center was collected. The spinal cord section was divided equally into five 1-cm segments (S1: distant rostral, S2: near rostral, S3: lesion or injury, S4: near caudal and S5: distant caudal) for analysis. Determination of mRNA levels by reverse transcriptase-polymerase chain reaction (RT-PCR) indicated that ratios of bax/bcl-2 and calpain/calpastatin were increased in spinal cord segments from injured rats compared to controls. Degradation of the 68-kD neurofilament protein and internucleosomal DNA fragmentation were also increased. All of these changes were maximally increased in the lesion and gradually decreased in the adjacent areas of SCI rats, while largely undetectable in E-64-d treated rats and absent in sham controls. The results indicate that apoptosis in rat SCI appears to be associated with calpain activity which can be attenuated by the calpain inhibitor E-64-d.
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PMID:E-64-d prevents both calpain upregulation and apoptosis in the lesion and penumbra following spinal cord injury in rats. 1083

SCH58500 (ACN53) is a replication-deficient, type 5 adenovirus (Ad) expressing human wild-type p53 tumor suppressor. It is currently undergoing clinical trials as a cancer therapeutic. Many SCH58500 clinical trials incorporate an arm comparing traditional chemotherapy against chemotherapy combined with SCH58500. Paclitaxel was chosen for combination therapy in the preclinical study reported here due to its extensive use as a first-line therapy in ovarian cancer, its synergy with SCH58500 in preclinical cancer models, and its activation of p53-independent apoptosis, which might result in a "lowered threshold" for tumor cell death. SCID mice bearing human tumor xenografts were dosed with intratumoral vehicle, control Ad vector, or SCH58500, with or without paclitaxel. Real-time quantitative reverse transcriptase polymerase chain reaction assays were developed and validated to quantitate expression of p53, the p53 downstream effector gene p21, and the apoptosis-related genes, bax, bcl-2, and survivin. Protein expression was confirmed using immunohistochemical assays for p53 and p21. Only tumors injected with SCH58500 had detectable levels of exogenous p53 DNA and mRNA. After SCH58500 treatment, 3-11-fold elevations of p21 expression were observed in tumor xenografts containing nonfunctional p53 (MDA-MB-468, MDA-MB-231, MIAPaCa2, DU-145, and SK-OV-3), but no change in p21 mRNA in wild-type p53 PA-1 tumors. Immunohistochemical assays confirmed induction of p21 protein in MDAMB-468 and SK-OV-3 cells, but not in PA-1 cells. Ad vector alone or paclitaxel alone had no effect on p21 mRNA levels in most tumors. However, paclitaxel suppressed p21 expression induced by SCH58500 4-fold in DU-145 and SK-OV-3 tumors. Paclitaxel also affected expression of the housekeeping gene gapdh. There was no consistent pattern to the changes in bax, bcl-2, or survivin after SCH58500 treatment with or without paclitaxel between tumor types, although there were consistent responses within individual tumor lines. The mRNA ratios for bax/bcl-2 and bax/survivin were also not informative across tumor types. Of the genes examined, only p21 gave a predictable response 24 hours after p53 gene therapy and therefore, p21 expression may be useful for confirming SCH58500 activity in human tumor biopsies.
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PMID:Development and validation of sensitive assays to quantitate gene expression after p53 gene therapy and paclitaxel chemotherapy using in vivo dosing in tumor xenograft models. 1112 89

Fumonisins are a family of mycotoxins produced by Fusarium moniliforme, which is the most common mold found on corn throughout the world. These compounds are both toxic and carcinogenic for animals, and perhaps humans, with the kidney being the most sensitive organ to fumonisin toxicity. The molecular mechanism of fumonisin toxicity appears to involve disruption of de novo biosynthesis of sphingolipids and accumulation of sphinganine. The goals of this study were to determine whether fumonisin B(1) kills LLC-PK(1) renal kidney epithelial cells by inducing apoptosis and to identify genes affected by sphinganine that mediate fumonisin B(1)-induced cell death. Fumonisin B(1) produced morphological changes (i.e., cell shrinkage, membrane blebbing) and time-dependent increases in DNA fragmentation demonstrating that the toxin induces apoptosis. Simultaneously, fumonisin B(1) blocked sphingolipid biosynthesis and caused accumulation of sphinganine. To further investigate the role of sphinganine in fumonisin B(1)-induced apoptosis, beta-fluoroalanine (betaFA) was used to inhibit serine palmitoyltransferase, which catalyzes an earlier step in the sphingolipid biosynthetic pathway. betaFA blocked sphinganine accumulation and prevented fumonisin B(1)-induced DNA fragmentation, confirming that apoptosis induced by fumonisin B(1) is dependent upon accumulation of sphinganine. To examine gene expression, differential display reverse transcriptase polymerase chain reaction (DDRT-PCR) was applied to RNA isolated after 16 h of exposure to fumonisin B(1). Differential expression in response to fumonisin B(1) of a gene identified as calmodulin has been verified by Northern analysis. Sphinganine appears to mediate the effect because betaFA reduces induction of calmodulin mRNA by fumonisin B(1). Fumonisin B(1) also increases calmodulin protein in a concentration-dependent manner and the calmodulin antagonist W7 blocks fumonisin B(1)-induced DNA fragmentation, supporting a role for calmodulin in fumonisin B(1)-induced apoptosis. In contrast, fumonisin B(1) had no effect on expression of bcl-2 family genes (bax, bcl-2, and bcl-x). These findings demonstrate that fumonisin B(1) kills LLC-PK(1) kidney cells by inducing apoptosis. Further, the results establish a sequence of events for fumonisin B(1)-induced apoptosis involving initial disruption of sphingolipid metabolism and accumulation of sphinganine (or a metabolite), which, in turn, induces expression of calmodulin.
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PMID:Fumonisin B(1) induces apoptosis in LLC-PK(1) renal epithelial cells via a sphinganine- and calmodulin-dependent pathway. 1160 88

One day old rats received daily injections of dexamethasone and were sacrificed 24 h after the 1st and 7th injections. Neuronal death by apoptosis in the hippocampus was investigated by immunohistochemistry using bcl2, bax and caspase3 antibodies. The immunoreactivity expressed by the pyramidal neurons and the dentate granule cells with these antibodies was comparable in the dexamethasone treated and control rats injected with saline. At the ultrastructural level, the dendrites showed vacuolation indicative of degeneration in the dexamethasone administered rats. Results of reverse transcriptase-polymerase chain reaction analysis showed that bcl2 and bax mRNA was constitutively expressed in the hippocampus of control rats and showed no significant change in the dexamethasone treated rats. The results of this study indicate that dexamethasone induces degeneration of the dendrites but does not induce neuronal apoptosis in the hippocampus of postnatal rats.
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PMID:Dexamethasone induces dendritic alteration but not apoptosis in the neurons of the hippocampus in postnatal rats. 1209 57


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