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 a healing wound, inflammatory cells undergo apoptosis immediately beneath the leading edge of migrating epithelium. A potential mediator of this apoptosis pattern is p53, a protein with antiproliferative effects. Another protein, bcl-2, is antagonistic to p53 and prevents apoptosis. The purpose of this study was to determine the expression and location of p53 and bcl-2 mRNA and protein in healing wounds of normal and genetically diabetic mice. At various time points, full-thickness skin wounds from nondiabetic and diabetic mice were evaluated for p53 and bcl-2 by immunohistochemistry and in situ hybridization. Apoptosis patterns were also determined using the TUNEL method. Messenger RNA for p53 and bcl-2 were quantitated by competitive reverse transcriptase-polymerase chain reaction. Protein and mRNA for p53 were expressed in the leading edge of migrating epithelium, with apoptosis patterns closely following those of p53 production. p53 mRNA levels decreased soon after wounding, but after a few days, levels increased to greater than baseline. bcl-2 was localized to the wound epithelium, but relative amounts tended to oppose levels of p53, i.e, when p53 increased, bcl-2 decreased and vice versa. Wounds in diabetic animals showed a delayed onset of p53 mRNA expression but had persistently greater levels for longer periods of time. bcl-2 mRNA expression was further delayed in diabetic mice and did not develop to levels as high as p53. Production of both proteins was delayed, consistent with the mRNA expression. Our data show that immediately after wounding, bcl-2 increases and p53 decreases to allow for the cellular proliferation that is required for tissue repair. Over time, bcl-2 levels decrease while p53 levels increase to shut down the inflammatory process and down-regulate the proliferative response. Diabetic animals appear to lose the indirect relationship between p53 and bcl-2. This loss may contribute to the altered apoptosis patterns observed in diabetic healing.
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PMID:Expression and localization of p53 and bcl-2 in healing wounds in diabetic and nondiabetic mice. 1076 Feb 14

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

Apoptosis - programmed death of a cell - is a natural mechanism that controls the number of cells in an organism. Neoplastic cells as many types of normal cells, may be the subject of spontaneous apoptosis as well as they may be induced by anti-neoplastic factors. Neoplastic cells' resistance to drugs is often correlated with impossible induction of apoptosis in those cells. Though the process of apoptosis is not fully explained, a possible involvement of many genes in regulation of this process is indicated. One of them is bcl-2 gene and its product - bcl-2 protein, which has the property of apoptosis process inhibition and stimulation of a cell towards outliving (survival). Increased expression of bcl-2 gene is present in many neoplastic cells and it suggests a possible pathogenic role of bcl-2 gene in oncogenesis. In this paper the expression of bcl-2 gene in the cells of papilloma in larynx is defined in six children operated in the Department of Paediatric Otolaryngology of Medical School in Lublin. Papillomas of larynx are neoplasm's of particular resistance to treatment. Complete, cellular RNA was isolated with Chomczynski and Sacchi method using guanidine thiocyanate. Gene expression was defined with the method of reverse transcription by cDNA synthesis and amplification of bcl-2 gene fragment with specific oligonucleotides in reverse transcriptase polymerase chain reaction (RT-PCR). The products were identified on agarose gel. Expression of bcl-2 gene in the investigated cells of laryngeal papilloma was confirmed in all the children. The presence of bcl-2 gene product in these cells may be the cause of apoptosis inhibition and stimulation of cells proliferation of the neoplasm.
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PMID:Evaluation of bcl-2 gene expression in papilloma of larynx in children. 1086 21

Ginseng is a medicinal herb widely used in Asian countries, and many of its pharmacological actions are attributed to the ginsenosides. In a study of the anti-proliferative activity of ginsenosides using human prostate carcinoma LNCaP cell line, ginsenoside Rg3 displayed growth inhibitory activity. The cells lost its adherent property after incubation in the presence of 250 microM of ginsenoside for 48h. The expression of biomarker genes, including prostate specific antigen (PSA), androgen receptor (AR) and 5alpha-reductase (5alphaR), and that of the proliferating cell nuclear antigen (PCNA), were suppressed. Ginsenoside Rg3 induced classic apoptotic morphology and interfered with the expression of apoptosis-related genes, bcl-2 and caspase-3, in LNCaP cells, as demonstrated by fluorescence microscopy, flow cytometry and reverse transcriptase-polymerase chain reaction. Taken our results together, we suggested that ginsenoside Rg3 activated the expression of cyclin-kinase inhibitors, p21 and p27, arrested LNCaP cells at G1 phase, and subsequently inhibited cell growth through a caspase3-mediated apoptosis mechanism.
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PMID:Anti-proliferative effect of ginseng saponins on human prostate cancer cell line. 1097 98

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

It was shown using complement-dependent cytolysis and monoclonal antibodies against CD4, CD8, and NK1.1 antigens that the cortisone-resistant CD3+4-8-NK1.1(-)-thymocytes spontaneously secreted a chemotactic transmitter inducing the release and directed migration of bone marrow cells. When estimating the general profile of the cytokines of these thymocytes by PCR with revertase, it was demonstrated the cells in question did not express cytokines with colony stimulating activities (SCF, IL-3, or GM-CSF) or cytokines affecting the migration of bone marrow stem elements (IL-2, 4, or 7). In addition, an active expression of gene bcl-2 was detected. Thus, the chemotactic cytokine inducing the release of bone marrow stem elements is a product of the cortisone-resistant long-living CD3+4-8-NK1.1(-)-T-cells of the thymus.
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PMID:[Characteristics of the cells producing thymic chemotactic factor for bone marrow stem cells]. 1196 78

Cisplatin (CDDP) exerts significant activity against a wide variety of human malignancies. However, sensitivity to CDDP differs among cancer cells. CDDP induces apoptosis in cancer cells. In the present study, to evaluate good markers of chemo-sensitivity or chemo-resistance of cancer cells, the correlation between occurrence of apoptosis and the changes in expression levels of messenger RNA (mRNA) of three genes (bax, bcl-2, and survivin) in cancer cell lines during CDDP treatment were investigated. Cells (MKN-45, LoVo, and PANC-1) were incubated with CDDP (10 microg/ml). The percentage of cells in sub-G1 fraction was measured by flow cytometry. The changes in expression levels of three genes (bax, bcl-2, and survivin) during CDDP treatment were evaluated by real-time reverse transcriptase-polymerase chain reaction (RT-PCR). The percentage of cells in sub-G1 fraction increased after a shorter incubation with CDDP in LoVo cells and also increased between 12 and 24 h CDDP treatment in MKN-45 cells. On the other hand, even with a 24 h incubation with CDDP, the percentage of cells in sub-G1 fraction did not change in PANC-1 cells. The expression level of bax mRNA significantly increased after 24 h treatment with CDDP in MKN-45 cells and it significantly increased after 12 h treatment with CDDP in LoVo cells. Also, in LoVo cells, the expression level of bcl-2 mRNA decreased after 24 h treatment with CDDP. On the other hand, during CDDP treatment, the expression levels of bcl-2 and survivin mRNA significantly increased in PANC-1 cells. These findings indicate that during chemotherapy, changes in expression levels of bax, bcl-2, and survivin may provide information about chemo-sensitivity or the chemo-resistance of tumors.
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PMID:Quantitative analysis of expression levels of bax, bcl-2, and survivin in cancer cells during cisplatin treatment. 1216 83

In order to evaluate the appearance of brain bcl-2 during development of the hydrocephalus, we measured levels of bcl-2 mRNA in the cortex and cerebellum of congenital hydrocephalic rats (LEW-HYR) at 1 and 2 weeks after birth using the quantified reverse transcriptase-polymerase chain reaction (RT-PCR) with TaqMan fluorogenic detection system. Normal and hydrocephalic siblings were killed 7 and 14 days after birth, and their cortices and cerebella were homogenized with the Isogen-chloroform mixtured solution. By means of the RT-PCR with genetic analyzer, the sequence of bcl-2 mRNA detected in the LEW-HYR was identified to be the same as that of the registered rat brain (L14680). During the development of normal siblings of LEW-HYR, the levels of bcl-2 mRNA detected in the cortex and cerebellum 7 days after birth were significantly higher than those seen on day 14 after birth. In the hydrocephalic rats, however, these levels were not significantly different during development. On days 7 and 14 after birth, the cortical levels of bcl-2 mRNA detected in the hydrocephalic rats were significantly higher than those in normal rats. In the cerebellum, these levels in the hydrocephalic rats were higher, but not significantly, than those of normal rats. These results indicate that the significant appearance of bcl-2 mRNA in the developing normal rat brain is related to sprouting and to the diminished number of neurons, whereas the significant increase of bcl-2 levels seen in the developing hydrocephalic rats is indicative of an excess activity of glutamate neurons in cerebral cortex and the protection of neurons from cell death induced by cerebral ventricular dilatation in the cortex after bcl-2 levels.
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PMID:Changes in cortical and cerebellar bcl-2 mRNA levels in the developing hydrocephalic rat (LEW-HYR) as measured by a real time quantified RT-PCR. 1220 63


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