Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.7.7.48 (transcriptase)
 9,479 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Recent observations demonstrated that interleukin-1beta converting enzyme family proteases, now referred to as caspase family, play central roles in apoptosis, or programmed cell death. In this study, we tried to isolate and characterize epidermal caspases. By DEAE-Sephacel anion-exchange chromatography, human cornified cell extract showed two caspase-like fractions (F-I and F-II) with different substrate specificities. These were further purified by Sephacryl S-200, Mono Q ion exchange and Superose 6 gel chromatography. F-I showed a molecular weight of 30 kDa and specifically hydrolyzed acetyl-Asp-Glu-Val-Asp-methylcoumarinamide, a fluorogenic substrate for caspase-3 (CPP32) with a Km value of 13.8 microM. F-I generated a characteristic 85 kDa fragment from poly(ADP-ribose) polymerase. Inhibitor susceptibility of F-I was very similar to that of caspase-3, further confirming the caspase-3-like properties of F-I. In contrast, the molecular weight of F-II was estimated to be 110 kDa, which was much higher than the other caspases. F-II equally hydrolyzed acetyl-Asp-Glu-Val-Asp-methylcoumarinamide, and acetyl-Tyr-Val-Ala-Asp-methylcoumarinamide, caspase-1 (interleukin-1beta converting enzyme)-specific substrate, and was inhibited by acetyl-Tyr-Val-Ala-Asp-aldehyde and acetyl-Tyr-Val-Ala-Asp-aldehyde. Affinity labeling using biotinylated YVAD-cmk demonstrated several positive bands ranging from 25 to 35 kDa, supporting the hypothesis that F-II is a complex of multiple caspases. Reverse transcriptase-polymerase chain reaction analysis demonstrated that among known caspases tested, caspase-1, -2, -3, -4, and -7 were expressed in cultured human keratinocytes. These results suggest that multiple caspases are synthesized in human keratinocytes and are involved in terminal differentiation.
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PMID:Partial purification and characterization of two distinct types of caspases from human epidermis. 974 Feb 25

Caspase-3/CPP32, a member of the interleukin-1 converting enzyme (ICE) family, is considered an executioner protease in mammalian cells during apoptosis. Although expression and activation of caspase-3/CPP32 protein have been studied in many tissues and leukemia cell lines, this has not been explored in primitive hematopoietic CD34(+) cells. In this study, we evaluated expression and activation of caspase-3/CPP32 protein in CD34(+) cells from cord blood (CB) during apoptosis induced by growth factor deprivation. Reverse transcriptase-polymerase chain reaction (RT-PCR), Western blot, and flow cytometry analysis were used in this study to determine the expression of caspase-3/CPP32 in CD34(+) CB cells during apoptosis. Our results demonstrated that caspase-3/CPP32 mRNA was constitutively expressed at a very low level in freshly isolated CD34(+) cells. Expression of caspase-3/CPP32 mRNA and protein was upregulated when these cells were first expanded in suspension culture with growth factors for 3 days. However, only the 32 kDa inactive caspase-3/CPP32 proenzyme was detected in the freshly isolated CD34(+) cells and after 3 days expansion with cytokines. Within 12 hours after growth factor withdrawal from expanded cells caspase-3/CPP32 was activated and a cleavage 20 kDa protein was detected; a poly(ADP-ribose) polymerase (PARP) was cleaved by activated caspase-3/CPP32. Activation of caspase-3/CPP32 and apoptosis upon growth factor withdrawal were inhibited/reduced by the caspase inhibitors, z-VAD-fmk and DEVD-CHO. These results demonstrate that caspase-3/CPP32 is involved in apoptosis of primitive CB CD34(+) cells but may not be the only mechanism involved.
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PMID:Expression and activation of caspase-3/CPP32 in CD34(+) cord blood cells is linked to apoptosis after growth factor withdrawal. 1098 91

Neutrophils aggravate cholestatic liver injury after bile duct ligation (BDL). Recently, it was suggested that hepatocellular apoptosis might be critical for liver injury in this model. To test the hypothesis that apoptosis could be a signal for neutrophil extravasation and injury, we assessed parameters of apoptosis and inflammation after BDL using 2 different approaches: (1) wild-type and Fas receptor-deficient lpr mice of the C57BL/6J or C3H/HeJ strains, and (2) treatment with the pancaspase inhibitor z-Val-Ala-Asp-fluoromethylketone (z-VAD-fmk)in C3HeB/FeJ mice. After BDL for 3 days, total cell death was estimated to be between 10% and 50% of all cells evaluated. However, less than 0.1% of hepatocytes showed apoptotic morphology in all 3 strains. Processing of procaspase-3, caspase-3 enzyme activities, and immunohistochemical staining for cytokeratin 18 cleavage products indicated no activation of caspases. Real-time reverse-transcriptase polymerase chain reaction analysis revealed increased expression of many inflammatory mediators but no effect on proapoptotic genes. More than 50% of all accumulated neutrophils were extravasated and colocalized with foci of oncotic hepatocytes and chlorotyrosine adducts. z-VAD-fmk treatment had no effect on apoptosis or liver injury after BDL but eliminated apoptosis after galactosamine/endotoxin in C3HeB/FeJ mice. In Fas receptor-deficient lpr mice (C57BL/6J), expression of inflammatory mediators, neutrophil accumulation and extravasation, chlorotyrosine adduct formation, and liver injury were reduced. This protection was not observed in lpr mice of the endotoxin-resistant C3H/HeJ strain. In conclusion, liver injury (oncotic necrosis) after BDL correlated with the severity of the inflammatory response. The minimal amount of apoptosis had no effect on inflammation or on the overall injury.
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PMID:Reduced oncotic necrosis in Fas receptor-deficient C57BL/6J-lpr mice after bile duct ligation. 1538 26

PDE4A11 is a novel cAMP-specific phosphodiesterase that is conserved in humans, mouse, rat, pig, and bat. Exon-1(4A11) encodes its unique, 81 amino acid N-terminal region. Reverse-transcriptase polymerase chain reaction performed across the splice junction, plus identification of expressed sequence tags, identifies PDE4A11 as a long isoform possessing UCR1 and UCR2 regulatory domains. Transcript analysis shows that PDE4A11 is widely expressed compared with PDE4A10 and PDE4A4B long isoforms. Truncation analysis identifies a putative promoter in a 250-base pair region located immediately upstream of the start site in Exon-1(4A11). Recombinant PDE4A11, expressed in COS-7 cells, is a 126-kDa protein localized predominantly around the nucleus and in membrane ruffles. PDE4A11 exhibits a K(m) for cAMP hydrolysis of 4 microM, with relative V(max) similar to that of PDE4A10 and PDE4A4B. PDE4A11 is dose-dependently inhibited by rolipram, 4-[(3-butoxy-4-methoxyphenyl)-methyl]-2-imidazolidinone (Ro 20-1724), cilomilast, roflumilast, and denbufylline, with IC(50) values of 0.7, 0.9, 0.03, 0.004, and 0.3 microM, respectively. Soluble and particulate PDE4A11 exhibit distinct rates of thermal inactivation (55 degrees C; T((0.5)) = 2.5 and 4.4 min, respectively). Elevating cAMP levels in COS-7 cells activates PDE4A11 concomitant with its phosphorylation at Ser119 by protein kinase A (PKA). PDE4A11 differs from PDE4A4 in sensitivity to cleavage by caspase-3, interaction with LYN SH3 domain, redistribution upon long-term rolipram challenge, and sensitivity to certain PDE4 inhibitors. PDE4A11, PDE4A10, and PDE4A4 all can interact with betaarrestin. PDE4A11 is a novel, widely expressed long isoform that is activated by PKA phosphorylation and shows a distinct intracellular localization, indicating that it may contribute to compartmentalized cAMP signaling in cells in which it is expressed.
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PMID:Identification and characterization of PDE4A11, a novel, widely expressed long isoform encoded by the human PDE4A cAMP phosphodiesterase gene. 1573 10

Exposure to environmental tobacco smoke has been epidemiologically linked to heart disease among nonsmokers. However, the molecular mechanism behind the pathogenesis of cardiac disease is unknown. In this study, we found that Wistar rats, exposed to tobacco cigarette smoke at doses of 5, 10, or 15 cigarettes for 30 min twice a day for 1 month, had a dose-dependently reduced heart weight to body weight ratio and enhanced interstitial fibrosis as identified by histopathologic analysis. The mRNA and activity of matrix metalloprotease-2 (MMP-2), representing the progress of cardiac remodeling, were also elevated in the heart. In addition, we used reverse-transcriptase polymerase chain reaction and Western blotting to demonstrate significantly increased levels of the apoptotic effecter caspase-3 in treated animal hearts. Dose-dependently elevated mRNA and protein levels of Fas, and promoted apoptotic initiator caspase-8 (active form), a molecule of a death-receptor-dependent pathway, coupled with unaltered or decreased levels of cytosolic cytochrome c and the apoptotic initiator caspase-9 (active form), molecules of mitochondria-dependent pathways, may be indicative of cardiac apoptosis, which is Fas death-receptor apoptotic-signaling dependent, but not mitochondria pathway dependent in rats exposed to second-hand smoke (SHS). With regard to the regulation of survival pathway, using dot blotting, we found cardiac insulin-like growth factor-1 (IGF-1) and IGF-1 receptor mRNA levels to be significantly increased, indicating that compensative effects of IGF-1 survival signaling could occur. In conclusion, we found that the effects of SHS on cardiomyocyte are mediated by the Fas death-receptor-dependent apoptotic pathway and might be related to the epidemiologic incidence of cardiac disease of SHS-exposed nonsmokers.
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PMID:Second-hand smoke-induced cardiac fibrosis is related to the Fas death receptor apoptotic pathway without mitochondria-dependent pathway involvement in rats. 1620 45

Recent data suggest that new treatment options for superficial bladder cancer are necessary, owing to the high recurrence rate after conventional treatment, especially in T1G3 and Bacillus Calmette-Guerin-refractory patients. Phase I and II studies have demonstrated that gemcitabine may represent a candidate for intravesical therapy in superficial bladder cancer. Despite clinical trials, the in-vitro cytotoxic and proapoptotic effects of gemcitabine have been poorly investigated. In the present study, we investigated how gemcitabine affects apoptosis in bladder cancer cell line 5637, which has the same molecular features of high-risk superficial bladder cancer. Apoptosis was evaluated by DNA fragmentation, flow cytometry and caspase activation. bcl-2, bcl-X, bax, survivin and fas gene expression were also evaluated by reverse-transcriptase polymerase chain reaction. Nuclear factor-kappa B activation was assessed by immunofluorescence. Gemcitabine induced apoptosis in 5637 cells in a time-dependent manner, with activation of caspase-3, -8 and -9. Expression of bcl-2, bax, survivin and bcl-X was not affected by treatment, whereas fas strongly increased after 24 h of treatment. After treatment, we failed to find any nuclear localization of nuclear factor-kappa B. As gemcitabine-induced apoptosis involves fas upregulation, these results may encourage the investigation of intravesical gemcitabine in fas-negative bladder tumors. Furthermore, as nuclear factor-kappa B activation by cisplatin, doxorubicin and adriamycin may result in enhanced proliferation, migration, immortality and inhibition of apoptosis, the observation that gemcitabine does not activate nuclear factor-kappa B may have implications in intravesical therapy of high-risk superficial bladder cancer.
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PMID:Gemcitabine-induced apoptosis in 5637 cell line: an in-vitro model for high-risk superficial bladder cancer. 1715 4

1. The aim of the present study was to investigate the changes in chemotherapeutic drug sensitivity of HepG2 cells transfected with Bcl-2 and Bcl-xl siRNA expression vectors. 2. Bcl-2 and Bcl-xl siRNA and negative siRNA expression vectors were constructed and stably transfected into HepG2 cells. Reverse transcriptase-polymerase chain reaction was used to detect the target gene expression, and the Bcl-2, Bcl-xl, Bax and caspase-3 protein levels were measured using western blots and immunofluorescence. The sensitivity of the cells to the chemotherapeutic drugs 5-fluorouracil (5-FU) and 10-hydroxycamptothecin (HCPT) was analysed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazoliumbromide (MTT) and flow cytometry. 3. The Bcl-2 and Bcl-xl gene expression and corresponding protein levels in Bcl-2 siRNA, Bcl-xl siRNA and Bcl-2/Bcl-xl siRNA transfected cells were reduced compared with negative siRNA transfected or untreated cells. The Bax protein level remained unaltered but the caspase-3 level was enhanced when Bcl-2 and Bcl-xl protein levels were reduced. The MTT results demonstrated that Bcl-2 and Bcl-xl transfected cells exhibited increased sensitivity to 5-FU or HCPT. Flow cytometry demonstrated that the sub G1 cell population increased in Bcl-2/Bcl-xl siRNA co-transfected and Bcl-xl siRNA and Bcl-2 siRNA transfected cells when compared with negative siRNA or untreated cells. The latter trend was strengthened further in the presence of 5-FU or HCPT. 4. Thus, Bcl-2 and Bcl-xl siRNA-mediated gene silencing, in combination with chemotherapy, may be a potential therapeutic strategy against human hepatoblastoma.
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PMID:siRNA-mediated Bcl-2 and Bcl-xl gene silencing sensitizes human hepatoblastoma cells to chemotherapeutic drugs. 1743 14

The distribution and density of pituitary adenylate cyclase-activating polypeptide (PACAP) binding sites have been investigated in the brain of the primates Jacchus callithrix (marmoset) and Macaca fascicularis (macaque) using [(125)I]-PACAP27 as a radioligand. PACAP binding sites were widely expressed in the brain of these two species with particularly high densities in the septum, hypothalamus and habenula. A moderate density of recognition sites was seen in all subdivisions of the cerebral cortex with a heterogenous distribution, the highest concentrations occurring in layers I and VI while the underlying white matter was almost devoid of binding sites. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis revealed intense expression of the mRNAs encoding the short and hop-1 variants of pituitary adenylate cyclase-activating polypeptide-specific receptor (PAC1-R) in the cortex of both marmoset and macaque, whereas vasoactive intestinal polypeptide/pituitary adenylate cyclase-activating polypeptide mutual receptor, subtype 1 (VPAC1-R) and vasoactive intestinal polypeptide/pituitary adenylate cyclase-activating polypeptide mutual receptor, subtype 2 (VPAC2-R) mRNAs were expressed at a much lower level. In situ hybridization histochemistry showed intense expression of PAC1-R and weak expression of VPAC1-R mRNAs in layer IV of the cerebral cortex. Incubation of cortical tissue slices with PACAP induced a dose-dependent stimulation of cyclic AMP formation, indicating that PACAP binding sites correspond to functional receptors. Moreover, treatment of primate cortical slices with 100 nM PACAP significantly reduced the activity of caspase-3, a key enzyme of the apoptotic cascade. The present results indicate that PACAP should exert the same neuroprotective effect in the brain of primates as in rodents and suggest that PAC1-R agonists may have a therapeutic value to prevent neuronal cell death after stroke or in specific neurodegenerative diseases.
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PMID:Distribution and functional characterization of pituitary adenylate cyclase-activating polypeptide receptors in the brain of non-human primates. 1923 5

Exposure to 17beta-estradiol prior to induction of apoptosis protects skeletal muscle cells against damage. The mechanism involved in this protective action of the hormone is poorly understood. In the present study, using the murine muscle cell line C2C12, evidence was obtained that inhibition of H(2)O(2)-induced apoptosis by the estrogen requires the participation of heat shock protein 27 (HSP27). Reverse transcriptase polymerase chain reaction, Western blot, and immunocytochemistry assays showed that 17beta-estradiol induces a time-dependent (5-60 min) increase in the expression of HSP27. In addition, in presence of quercetin, an inhibitor of HSPs, the antiapoptotic effect of the hormone was diminished. More specifically, blockage experiments with short interference RNA targeting HSP27 confirmed the role of this chaperone in the protective effect of the steroid. 17beta-Estradiol abolished caspase-3 cleavage elicited by H(2)O(2). Coimmunoprecipitation assays suggested physical interaction of HSP27 with caspase-3 in presence of estradiol. Furthermore, we observed that this chaperone interacts with estrogen receptors (ER) beta in mitochondria. Then, this study suggests that HSP27 plays a new role in the antiapoptotic action triggered by 17beta-estradiol by modulating caspase-3 activity and stabilizing ERbeta in skeletal muscle cells.
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PMID:Participation of HSP27 in the antiapoptotic action of 17beta-estradiol in skeletal muscle cells. 1962 Dec 76

Integrin alpha(v)beta(8) plays an important role in cerebral vascular development. It has been proven that alpha(v)beta(8) is a key factor for transforming growth factor-beta1 (TGF-beta1) activation in epithelial cells. However, it is not clear whether alpha(v)beta(8) can activate TGF-beta1 and play a role in protection during neonatal hypoxic-ischemic brain injury. In this study, we investigated the relationship between alpha(v)beta(8) and TGF-beta1 activation, and thus the effects of TGF-beta1 activation in the protection of neurons after hypoxia-ischemia (HI). Astrocytes and neurons from rat brains were cultured and then subjected to oxygen-glucose deprivation to generate HI model in vitro. beta(8) expression was determined using immunocytochemistry, western blot, and reverse-transcriptase polymerase chain reaction. TGF-beta1 activation was determined by TGF-beta bioassay in a tested cell (astrocyte) and a reporter cell co-culture system. The pro-apoptotic protein, cleaved caspase-3, and the anti-apoptotic protein, Bcl-2 and Bcl-xL, were detected using western blot. Cellular apoptosis was detected with TUNEL. We found that beta(8) expression was stronger in astrocytes than that in neurons under normoxia. HI resulted in a rapid and persistent increase of beta(8) expression in astrocytes, but only in a slight and transient increase in neurons. Astrocytes beta(8) could induce TGF-beta1 leading to upregulation of Bcl-2 and Bcl-xL, and thus attenuated neuronal apoptosis. The present findings suggest that beta(8) protecting the brain against neonatal HI injury through TGF-beta1 signaling pathway, which may have implications for the treatment of HI brain injury.
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PMID:The role of integrin alpha(v)beta (8) in neonatal hypoxic-ischemic brain injury. 1977 86


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