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)

Cystathionine-beta-synthase (CBS) catalyzes the condensation of serine and homocysteine to form cystathionine, an intermediate step in the synthesis of cysteine. We previously described essential transactivating roles for specificity protein 1 (Sp1), Sp3, nuclear factor Y (NF-Y), and USF-1 in the regulation of the CBS-1b promoter. Differential binding of Sp1/Sp3 to the CBS-1b promoter due to differences in Sp1/Sp3 phosphorylation, and Sp1/Sp3 synergism with NF-Y might, in part, explain cell-specific patterns of CBS expression. In this report, the roles of various NF-YA isoforms in influencing cell-specific differences in CBS gene expression were determined in HT1080 and HepG2 cells. Seven unique NF-YA isoforms were detected in HT1080 by reverse transcriptase-PCR (RT-PCR) and DNA sequencing, characterized by deletions in the glutamine-rich and/or serine/threonine-rich domains. Only four of the seven NF-YA isoforms were found in HepG2 cells. The six alternatively spliced NF-YA isoforms all showed significantly less synergistic transactivation of the CBS-1b promoter with Sp1 than wild-type NF-YA, as determined by cotransfections in Drosophila SL2 cells with NF-YB and NF-YC. Further, all six alternatively spliced NF-YA isoforms inhibited the synergistic transactivation of the CBS-1b promoter by wild-type NF-Y and Sp1. Thus, the cellular distributions of these alternatively spliced NF-YA isoforms could impart an important cell-specific component to CBS transcriptional regulation, by virtue of their abilities to directly synergize with Sp1/Sp3 and interfere with transactivation of the CBS-1b promoter by wild-type NF-Y. Characterization of CBS promoter structure and function should clarify the molecular bases for variations in CBS gene expression in genetic diseases and the relationship between CBS and Down's syndrome (DS).
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PMID:Synergistic regulation of human cystathionine-beta-synthase-1b promoter by transcription factors NF-YA isoforms and Sp1. 1242 42

Mixed lineage kinases (MLKs) belong to the family of mitogen activated protein kinase kinase kinase (MAPKKK) and cause neuronal cell death mediated through c-Jun, N-terminal kinase (JNK) pathway. Recently, genetic studies in Drosophila revealed the presence of an MLK termed slipper (slpr). However, its biochemical features like physiological substrate, role in different MAPK pathways and developmental and tissue-specific expression pattern were not reported. Here, we report cDNA cloning, expression analysis and biochemical characterization of a Drosophila mixed lineage kinase (dMLK) that is also known as slipper. The protein structure analysis of dMLK/slipper revealed, in addition to the conserved domains, a stretch of glutamine in the amino terminus and an asparagine-threonine stretch at the carboxy-terminus. In situ hybridization and reverse transcriptase polymerase chain reaction (RT-PCR) analysis revealed that dMLK is expressed in early embryonic stages, adult brain and thorax. Ectopic expression of dMLK either in Drosophila S2 or in mammalian HEK293 cells leads to activation of JNK, p38 and extracellular signal regulated kinase (ERK) pathways. Further, dMLK directly phosphorylates Hep, dMKK4 and also their mammalian counterparts, MKK7 and SEK1, in an in vitro kinase assay. Taken together, our results provide for the first time a comprehensive expression profile and new biochemical insight of dMLK/slipper.
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PMID:Drosophila mixed lineage kinase/slipper, a missing biochemical link in Drosophila JNK signaling. 1267 57

The alpha-ketoisocaproic acid (KIC) is a short branched-chain monocarboxylate, which accumulates in neural cells. It plays an important role in maintaining nitrogen balance in the brain, a process of a great importance for shuttling of glutamine and glutamate between astrocytes and neurons. Higher accumulation of KIC in isolated cerebral cortex neurons at lower external pH, as well as sensitivity of this process to alpha-cyano-4-hydroxycinnamate indicate an involvement of a transporter, belonging to the family of monocarboxylate transporters (MCT).The expression of MCT1 and MCT2 isoforms in the brain cells was studied using reverse transcriptase-polymerase chain reaction (RT-PCR) method. The mRNA coding MCT1 was detected in astrocytes, brain endothelial cells, tumour cells (neuroblastoma and glioma) and in cortex neurons of newborn rats, but not in adult ones. MCT2, which is less abundant isoform than MCT1, was expressed in astrocytes, in brain endothelial cells and at low level in newborn rats' neurons, being absent in neurons from adult brain.The observed sensitivity of KIC accumulation towards SH-groups reagents did not fit to the known characteristics of MCT1 and MCT2. Therefore, the change of MCT expression during brain development, as well as lack of MCT1 and MCT2 in neurons of adults, point to another MCT isoform being involved in alpha-ketoisocaproic acid accumulation. This could be either one of other known MCT isoforms or a new member of family MCT, specific towards branched chain alpha-ketoacids.
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PMID:Expression of monocarboxylic acid transporters (MCT) in brain cells. Implication for branched chain alpha-ketoacids transport in neurons. 1274 73

The expression of glutamine synthetase (GS), catalysing the ATP-dependent conversion of glutamate and ammonia into glutamine, is transcriptionally and post-transcriptionally regulated. The genomic structure of dog GS shown in the present study is basically similar to that of other mammals in that it is composed of seven exons and six introns. Using 5'-cRACE (where cRACE stands for circular rapid amplification of cDNA ends) and reverse transcriptase-PCR, we identified an additional exon (120 bp) in the first intron, designated in the present study as exon 1'. By means of alternative splicing, the GS gene produces an altered form of GS transcript with 5'-untranslated region (UTR) containing the exon 1'. This alternative transcript is abundantly expressed in brain, whereas it is found at lower levels in other tissues. In the human and mouse GS genes, extra exons are also found at the corresponding site of the intron 1 but with different sizes. An exon-trapping experiment for the GS gene in COS-7, Madin-Darby canine kidney and SK-N-SH cells revealed that the pattern of alternative splicing is variable in different cell types. The propensity of forming a secondary structure is predicted to be considerably higher in the presence of extra 5'-UTR, suggesting the possibility of a translational effect. To test this, we performed a reporter assay for fusions with different 5'-UTRs, demonstrating that the long form with extra 5'-UTR was translated 20- and 10-fold less than the short one in SK-N-SH and Neuro-2A cells respectively. Similarly, translations of human and mouse transcripts with extra 5'-UTRs were less efficient, showing 6-8-fold reductions in SK-N-SH cells. Furthermore, when we mutated an ATG sequence contained in the exon 1', the suppression of translation was partially relieved, suggesting that the negative regulation by an extra 5'-UTR is, to some extent, due to an abortive translation from the upstream ATG.
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PMID:A splice variant acquiring an extra transcript leader region decreases the translation of glutamine synthetase gene. 1274 66

The objective of the present study was to determine the effects of follistatin addition on myostatin and follistatin gene expression patterns in C2C12 muscle cells. C2C12 cells were administered with 100 ng/ml recombinant human (rh) follistatin in Dulbecco's modified Eagle medium (DMEM) containing 10% fetal bovine serum (FBS), 4 mM glutamine and antibiotics daily for three days. Rh follistatin was not added in the control wells. Follistatin and myostatin gene cDNAs were synthesised by reverse transcriptase polymerase chain reaction (RT-PCR). The time course of follistatin gene expression pattern was similar in both the control and the follistatin-treated group. Myostatin mRNA level significantly increased in the follistatin-treated group after 24 h of culture (Fig. 3, P < 0.01). Amounts then sharply decreased (Fig. 3, P < 0.01) at 48 h of culture, whereas there was no significant difference between the control and the follistatin-treated group at 72 h of culture. Our results demonstrated that myostatin and follistatin mRNA were expressed in C2C12 cells and rh follistatin changed the myostatin expression pattern.
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PMID:Follistatin alters myostatin gene expression in C2C12 muscle cells. 1516 44

We reported previously that insulin inhibits the stimulatory effect of high glucose on the expression of angiotensinogen (ANG) gene in both rat immortalized renal proximal tubular cells (IRPTCs) and non-diabetic rat renal proximal tubular cells (RPTCs), but has no effect in diabetic rat RPTCs. In the present study we investigated whether hyperglycaemia-induced resistance to the insulin-induced inhibition of expression of the ANG gene is mediated via the generation of reactive oxygen species (ROS) in RPTCs. Rat IRPTCs were cultured for 2 weeks in high-glucose (25 mM) or normal-glucose (5 mM) medium plus angiotensin II (Ang II) with or without a superoxide scavenger (tiron), or inhibitors of: NADPH oxidase (diphenylene iodinium, DPI), Ang II type 1 and 2 receptors (losartan and PD123319), angiotensin-converting enzyme (perindopril), protein kinase C (GF 109203X), or glutamine:fructose-6-phosphate amino-transferase (azaserine). Cellular generation of ROS, and ANG and renin mRNA levels were assessed by lucigenin assay and specific reverse transcriptase-PCR respectively. Phosphorylation of p44/42 mitogen-activated protein kinase (p44/42 MAPK) was evaluated by western blotting. Prolonged exposure of IRPTCs to high concentrations of glucose or Ang II evoked generation of ROS and resistance to the insulin-induced inhibition of expression of the ANG gene and of p44/42 MAPK phosphorylation. Co-incubation of IRPTCs with tiron, DPI, losartan, PD123319, perindopril, GF 109203X or azaserine prevented ROS generation, restoring the inhibitory action of insulin on ANG gene expression and on p44/42 MAPK phosphorylation. In conclusion, our studies demonstrate that blockade of both ROS generation and activation of the intrarenal renin-angiotensin system improves the inhibitory action of insulin on ANG gene expression in IRPTCs in conditions of high glucose.
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PMID:Reactive oxygen species blockade and action of insulin on expression of angiotensinogen gene in proximal tubular cells. 1559 Sep 80

Glutamine (Gln), a conditionally essential amino acid, can be a potential enhancer of the heat stress response. And glucose-regulated protein 75(grp75) is a member of the hsp family. To evaluate the effect of glutamine on the expression of grp75, PC12 cell was cultured with DMEM, glucose-free DMEM, DMEM within Gln, and glucose-free DMEM within Gln, and the expression of grp75 was detected by immunocytochemistry and western blot, the mRNA level of grp75 in the cells was detected by reverse transcriptase-polymerase chain reaction (RT-PCR). Data indicated that Gln can upregulated the expression of grp75 in PC12 cell line, and the effect is more significantly in PC12 cell which was glucose deprivation than the normal cell. To investigate the effect of Gln on PC12 cells under glucose deprivation, MTT method was used to monitor the cell viability after Gln treatment for the cell under glucose depriving. The experiments results showed that glutamine at a concentration range of 0.2-40 mmol/L significantly enhanced the cell viability in glucose-free DMEM. And the protective effect to grp75 low-expresssion on PC12 cells is markedly decreased.
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PMID:[Effect of glutamine on the expression of grp75 in PC12 cells]. 1636 23

Formation of tooth enamel is a very complex process in which a specific set of proteins secreted by ameloblasts play a primordial role. As part of a screening procedure to identify novel proteins secreted by EO (enamel organ) cells of rat incisors, we isolated a partial cDNA fragment (EO-017) that is the homologue of the recently described mouse Amtn (amelotin) gene [Iwasaki, Bajenova, Somogyi-Ganss, Miller, Nguyen, Nourkeyhani, Gao, Wendel and Ganss (2005) J. Dent. Res. 84, 1127-1132]. Presented herein is the cloning of rat and pig full-length cDNAs with their deduced protein sequences. Detailed expression profiling by Northern-blot analysis and RT (reverse transcriptase)-PCR on rat and mouse tissues revealed highest expression in the mandible, more specifically in the maturation stage of the EO. Among all tissues tested, low expression was detected only in periodontal ligament, lung, thymus and gingiva. In silico analyses revealed that the Amtn gene is highly conserved in seven other mammals, but is absent from fish, birds and amphibians. The Amtn protein is enriched in proline, leucine, glutamine and threonine (52% of total) and contains a perfectly conserved protein kinase CK2 phosphorylation site. Transient transfection experiments in HEK-293 cells (human embryonic kidney cells) showed that secreted Amtn is post-translationally modified possibly through O-linked oligosaccharides on threonine residues. In concordance with its predominant expression site, immunofluorescence localization within the rat and mouse mandibles revealed Amtn localized to the basal lamina of maturation stage ameloblasts of incisors and unerupted molars. Intense Amtn protein expression was also detected in the internal basal lamina of junctional epithelium in molars. The peculiar and unique cellular localization of Amtn suggests a role in cell adhesion.
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PMID:Cloning of rat amelotin and localization of the protein to the basal lamina of maturation stage ameloblasts and junctional epithelium. 1678 91

We present a protocol that has been developed for induction of the differentiation of murine embryonic stem (ES) cells to alveolar type II cells. With this protocol, undifferentiated murine ES cells are induced to form embryoid bodies (EBs). The 10-d-old EBs are transferred to adherent culture conditions and are fed with high-glucose Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% v/v fetal bovine serum, 2 mM L-glutamine, and 0.1 mM 2-mercaptoethanol for 20 d without splitting. Then, the cells are fed with a medium designed for the maintenance and growth of mature distal airway epithelial cells (small airway growth medium, SAGM) for 3 d. Characterization of the alveolar type II cells was done using real-time reverse transcriptase polymerase chain reaction detection of surfactant protein C mRNA and immunocytochemical detection of prosurfactant protein C. Real-time reverse transcriptase polymerase chain reaction revealed that SAGM increases the mRNA expression level of SPC by a factor of 8 when compared to that of cells grown in supplemented high-glucose DMEM (p < 0.05, Student t-test). Immunocytochemistry revealed that proSPC-expressing cells comprised 2.8 +/- 0.23% of the total cell population in SAGM-treated samples and 0.5 +/- 0.1% in samples treated with supplemented high-glucose DMEM (p < 0.05, chi2 test).
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PMID:Derivation and characterization of alveolar epithelial cells from murine embryonic stem cells in vitro. 1684 28

Understanding how the various host cells respond to probiotic bacteria in vitro may provide important insight into elaborate immune responses triggered by beneficial bacteria. The aim of this study was to investigate the detailed pattern of the mRNA expression of cytokines (IL-1beta, IL-8, TNF-alpha and TGF-beta) in head kidney (HK) leucocytes and gut cells isolated from rainbow trout (Oncorhynchus mykiss Walbaum) after co-culturing with live probiotics. HK leucocytes and gut cells adjusted to 5 x 10(6) and 2 x 10(6) ml(-1), respectively, in L-15 medium containing 25% decomplemented FCS and 300 mg l(-1) L-glutamine were co-cultured with Carnobacterium maltaromaticum B26 and C. divergens B33 at an multiplicity of infection of 25 for 6 and 12 h. Quantitative real-time reverse transcriptase polymerase chain reaction using SYBR Green I was employed to determine the mRNA expression of studied genes. Although neither probiotic strains significantly induced mRNA of the cytokines in gut cells, expression ratios of IL-1beta and TNF-alpha of HK cells were significantly higher, suggesting that these bacteria can stimulate innate immunity in rainbow trout.
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PMID:Cytokine expression in leucocytes and gut cells of rainbow trout, Oncorhynchus mykiss Walbaum, induced by probiotics. 1701 Oct 45


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