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:2.7.7.48 (transcriptase)
9,479 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

In excitable cells, oscillations in intracellular free calcium concentrations ([Ca(2+)](i)) can arise from action-potential-driven Ca(2+) influx, and such signals can have either a localized or global form, depending on the coupling of voltage-gated Ca(2+) influx to intracellular Ca(2+) release pathway. Here we show that rat pituitary somatotrophs generate spontaneous [Ca(2+)](i) oscillations, which rise from fluctuations in the influx of external Ca(2+) and propagate within the cytoplasm and nucleus. The addition of caffeine and ryanodine, modulators of ryanodine-receptor channels, and the depletion of intracellular Ca(2+) stores by thapsigargin and ionomycin did not affect the global nature of spontaneous [Ca(2+)](i) signals. Bay K 8644, an L-type Ca(2+) channel agonist, initiated [Ca(2+)](i) signaling in quiescent cells, increased the amplitude of [Ca(2+)](i) spikes in spontaneously active cells, and stimulated growth hormone secretion in perifused pituitary cells. Nifedipine, a blocker of L-type Ca(2+) channels, decreased the amplitude of spikes and basal growth hormone secretion, whereas Ni(2+), a blocker of T-type Ca(2+) channels, abolished spontaneous [Ca(2+)](i) oscillations. Spiking was also abolished by the removal of extracellular Na(+) and by the addition of 10 mM Ca(2+), Mg(2+), or Sr(2+), the blockers of cyclic nucleotide-gated channels. Reverse transcriptase-polymerase chain reaction and Southern blot analyses indicated the expression of mRNAs for these channels in mixed pituitary cells and purified somatotrophs. Growth hormone-releasing hormone, an agonist that stimulated cAMP and cGMP productions in a dose-dependent manner, initiated spiking in quiescent cells and increased the frequency of spiking in spontaneously active cells. These results indicate that in somatotrophs a cyclic nucleotide-controlled plasma membrane Ca(2+) oscillator is capable of generating global Ca(2+) signals spontaneously and in response to agonist stimulation. The Ca(2+)-signaling activity of this oscillator is dependent on voltage-gated Ca(2+) influx but not on Ca(2+) release from intracellular stores.
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PMID:Characterization of a plasma membrane calcium oscillator in rat pituitary somatotrophs. 1058 49

Osteoblasts express protease-activated receptor-1 (PAR-1), which is activated by thrombin or by synthetic peptides corresponding to the new "tethered ligand" N-terminus of PAR-1 created by receptor cleavage. Both thrombin and human PAR-1-activating peptide stimulate an elevation of [Ca2+]i in the human SaOS-2 osteoblast-like cell line, but the peptide stimulates receptor-mediated Ca+ entry, whereas thrombin does not. Stimulation of proliferation in rat primary osteoblast-like cells is greater in response to rat PAR-1-activating peptide than to thrombin. Because the PAR-1-activating peptides are now known to activate PAR-2, the current study was undertaken to investigate whether osteoblasts express this receptor and, if so, whether this could account for the observed discrepancies between responses of osteoblasts to thrombin and to PAR-1-activating peptides. Reverse transcriptase-polymerase chain reaction (RT-PCR) and immunocytochemical studies demonstrated expression of PAR-2 by primary cultures of rat calvarial osteoblast-like cells. In immunohistochemical studies of embryonic mouse bones, osteoblasts showed positive staining for the presence of PAR-2. Activators of PAR-2 include trypsin, mast cell tryptase, gingipain-R, and synthetic peptides corresponding to the PAR-2 tethered ligand sequence. Treatment of primary rat osteoblast-like cells with rat PAR-2-activating peptide (SLIGRL), or SaOS-2 cells with human PAR-2-activating peptide (SLIGKV), caused a dose-dependent increase in [Ca2+]i. Trypsin or gingipain-R also induced an increase in intracellular calcium concentration, and caused reciprocal cross desensitization. Activators of PAR-2 caused a sharp peak in [Ca2+]i followed by a sustained plateau; [Ca2+]i returned to baseline levels upon treatment with ethylene-glycol tetraacetic acid (EGTA). Treatment of rat osteoblast-like cells in vitro with SLIGRL did not affect thymidine incorporation or endogenous alkaline phosphatase activity. The results presented here demonstrate that osteoblasts express PAR-2, and that such expression is able to account for the observed discrepancies between thrombin and PAR-1-activating peptides in their ability to evoke calcium entry, but not proliferative responses.
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PMID:Expression of protease-activated receptor-2 by osteoblasts. 1061 51

The activation of osteoblast calcium channels by many bone regulatory factors suggests an important role for intracellular calcium signaling in the control of bone remodeling. At least six different genes for the alpha 1 subunit of voltage-gated calcium channels have been cloned including L-type (alpha 1S, alpha 1C, and alpha 1D) and non-L-type (alpha 1A, alpha 1B, and alpha 1E) isoforms. The goal of the present study was to identify which of these calcium channel isoforms are transcribed in human osteoblast-like cell lines (hFOB, MG-63, SAOS-2, TE-85, G-292) and in cultures of normal human osteoblasts. Reverse transcriptase-PCR was used to amplify sequences corresponding to each of the alpha 1 subunits using isoform specific primers. The products of the PCR reaction were cloned and sequenced to verify their identify and used to probe southern blots of the PCR reactions. The results indicate that among the different types of osteoblast-like cells examined, two calcium channel isoforms were always expressed (alpha 1C and alpha 1A), three isoforms were variably expressed (alpha 1S, alpha 1D and alpha 1B), and one isoform was not expressed in any of the osteoblast-like cells (alpha 1E) but was easily detected in human brain controls. Our results indicate that mRNAs for multiple calcium channel alpha 1 subunits are expressed in human osteoblasts, including both L-type and non-L-type isoforms. In addition, significant heterogeneity exists between the different osteoblast cell models examined in the type and mRNA abundance of the different calcium channel isoforms.
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PMID:Expression of mRNAs for the alpha 1 subunit of voltage-gated calcium channels in human osteoblast-like cell lines and in normal human osteoblasts. 1065 63

We previously demonstrated that iodothyronine 5'-deiodination (5'D) activity is present and increased by triiodothyronine (T3) and angiotensin II (Ang II) in cultured rat cardiac myocytes. To further elucidate the stimulatory mechanism of Ang II, we investigated the effect of intracellular Ca2+ and protein kinase C on myocardial 5'D activity. Moreover, to elucidate the molecular mechanism of the stimulatory effect of T3 and Ang II, we detected the mRNA levels by means of a reverse-transcriptase polymerase chain reaction (RT-PCR). 5'D activity was increased by adding Bay-k 8644, Ca2+ channel agonist and the effect of Bay-k 8644 was completely blocked by nifedipine, a Ca2+ channel antagonist. 12-O-tetradecanoylphorbol-13-acetate, a protein kinase C activator, similarly stimulated 5'D activity. The addition of a high concentration (20-40 mM) of K+, which caused the depolarization of the membrane had significant stimulatory effects on 5'D activity. Type 1 deiodinase (D1) mRNA was evident in myocardial cells by RT-PCR in a single 758 bp band similar to that in the liver. Cardiac fibroblasts did not express the D1 mRNA. A significant increase in D1 mRNA was also evident after adding T3 and Ang II. These findings indicate that 5'D activity in myocardial cells is increased by activating the voltage sensitive Ca2+ channel, protein kinase C, and membrane depolarization, and that the D1 mRNA is present in cardiac myocytes and is increased by T3 and Ang II. This study therefore suggests that Ang II could affect the action of thyroid hormone on the heart by increasing the D1 gene expression.
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PMID:Type 1 iodothyronine deiodinase in heart --effects of triiodothyronine and angiotensin II on its activity and mRNA in cultured rat myocytes. 1067 Jul 46

These studies were performed to establish whether functional receptors for calcitonin gene-related peptide (CGRP) are present on human dendritic cells (DCs) and to investigate potential immunomodulatory effects of CGRP on DCs other than Langerhans cells. Reverse transcriptase-PCR revealed expression of mRNA for a type 1 CGRP receptor by mature and immature blood-derived DCs. Sequence analysis confirmed the identity of the type 1 CGRP receptor (CGRP-R1). Addition of CGRP (10-7 M) to mature and immature DCs resulted in mobilization of intracellular calcium. Treatment of immature DCs with CGRP (10-7 M), before and after maturation in monocyte-conditioned medium, resulted in decreased cell surface expression of HLA-DR MHC class II and the costimulatory molecule, CD86. Treatment of immature DCs with CGRP (10-7 M) also resulted in decreased expression of CD86, but expression of HLA-DR was unchanged. When CGRP-treated mature DCs were used to stimulate allogeneic T cells, proliferative responses were dampened (approximately 50%), especially at low DC:T cell ratios (1:360). This effect was not observed with CGRP-treated, immature DCs. In contrast, CGRP-treated mature or immature DCs were no less efficient than untreated DCs in driving syngeneic T cell-proliferative responses to staphylococcal enterotoxin B. We conclude that mature and immature DCs express type 1 CGRP receptors and that signaling through these receptors may dampen mature DC-driven T cell proliferation most likely via down-regulation of CD86 and HLA-DR.
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PMID:Calcitonin gene-related peptide decreases expression of HLA-DR and CD86 by human dendritic cells and dampens dendritic cell-driven T cell-proliferative responses via the type I calcitonin gene-related peptide receptor. 1072 2

The ability of metabotropic glutamate receptor activation to mobilise intracellular calcium was investigated in cultured dorsal root ganglion (DRG) neurones from neonatal rats using the calcium sensitive fluorescent dye Fura-2. L-glutamate (10 microM) caused sustained and oscillatory increases in intracellular calcium concentration ([Ca2+]i) in a subpopulation of cultured DRG neurones. The oscillatory responses were not blocked by combined application of the ionotropic glutamate receptor antagonists MK 801 (2 microM) and CNQX (20 microM). Oscillations in [Ca2+]i were also observed following application of the nonselective metabotropic glutamate receptor (mGluR) agonist, trans-(1S,3R)-1-aminocyclopentane-1S, 3R-dicarboxylic acid (1S,3R)-ACPD, 20 microM) and the mGluR5 agonist (RS)-2-chloro-5-hydroxyphenylglycine (CHPG, 500 microM). These responses were blocked by the selective Group I mGluR antagonist (RS)-1-aminoindan-1,5-dicarboxylic acid (AIDA) (100 microM) and Ca2+ release channel inhibitors ryanodine (100 microM) and dantrolene (10 microM). The predominantly Group II agonist (2S,2'R,3'R)-2-(2'3'-dicarboxy-cyclopropyl)glycine (DCG-IV, 100 microM) failed to produce Ca2+ transients alone but suppressed responses to CHPG. Reverse transcriptase PCR techniques, using primers specific to Group I mGluRs, revealed the presence of mGluR5 but not mGluR1 mRNA in these cells. Therefore, glutamate can cause a slowly activating and reversible mobilisation of [Ca2+]i in sensory neurones by activation of ionotropic receptors, and can induce oscillatory calcium transients by selectively activating metabotropic glutamate receptors that are likely to be of the mGluR5 subtype.
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PMID:Mobilisation of intracellular Ca2+ by mGluR5 metabotropic glutamate receptor activation in neonatal rat cultured dorsal root ganglia neurones. 1072 83

Humans are exposed occupationally and environmentally to metal aerosols including lead (Pb2+) and cadmium (Cd2+). These toxicants accumulate in male reproductive organs. Epidemiological studies have been equivocal about effects of Pb2+ and Cd2+ on hormone concentrations, male fertility and sperm parameters. Comparison of Pb2+ and Cd2+ concentrations in fertile and infertile men are problematic. Problem areas include failure to control confounding variables, but genetic polymorphisms as in somatic diseases may modulate Pb2+ and Cd2+ damage. Multiple calcium (Ca2+) and potassium (K+) channel isoforms have been identified in human testes and spermatozoa. These Ca2+ and K+ channels are involved in early events of acrosome reactions. Ca2+ channel are susceptible to Cd2+ poisoning and K+ channels to Pb2+. These channels offer entry paths for metallic toxicants into mature spermatozoa. Ion channel polymorphisms may cause differential sensitivities to Cd2+ and Pb2+, explaining in part prospective blinded studies showing high Cd2+ in varicocele-related human infertility and high Pb2+ in unexplained infertility. In both forms of male infertility the ability to undergo an acrosome reaction decreases. Reverse transcriptase-polymerase chain reaction assays for Ca2+ and K+ channel isoforms may identify susceptibility subgroups with lower resistance to environmental exposures.
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PMID:Male infertility and environmental exposure to lead and cadmium. 1078 69

Functional chemokine receptors and chemokines are expressed by glial cells within the CNS, though relatively little is known about the patterns of neuronal chemokine receptor expression and function. We developed monoclonal antibodies to the CCR1, CCR2, CCR3, CCR6, CXCR2, CXCR3 and CXCR4 chemokine receptors to study their expression in human fetal neurons cultured from brain tissue as well as the clonally derived NT2.N human neuronal cell line (NTera 2/cl.D1). Specific monoclonal antibody labeling demonstrated expression of CCR2, CXCR2, CXCR3 and CXCR4 on neurons from both sources. Co-labeling studies revealed strong expression of CXCR3 and CXCR4 on both dendritic and axonal processes, with a weaker expression of CXCR2 and CCR2. Reverse transcriptase-polymerase chain reaction analysis of pure NT2.N neurons confirmed RNA expression for CCR2, CXCR2, CXCR3 and CXCR4. No changes in the neuronal labeling pattern of chemokine receptor expression were noted when NT2.N neurons were grown on a supporting layer of astrocytes, again consistent with similar patterns seen in primary human fetal brain cultures. Analysis of single-cell calcium transients revealed a robust response to stromal derived factor-1alpha (CXCR4) and melanocyte growth-stimulating activity (CXCR2), and variable response to monocyte chemoattractant protein-1 (CCR2) or interferon-gamma inducible protein-10 (CXCR3). Finally, we detected the release of monocyte chemoattractant protein-1 from pure cultures of NT2.N neurons, but not undifferentiated NT2 cells. These data indicate that individual neurons may not only co-express multiple functional chemokine receptors, but also that neurons themselves produce chemokines which may influence cellular function within the central nervous system.
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PMID:Expression of multiple functional chemokine receptors and monocyte chemoattractant protein-1 in human neurons. 1082 41

The full-length cDNAs of two different alpha-subunits (Kir6.1 and Kir6.2) and partial cDNAs of three different beta-subunits (SUR1, SUR2A and SUR2B) of ATP-sensitive potassium (KATP) channels of the guinea-pig (gp) were obtained by screening a cDNA library from the ventricle of guinea-pig heart. Cell-specific reverse-transcriptase PCR with gene-specific intron-spanning primers showed that gpKir6.1, gpKir6.2 and gpSUR2B were expressed in a purified fraction of capillary endothelial cells. In cardiomyocytes, gpKir6.1, gpKir6.2, gpSUR1 and gpSUR2A were detected. Patch-clamp measurements were carried out in isolated capillary fragments consisting of 3-15 endothelial cells. The membrane capacitance measured in the whole-cell mode was 19.9 +/- 1.0 pF and was independent of the length of the capillary fragment, which suggests that the endothelial cells were not electrically coupled under our experimental conditions. The perforated-patch technique was used to measure the steady-state current-voltage relation of capillary endothelial cells. Application of K+ channel openers (rilmakalim or diazoxide) or metabolic inhibition (250 microM 2,4-dinitrophenol plus 10 mM deoxyglucose) induced a current that reversed near the calculated K+ equilibrium potential. Rilmakalim (1 microM), diazoxide (300 microM) and metabolic inhibition increased the slope conductance measured at -55 mV by a factor of 9.0 (+/-1.8), 2.5 (+/-0.2) and 3.9 (+/-1.7), respectively. The effects were reversed by glibenclamide (1 microM). Our results suggest that capillary endothelial cells from guinea-pig heart express KATP channels composed of SUR2B and Kir6.1 and/or Kir6.2 subunits. The hyperpolarization elicited by the opening of KATP channels may lead to an increase in free cytosolic Ca2+, and thus modulate the synthesis of NO and the permeability of the capillary wall.
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PMID:ATP-sensitive potassium channels in capillaries isolated from guinea-pig heart. 1083 35

LALP70 is a novel lysosomal membrane protein belonging to the apyrase protein family. The apyrase protein family comprises enzymes capable of cleaving nucleotide tri- and diphosphates in a calcium- or magnesium-dependent manner, not being altered by P-type, F-type, or V-type NTPase inhibitors. In this study we have cloned and sequenced the human LALP70 gene to determine the genomic structure. The gene is organized in 11 introns and 12 exons covering a genomic region of approximately 16 kilobase pairs. By fluorescence in situ hybridization analysis, the hLALP70 gene was mapped to the human chromosome 8p21.1-p21.3. We further show that there is at least one alternatively spliced variant, hLALP70v, which can be generated via an alternative splice side at the 3'-end of exon 7, leading to a protein variant differing in 8 amino acids (VSFASSQQ). This is the first splice variant that has been described in the apyrase protein family. Reverse transcriptase polymerase chain reaction analysis showed an ubiquitous expression of both variants, with different relative mRNA expression levels in different tissues. Comparison of the enzymatic properties of the splice variants revealed a broader substrate specificity for hLALP70v with CTP, UDP, CDP, GTP, and GDP as preferred substrates, while hLALP70 utilized UTP and TTP preferentially. Furthermore, enzyme activity of hLALP70v was equally dependent on Ca(2+) and Mg(2+), being saturated already at 1 mm concentration. In contrast, hLALP70 enzymatic activity were unsaturated up to 10 mm Ca(2+), while Mg(2+) showed a saturation at already 1 mm concentration with 2-3-fold lower enzymatic activity as observed with Ca(2+). Our data suggest that the presence or absence of the 8-amino acid motif VSFASSQQ provoke differences in substrate specificity and divalent cation dependence of hLALP70/hLALP70v.
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PMID:First apyrase splice variants have different enzymatic properties. 1085 52


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