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)

Human blood basophils selectively express Fc gamma RII (CDw32) among IgG receptor subtypes, but its functional role in allergic reactions remains unknown. Using the human basophilic leukemia cell line KU812F as a model system, we investigated cellular signaling events mediated through IgG receptor stimulation. KU812F cells express Fc gamma RII on their surface. mRNAs for both Fc gamma RIIA and IIB subtypes were detected by reverse transcriptase-PCR analysis. In this cell line, Fc gamma RII stimulation induced mobilization of free intracellular calcium and actin polymerization. Yet, no significant histamine release was observed, nor did blood basophils stimulated by anti-Fc gamma RII monoclonal antibody IV.3 and by a secondary antibody release histamine. These data indicate that Fc gamma RII stimulation induces cellular signaling events such as calcium mobilization in human basophils. However, these events do not lead to histamine release.
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PMID:Signal transduction by IgG receptors induces calcium mobilization, but not histamine release, in the human basophilic cell line KU812F. 852 47

Two cDNA clones homologous with human neuropeptide (NP) Y-Y1 receptor have been isolated from a mouse bone marrow cDNA library. One was thought to be the cognate of the human NPY-Y1 receptor, termed Y1 alpha receptor, and the other form, termed Y1 beta receptor, differed from the Y1 alpha receptor in the seventh transmembrane domain and C-terminal tail. Analysis of the mouse genomic DNA showed that both receptors originated from a single gene. The different peptide sequences of the Y1 beta receptor were encoded by separate exons, hence, these receptors were generated by differential RNA splicing. High affinity binding of [125I]NPY to each receptor expressed in Chinese hamster ovary (CHO) cells and sequestration of [125I]NPY after binding to each receptor were observed. In the CHO cells expressing the Y1 alpha receptor, intracellular Ca2+ increase, inhibition of forskolin-induced cAMP accumulation, and mitogen-activated protein kinase (MAPK) activation were observed by stimulation of NPY, and these responses were abolished by pretreatment with pertussis toxin. Since wortmannin completely inhibited NPY-elicited MAPK activation, we speculate that wortmannin-sensitive signaling molecule(s) such as phosphoinositide 3-kinase may lie between pertussis toxin-sensitive G-protein and MAPK. In contrast, these intracellular signals were not detected in CHO cells expressing the Y1 beta receptor. Northern blots and reverse transcriptase-polymerase chain reaction analyses indicated that the Y1 alpha receptor was highly expressed in the brain, heart, kidney, spleen, skeletal muscle, and lung, whereas the Y1 beta receptor mRNA was not detected in these tissues. However, the Y1 beta receptor was expressed in mouse embryonic developmental stage (7 and 11 days), bone marrow cells and several hematopoietic cell lines. These results suggest that the Y1 beta receptor is an embryonic and a bone marrow form of the NPY-Y1 receptor, which decreases in the expression during development and differentiation.
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PMID:Identification of two isoforms of mouse neuropeptide Y-Y1 receptor generated by alternative splicing. Isolation, genomic structure, and functional expression of the receptors. 853 Apr 15

Since mast cells and basophils are thought to play a central role in several types of cutaneous inflammatory and allergic reactions, and since interleukin-6 (IL-6) is an important mediator in these processes, we have studied the ability of the human mast cell line HMC-1, the human basophilic cell line KU812, and human skin mast cells to produce IL-6. All three cell types proved to be potent sources of this cytokine after appropriate stimulation. Transcription of IL-6 mRNA was first detectable 2 h after stimulation with the ester phorbol myristate acetate (PMA) and the calcium ionophore A23187 in both cell lines, as evidenced by semiquantitative reverse transcriptase polymerase chain reaction analysis. Whereas resting cells did not produce IL-6 protein, PMA/A23187-stimulated cells released immunoreactive and biologically active IL-6, as demonstrated and quantitated by enzyme-linked immunosorbent assay and by the use of TEPC 1033 cells, an IL-6-dependent murine plasmacytoma cell line. Stimulated KU812 cells secreted sevenfold more IL-6 (up to 15 ng/ml) than HMC-1 cells (up to 2.4 ng/ml). Immunoblotting of HMC-1- and KU812 cell-derived IL-6 revealed several IL-6 forms in the molecular weight range of 21 to 30 kDa. Immunoelectron microscopic studies of human skin biopsies provided evidence that unstimulated mast cells do not contain preformed IL-6 but accumulate IL-6 in cytoplasmic and extruded granules after IgE-dependent stimulation. These findings suggest that IL-6 secreted by human mast cells and basophils potentially contributes to allergic, other immunologically mediated and nonspecific inflammatory responses.
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PMID:Production of interleukin-6 by human mast cells and basophilic cells. 859 85

Animal studies and cell culture experiments demonstrated that posttranscriptional editing of the transcript of the GluR-2 gene, resulting in substitution of an arginine for glutamine in the second transmembrane region (TM II) of the expressed protein, is associated with a reduction in Ca2+ permeability of the receptor channel. Thus, disturbances in GluR-2 RNA editing with alteration of intracellular Ca2+ homeostasis could lead to neuronal dysfunction and even neuronal degeneration. The present study determined the proportions of edited and unedited GluR-2 RNA in the prefrontal cortex of brains from patients with Alzheimer's disease, in the striatum of brains from patients with Huntington's disease, and in the same areas of brains from age-matched schizophrenics and controls, by using reverse transcriptase-polymerase chain reaction, restriction endonuclease digestion, gel electrophoresis and scintillation radiometry. In the prefrontal cortex of controls, < 0.1% of all GluR-2 RNA molecules were unedited and > 99.9% were edited; in the prefrontal cortex both of schizophrenics and of Alzheimer's patients approximately 1.0% of all GluR-2 RNA molecules were unedited and 99% were edited. In the striatum of controls and of schizophrenics, approximately 0.5% of GluR-2 RNA molecules were unedited and 99.5% were edited; in the striatum of Huntington's patients nearly 5.0% of GluR-2 RNA was unedited. In the prefrontal white matter of controls, approximately 7.0% of GluR-2 RNA was unedited. In the normal human prefrontal cortex and striatum, the large majority of GluR-2 RNA molecules contains a CGG codon for arginine in the TMII coding region; this implies that the corresponding AMPA receptors have a low Ca2+ permeability, as previously demonstrated for the rat brain. The process of GluR-2 RNA editing is compromised in a region-specific manner in schizophrenia, in Alzheimer's disease and Huntington's Chorea although in each of these disorders there is still a large excess of edited GluR-2 RNA molecules. Disturbances of GluR-2 RNA editing leading to excessive Ca2+ permeability, may contribute to neuronal dysfunction in schizophrenia and to neuronal death in Alzheimer's disease and Huntington's disease.
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PMID:Editing for an AMPA receptor subunit RNA in prefrontal cortex and striatum in Alzheimer's disease, Huntington's disease and schizophrenia. 861 34

We have used reverse transcriptase-polymerase chain reaction to investigate the expression of ryanodine receptors in several excitable and nonexcitable cell types. Consistent with previous reports, we detected ryanodine receptor expression in brain, heart, and skeletal muscle. In addition, we detected ryanodine receptor expression in various other excitable cells including PC 12 and A7r5 cells. Several muscle cell lines (BC3H1, C2C12, L6, and Sol8) weakly expressed ryanodine receptor when undifferentiated but strongly expressed type 1 and type 3 ryanodine receptor isoforms when differentiated into a muscle phenotype. Only 2 (HeLa and LLC-PK1 cells) out of 11 nonexcitable cell types examined expressed ryanodine receptors. Expression of ryanodine receptors at the protein level in these cells was confirmed using [3H]ryanodine binding. We also investigated the function of ryanodine receptors in Ca2+ signaling in HeLa cells using single-cell Fura-2 imaging. Neither caffeine nor ryanodine caused a detectable elevation of cytoplasmic Ca2+ in single HeLa cells. However, ryanodine caused a significant decrease in the amplitude of Ca 2+ signals evoked by repetitive stimulation with ATP. These studies show that ryanodine receptors are expressed in some nonexcitable cell types and furthermore suggest that the ryanodine receptors may be involved in a subtle regulation of intracellular Ca2+ responses.
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PMID:Expression and function of ryanodine receptors in nonexcitable cells. 862 32

Organelles in the axoplasm from the squid giant axon move along exogenous actin filaments toward their barbed ends. An approximately 235-kDa protein, the only band recognized by a pan-myosin antibody in Western blots of isolated axoplasmic organelles, has been previously proposed to be a motor for these movements. Here, we purify this approximately 235-kDa protein (p235) from axoplasm and demonstrate that it is a myosin, because it is recognized by a pan-myosin antibody and has an actin-activated Mg-ATPase activity per mg of protein 40-fold higher than that of axoplasm. By low-angle rotary shadowing, p235 differs from myosin II and it does not form bipolar filaments in low salt. The amino acid sequence of a 17-kDa protein that copurifies with p235 shows that it is a squid optic lobe calcium-binding protein, which is more similar by amino acid sequence to calmodulin (69% identity) than to the light chains of myosin II (33% identity). A polyclonal antibody to this light chain was raised by using a synthetic peptide representing the calcium binding domain least similar to calmodulin. We then cloned this light chain by reverse transcriptase-PCR and showed that this antibody recognizes the bacterially expressed protein but not brain calmodulin. In Western blots of sucrose gradient fractions, the 17-kDa protein is found in the organelle fraction, suggesting that it is a light chain of the p235 myosin that is also associated with organelles.
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PMID:An axoplasmic myosin with a calmodulin-like light chain. 865 Feb 20

The highly regulated intracellular concentration of calcium (Ca2+) is a well-described regulator of diverse cellular events, including cell cycle control. In the present study we have addressed the regulation of cytosolic Ca2+ in differentiation events in the life cycle of the protozoan parasite Trypanosoma brucei. Bloodstream form (BSF) trypanosomes include the mitotically active long slender forms (LS) which differentiate to two nondividing stages--intermediate (INT) which transform into short stumpy (SS) forms. An axenic in vitro culture system was used to cultivate LS to a density greater than 1.0 x 10(6) cells/ml/day. Populations of the intermediate BSF (INT) and SS were derived from cultured LS by treatment with difluoromethyl ornithine (DFMO, 100 microM) for 2 and 4 days, respectively. A semiquantitative reverse transcriptase-coupled polymerase chain reaction protocol (SQ-RT-PCR) was developed to objectively distinguish the three BSF by monitoring the relative levels of stage-specific mRNAs--cytochrome oxidase II (COXII), variant surface glycoprotein, and procyclin during the differentiation of LS to SS, showing an increase in COXII and procyclin mRNA expression during this process of differentiation. Basal cytosolic Ca2+ levels [Ca2+]i of populations of LS, INT, and SS were studied using Indo-1 dual emission fluorometry. [Ca2+]i was maximal in dividing LS cells and was shown to decrease coincidentally with early events in the process of differentiation to INT and SS. Thapsigargin (1 microM), reported to cause the release of Ca2+ from the endoplasmic reticulum, elevated [Ca2+]i by about 30-60 nM in all BSF; however, the total thapsigargin-releasable stores decreased in parallel with the decrease in basal [Ca2+]i. Control treatments verified that elevations in [Ca2+]i in response to thapsigargin were intracellular in origin. These results may reflect the cessation of cytosolic Ca2+ transients involved in the regulation of mitosis as the parasite exits from the cell cycle and differentiates from rapidly dividing LS to the nondividing SS.
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PMID:Trypanosoma brucei: analysis of cytoplasmic Ca2+ during differentiation of bloodstream stages in vitro. 865 42

The effects of the trichothecene vomitoxin (VT) on the kinetics of cell proliferation and cytokine production were evaluated in murine CD4+ T cells. The CD4+ cultures were stimulated with phorbol 12-myristate 13-acetate (PMA) and ionomycin to activate protein kinase C and increase cytoplasmic free calcium, respectively, in a range of VT concentrations. Total and viable cell counts at 3, 5, 7, 9, and 11 days revealed delayed or impaired cell proliferation in cultures containing between 50 and 100 ng/ml VT, with complete inhibition being observed at 250 and 500 ng/ml of VT. The VT concentration required to inhibit protein synthesis in a 3-day culture by 50% in this model was 40 ng/ml. When enzyme-linked immunosorbent assay (ELISA) was used to quantitate cytokines, IL-2 levels in control cultures were highest at Day 1 and declined rapidly thereafter, whereas, in VT groups, IL-2 levels were highest at Day 3 and remained elevated up to 11 days. IL-2 levels were elevated by continuous exposure to 100-500 ng/ml of VT with more than 100-fold differences being observed between control and 250 ng/ml VT from Days 5 to 11. When IL-2 levels were expressed on a per viable cell basis, increases were even more marked with as much as 6 log differences being observed between the treatments at 250-500 ng/ml VT and control cultures at Day 7. Supernatant IL-4 and IL-5 levels were also elevated by 100 and 250 ng/ml VT in a dose- and time-dependent fashion compared to control cultures, whereas 500 ng/ml VT was inhibitory. When relative IL mRNA abundance was analyzed during the first 3 days of culture by reverse transcriptase-polymerase chain reaction (RT-PCR) in conjunction with Southern hybridization analysis, IL-2 mRNA levels in Days 1, 2 and 3 in cultures containing 100 and 250 ng/ml VT were greater than corresponding controls. IL-2 mRNA abundance in both control and VT-treated cultures was maximal at Day 1 and decreased rapidly thereafter in controls, whereas much slower rates of IL-2 disappearance were noted in 100 and 250 ng/ml of VT. IL-4 and IL-5 mRNA levels at VT doses of 50 and 100 ng/ml were also elevated compared to controls. Pulsed VT (8 to 48 hr) or cycloheximide (4 to 48 hr) exposure of CD4+ cells enhanced supernatant levels of IL-2 but not IL-4 upon incubation for 24 hr in fresh medium. This effect was not persistent. Taken together, VT enhanced and/or delayed peak IL-2, IL-4, and IL-5 gene expression and secretion in CD4+ T cells stimulated with PMA and ionomycin. Remarkably, cytokine superinduction occurred simultaneously with partial or maximal inhibition of cell proliferation.
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PMID:Vomitoxin-mediated IL-2, IL-4, and IL-5 superinduction in murine CD4+ T cells stimulated with phorbol ester calcium ionophore: relation to kinetics of proliferation. 865 34

The effects of the trichothecene vomitoxin (VT) on the kinetics of cell proliferation and cytokine production were evaluated in murine CD4(+) T cells. The CD4(+) cultures were stimulated with phorbol 12-myristate 13-acetate (PMA) and ionomycin to activate protein kinase C and increase cytoplasmic free calcium, respectively, in a range of VT concentrations. Total and viable cell counts at 3, 5, 7, 9, and 11 days revealed delayed or impaired cell proliferation in cultures containing between 50 and 100 ngsolidusml VT, with complete inhibition being observed at 250 and 500 ngsolidusml of VT. The VT concentration required to inhibit protein synthesis in a 3-day culture by 50% in this model was 40 ngsolidusml. When enzyme-linked immunosorbent assay (ELISA) was used to quantitate cytokines, IL-2 levels in control cultures were highest at Day 1 and declined rapidly thereafter, whereas, in VT groups, IL-2 levels were highest at Day 3 and remained elevated up to 11 days. IL-2 levels were elevated by continuous exposure to 100-500 ngsolidusml of VT with more than 100-fold differences being observed between control and 250 ngsolidusml VT from Days 5 to 11. When IL-2 levels were expressed on a per viable cell basis, increases were even more marked with as much as 6 log differences being observed between the treatments at 250-500 ngsolidusml VT and control cultures at Day 7. Supernatant IL-4 and IL-5 levels were also elevated by 100 and 250 ngsolidusml VT in a dose- and time-dependent fashion compared to control cultures, whereas 500 ngsolidusml VT was inhibitory. When relative IL mRNA abundance was analyzed during the first 3 days of culture by reverse transcriptase-polymerase chain reaction (RT-PCR) in conjunction with Southern hybridization analysis, IL-2 mRNA levels in Days 1, 2 and 3 in cultures containing 100 and 250 ngsolidusml VT were greater than corresponding controls. IL-2 mRNA abundance in both control and VT-treated cultures was maximal at Day 1 and decreased rapidly thereafter in controls, whereas much slower rates of IL-2 disappearance were noted in 100 and 250 ngsolidusml of VT. IL-4 and IL-5 mRNA levels at VT doses of 50 and 100 ngsolidusml were also elevated compared to controls. Pulsed VT (8 to 48 hr) or cycloheximide (4 to 48 hr) exposure of CD4(+) cells enhanced supernatant levels of IL-2 but not IL-4 upon incubation for 24 hr in fresh medium. This effect was not persistent. Taken together, VT enhanced andsolidusor delayed peak IL-2, IL-4, and IL-5 gene expression and secretion in CD4(+) T cells stimulated with PMA and ionomycin. Remarkably, cytokine superinduction occurred simultaneously with partial or maximal inhibition of cell proliferation.
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PMID:Vomitoxin-Mediated IL-2, IL-4, and IL-5 Superinduction in Murine CD4+ T Cells Stimulated with Phorbol Ester and Calcium Ionophore: Relation to Kinetics of Proliferation 866 68

To verify the hypothesis that eosinophils produce interleukin-2 (IL-2), a cytokine essential for lymphocyte activation, the expression of IL-2 was examined in peripheral blood eosinophils obtained from normal, atopic, asthmatic and hypereosinophilic subjects. Purified blood cell preparations were > 95% eosinophils, the remaining cells being neutrophils. Based on morphological observations and on CD3 expression, no lymphocytes were detected in these eosinophil preparations. The expression of IL-2 mRNA was detected by reverse transcriptase polymerase chain reaction (RT-PCR) in total RNA extracted from purified eosinophils stimulated with granulocyte-macrophage colony-stimulating factor (GM-CSF), with or without calcium ionophore (A23187). In-cell RT-PCR combined with in situ hybridization further confirmed that it was the eosinophils that expressed IL-2 mRNA. Moreover, in this experiment IL-2 mRNA expression increased upon costimulation with A23187 and GM-CSF suggesting that a steady-state level of IL-2 mRNA was inducible. Finally, IL-2 was detected in purified eosinophils by immunochemistry. These data, obtained by different techniques, demonstrate that eosinophils can express IL-2. An IL-2-mediated eosinophil-lymphocyte interaction could contribute to the chronic state of cell activation in inflamed tissues where these cells are implicated.
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PMID:Gene expression of interleukin-2 in purified human peripheral blood eosinophils. 866 27


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