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:3.1.27.1 (RNase)
16,360 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Protein phosphorylation is considered an early cellular mechanism of signal transduction by surface immunoglobulins (sIg) and other receptors of B cells. Using intact human peripheral blood B cells of young subjects labeled with orthophosphate, increased phosphorylation levels of serine/threonine and tyrosine substrates were demonstrated on indicator phosphoproteins corresponding to the CD20 isoforms and microtubule-associated protein 2 kinase after cross-linking sIg and costimulation with phorbol diesters. By contrast, stimulated B cells from certain elderly subjects displayed substantial alterations in the phosphorylation patterns of serine/threonine or tyrosine indicator phosphoproteins. Also, age-related impairments in sIg stimulated mobilization of cytosolic protein kinase C (PKC) enzymatic activity and in cytosolic calcium [Ca2+]i responses of B cells were observed with the altered phosphorylation reactions. Comparison of the substrate phosphorylation profiles to the proliferative responses of stimulated B cells from individual elderly subjects suggested a model of signal transduction in which differing stimuli have different dependencies on phosphorylation reactions. Diminished proliferative responses after sIg ligation coincided with decreased phosphorylations of either tyrosine or serine/threonine indicator substrates. However, the decreased proliferative responses of B cells from elderly subjects with substantial reductions of tyrosine phosphorylation after sIg ligation were enhanced by the direct stimulation of serine/threonine kinase activity with phorbol diesters or CD40 ligation. Experiments with kinase inhibitors evaluated the relative dependency of different B cell stimuli on tyrosine and serine/threonine phosphorylation reactions. The proliferative responses of normal B cells to sIg ligation were quite sensitive to the tyrosine kinase inhibitor genistein whereas those observed following costimulations with phorbol diesters or CD40 ligation were more resistant. However, treatment of B cells with H7, an inhibitor of PKC activity, led to a more uniform reduction of B-cell responses after different stimuli. Results from RNase protection assays of c-myc expression also suggested that different B-cell stimuli might utilize distinct intracellular signaling pathways. Both the type of stimuli and mode of sIg ligation were important in determining the stimulated levels of c-myc mRNA expression. Thus, the current findings suggest that age-related defects are present in human B cell signaling pathways as reflected by tyrosine and serine/threonine phosphorylation reactions. Also, these age-related defects can coexist with altered mobilization of PKC enzymatic activity and with alterations in [Ca2+]i and proliferative responses.
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PMID:Signal transduction in human B cells during aging: alterations in stimulus-induced phosphorylations of tyrosine and serine/threonine substrates and in cytosolic calcium responsiveness. 180 9

X-linked hyper IgM syndrome (XHIM) is a primary immunodeficiency disorder caused by mutations of the gene encoding CD40 ligand (CD40L). We correlated mutations of the CD40L gene, CD40L expression, and the clinical manifestations observed in XHIM patients from 30 families. The 28 unique mutations identified included 9 missense, 5 nonsense, 9 splice site mutations, and 5 deletions/insertions. In 4 of 9 splice site mutations, normally spliced and mutated mRNA transcripts were simultaneously expressed. RNase protection assay demonstrated that 5 of 17 mutations tested resulted in decreased levels of transcript. The effect of the mutations on CD40L expression by activated peripheral blood mononuclear cells (PBMC) and T-cell lines or clones was assessed using one polyclonal and four monoclonal antibodies and a CD40-Ig fusion protein. In most patients, the binding of at least one antibody but not of CD40-Ig was observed, suggesting nonfunctional CD40L. However, activated PBMC from three patients and activated T-cell lines from two additional patients, each with different genotype, bound CD40-Ig at low intensity, suggesting functional CD40L. Thus, failure of activated PBMC to bind CD40-Ig is not an absolute diagnostic hallmark of XHIM and molecular analysis of the CD40L gene may be required for the correct diagnosis. Patients with genotypes resulting in diminished expression of wild-type CD40L or mutant CD40L that can still bind CD40-Ig appear to have milder clinical consequences.
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PMID:Mutations of the CD40 ligand gene and its effect on CD40 ligand expression in patients with X-linked hyper IgM syndrome. 1048 40

Rapid advances in our ability to localize and quantify macromolecular changes in health and disease are being brought about by the availability of genetically altered animals (mutants), purified reagents such as monoclonal antibodies, and new molecular methods. Targeted gene deletion (knockouts) and gene insertions (transgenics) in animals can allow identification of the importance and function of macromolecules. Monoclonal antibodies and fluorescent labels coupled with advances in microscopy provide exacting and multi-dimensional information about localization and cellular changes in proteins, carbohydrates, and lipids using immunohistochemistry, fluorescent activated cell sorting, and immunoprecipitation. Similarly, new applications of molecular methods can be used to identify and localize nucleic acids in tissues via in situ hybridization, polymerase chain reaction (PCR), reverse transcription (RT) PCR, differential display RT-PCR, RNase protection assays, and microchip arrays. The ligand for CD40 (CD40L), an important immunoregulatory molecule, is an example of the successful application of mutants, monoclonal antibodies, and molecular methods to cloning and biological characterization of new molecules. CD40L knockout mice, monoclonal antibodies, and several molecular methods were used to identify mutations in CD40L as the genetic basis for hyper-IgM syndrome in humans, to provide new insights into the pathobiology of Pneumocystis carinii infection, and to evaluate CD40L for immunotherapy of tumors and opportunistic infections.
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PMID:Mechanisms of disease and injury: utilization of mutants, monoclonals, and molecular methods. 1036 85

To understand how the TNF receptor-associated factor 1 (TRAF1) is transcriptionally regulated, in vitro DNA binding assays, promoter-reporter gene assays, and RNase protection assays were performed with the human TRAF1 gene. Binding of NF-kappaB to three of five putative binding sites within the human TRAF1 promoter was found in electrophoretic mobility shift assay studies, and analysis of TRAF1 gene promoter luciferase constructs confirmed the functional importance of these elements. Moreover, triggering of TNF-R1, CD40, and the interleukin-1 receptor resulted in transcription of the TRAF1 gene, whereas receptors that are not activators or only poor activators of NF-kappaB in HeLa cells failed to show a significant TRAF1 induction. Because it has been shown that members of the TRAF family are involved in activation of NF-kappaB and the c-Jun N-terminal kinase (JNK) by the interleukin-1 receptor and members of the TNF receptor superfamily, a role of TRAF1 in receptor cross-talk and/or feedback regulation of activated receptor signaling complexes can be suggested. In fact, we found that TNF-induced activation of JNK is prolonged in transfectants overexpressing TRAF1, whereas overexpression of a deletion mutant of TRAF1 in which the N-terminal part had been replaced by the green fluorescent protein interfered with TNF-induced activation of NF-kappaB and JNK.
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PMID:The human tumor necrosis factor (TNF) receptor-associated factor 1 gene (TRAF1) is up-regulated by cytokines of the TNF ligand family and modulates TNF-induced activation of NF-kappaB and c-Jun N-terminal kinase. 1038 49

Imiquimod (R-837) and its more potent derivative (R-848) are imidazoquinolines that have adjuvant activity in cultured human mononuclear cells. Its mechanism of action on epidermal antigen-presenting cells is not known. The purpose of the present investigation was to determine whether imiquimod and R-848 affect human epidermal Langerhans' cells' (LC) in vitro maturation. Pulse incubations (6-16 h) of cultured unfractionated epidermal cells or highly enriched LC suspensions with either imiquimod or R-848 (0. 05-5.0 microg/ml of culture medium) reproducibly enhanced their ability to induce T-cell proliferation in a primary mixed lymphocyte reaction. There was a 30 to 300% increase in T-lymphocyte proliferation induced by either imiquimod- or R-848-treated LC when compared to control, untreated LC. IFN-gamma secretion by T-lymphocytes stimulated by imiquimod- or R-848-treated LC was increased compared to control, untreated LC. After a 6-h incubation, phenotypic analysis of control-, imiquimod-, or R-848-treated LC indicated that such antigen-presenting cells were in an "intermediate" state of maturation (CD1a(+), HLA-DR, DP, DQ(bright+), CD40(low+), CD86(high+), and CD80(low+)). RNase protection assays demonstrated that either imiquimod or R-848 treatments increased steady-state transcripts encoding for IL-12 p40, IL-1beta, TNF-alpha, and IL-1 receptor antagonist by LC. These data indicate that imiquimod and R-848 dissociate the functional maturation (cytokine-mediated) and phenotypic maturation of epidermal LC. These data warrant further exploration for the use of imidazoquinoline-treated LC or other DC subsets for processing and presentation of viral peptides to Th-lymphocytes as a novel vaccine strategy to induce protective antiviral responses.
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PMID:The imidazoquinolines, imiquimod and R-848, induce functional, but not phenotypic, maturation of human epidermal Langerhans' cells. 1060 86

CD40 activation is necessary for thymus-dependent humoral immune responses and rescuing both phenotypically immature WEHI-231 B lymphoma cells from B cell antigen receptor-induced cell death and germinal center B cells from spontaneous apoptosis. As some effects of CD40 are probably mediated by differences in gene expression, cDNA expression arrays and RNase protection assays were used to identify the anti-apoptotic Bcl-2 homolog A1 as a CD40-inducible gene in B cell lines and purified germinal center B cells. Sustained CD40-induced A1 upregulation correlated with CD40-mediated rescue of WEHI-231 cells from anti-IgM-induced apoptosis. Moreover, overexpression of A1 specifically protected WEHI-231 cells from anti-IgM-induced apoptosis but not cell death triggered by certain other stimuli.
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PMID:The CD40-inducible Bcl-2 family member A1 protects B cells from antigen receptor-mediated apoptosis. 1071 83

We previously reported an increased percentage of CD14+CD16++ monocytes in the peripheral blood of HIV-infected patients but the physiopathological role of this monocyte subset remains unclear. Cells with a CD14+CD16++ phenotype may be obtained in vitro by culturing human peripheral blood monocytes in the presence of GM-CSF, IL-4 and IL-10. In the present study, we compared the phenotypic and functional characteristics of monocytes-derived CD14+CD16++ cells with those of macrophages and dendritic cells. We show that the CD14+CD16++ cells express dendritic cell markers: CD40, CD80, CD86, HLA-DR, CD11b, CD11c, CD18, CD1a, and CD83. Using RNase protection assay, we demonstrate that CD14+CD16++ cell subset expresses a low ratio of IL-1beta/IL-1ra mRNA and expresses IL-6, MIP-1alpha, MIP-1beta, MCP-1, IL-8, RANTES and I-309 transcripts, similar to dendritic cells. CD14+CD16++ cells produce IL-12, MCP-1 and IL-8, as assessed by flow cytometry. Moreover, CD14+CD16++ cells pulsed with different recall antigens induce a potent autologous T cell proliferation. Altogether, these results provide evidence that CD14+CD16++ cells differentiated in vitro from peripheral blood monocytes exhibit dendritic cell characteristics.
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PMID:CD14+CD16++ cells derived in vitro from peripheral blood monocytes exhibit phenotypic and functional dendritic cell-like characteristics. 1094 Aug 76

This study addresses a mechanism by which lymphocytes may promote vascular endothelial growth factor (VEGF) expression and angiogenesis in immune inflammation. Resting human umbilical endothelial cells (HUVECs) were found to express low levels of VEGF messenger RNA (mRNA) by reverse transcription polymerase chain reaction and ribonuclease protection assay with little or no change in expression following activation by cytokines, including tumor necrosis factor-alpha, interleukin (IL)-1, interferon gamma, or IL-4. In contrast, treatment of HUVECs and monocytes with soluble CD40 ligand (sCD40L) resulted in a marked dose-dependent induction of VEGF mRNA (approximately 4-fold), which peaked between 1 and 5 hours post-stimulation. Transient transfection of HUVECs was performed with a luciferase reporter construct under the control of the human VEGF promoter. Treatment of transfected HUVECs with sCD40L was found to enhance luciferase activity (approximately 4-fold) compared with controls, similar to the relative fold induction in mRNA expression in parallel cultures. Thus, CD40-dependent VEGF expression was a result of transcriptional control mechanisms. Treatment of HUVECs with sCD40L was also found to function in vitro to promote growth and proliferation in a VEGF-dependent manner, and CD40-dependent HUVEC growth was comparable to that found following treatment with recombinant human VEGF. Furthermore, subcutaneous injection of sCD40L in severe combined immunodeficient and nude mice induced VEGF expression and marked angiogenesis in vivo. Taken together, these findings are consistent with a function for CD40L-CD40 interactions in VEGF-induced angiogenesis and define a mechanistic link between the immune response and angiogenesis. (Blood. 2000;96:3801-3808)
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PMID:Ligation of CD40 induces the expression of vascular endothelial growth factor by endothelial cells and monocytes and promotes angiogenesis in vivo. 1109 63

Murine bone marrow cultured with GM-CSF produced dendritic cells (DCs) expressing MHC class II (MHC-II) but little CD40, CD80, or CD86. Oligodeoxynucleotides (ODN) containing CpG motifs enhanced DC maturation, increased MHC-II expression, and induced high levels of CD40, CD80, and CD86. When added with Ag to DCs for 24 h, CpG ODN enhanced Ag processing, and the half-life of peptide:MHC-II complexes was increased. However, Ag processing was only transiently enhanced, and exposure of DCs to CpG ODN for 48 h blocked processing of hen egg lysozyme (HEL) to HEL(48-61):I-A(k) complexes. Processing of this epitope required newly synthesized MHC-II and was blocked by brefeldin A (BFA), suggesting that reduced MHC-II synthesis could explain decreased processing. Real-time quantitative PCR confirmed that CpG ODN decreased I-A(beta)(k) mRNA in DCs. In contrast, RNase(42-56):I-A(k) complexes were generated via a different processing mechanism that involved recycling MHC-II and was partially resistant to BFA. Processing of RNase(42-56):I-A(k) persisted, although at reduced levels, after CpG-induced maturation of DCs, and this residual processing by mature DCs was completely resistant to BFA. Changes in endocytosis, which was transiently enhanced and subsequently suppressed by CpG ODN, may affect Ag processing by both nascent and recycling MHC-II mechanisms. In summary, CpG ODN induce DC maturation, transiently increase Ag processing, and increase the half-life of peptide-MHC-II complexes to sustain subsequent presentation. Processing mechanisms that require nascent MHC-II are subsequently lost, but those that use recycling MHC-II persist even in fully mature DCs.
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PMID:CpG DNA induces maturation of dendritic cells with distinct effects on nascent and recycling MHC-II antigen-processing mechanisms. 1112 Aug 13

Leukocytes resident in the liver may play a role in immune responses. We describe a cell population propagated from mouse liver nonparenchymal cells in IL-3 and anti-CD40 mAb that exhibits a distinct surface immunophenotype and function in directing differentiation of naive allogeneic T cells. After culture, such cells are DEC-205(bright)B220+CD11c-CD19-, and negative for T (CD3, CD4, CD8alpha), NK (NK 1.1) cell markers, and myeloid Ags (CD11b, CD13, CD14). These liver-derived DEC205+B220+ CD19- cells have a morphology and migratory capacity similar to dendritic cells. Interestingly, they possess Ig gene rearrangements, but lack Ig molecule expression on the cell surface. They induce low thymidine uptake of allogeneic T cells in MLR due to extensive apoptosis of activated T cells. T cell proliferation is restored by addition of the common caspase inhibitor peptide, benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (zVAD-fmk). T cells stimulated by liver-derived DEC205+B220+D19- cells release both IL-10 and IFN-gamma, small amounts of TGF-beta, and no IL-2 or IL-4, a cytokine profile resembling T regulatory type 1 cells. Expression of IL-10 and IFN-gamma, but not bioactive IL-12 in liver DEC205+B220+CD19- cells was demonstrated by RNase protection assay. In vivo administration of liver DEC205+B220+CD19- cells significantly prolonged the survival of vascularized cardiac allografts in an alloantigen-specific manner.
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PMID:Liver-derived DEC205+B220+CD19- dendritic cells regulate T cell responses. 1139 Apr 48


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