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:3.1.26.9 (ribonuclease)
6,589 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Hammerhead ribozymes were used as substrates to examine endoribonucleolytic activities in cell extracts and cultured human cells. Primer-extension analyses showed that ribozymes directed against tumor necrosis factor-alpha mRNA and human immunodeficiency virus type 1 tat mRNA were cleaved at UA and CA dinucleotides by extracts. Preferred cleavage sites were similar to those observed following digestion with RNase A, and cleavage was blocked by RNasin, an inhibitor of pyrimidine-specific ribonucleases. Removal of UA and CA dinucleotides rendered ribozymes more stable when incubated in cell extracts that were not significantly contaminated by extracellular nucleases. Placement of UA dinucleotides adjacent to a ribozyme in mRNA led to excision of the ribozyme from long transcripts during incubation in extracts. UA dinucleotides also made mRNA more labile than a control RNA when expressed from an endogenous plasmid gene in the human myeloid cell line U937. Similarly, UA and CA dinucleotides caused ribozymes to have a shorter half-life when delivered to U937 cells by lipofectin-mediated transformation. Taken together, these data indicate that one or more members of the pyrimidine-specific ribonuclease family is involved in the intracellular degradation of RNA, and they explain the paucity of UA dinucleotides in eukaryotic mRNA. Judicious manipulation of preferred target sequences of pyrimidine-specific ribonucleases may be useful in designing effective hammerhead ribozymes.
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PMID:Degradation of hammerhead ribozymes by human ribonucleases. 964 39

We examined the potential of several epithelial-derived factors to enhance neutrophil activation and survival. Neutrophils incubated in the presence of supernatants from nasal-derived primary epithelial cultures had significantly increased survival compared with neutrophils cultured in media alone. Of the cytokines reported to enhance neutrophil survival, transcripts for interleukin (IL)-1alpha, IL-1beta, IL-6, and granulocyte macrophage colony-stimulating factor (GM-CSF) (but not interferon-gamma or granulocyte colony-stimulating factor [G-CSF]) were detected by ribonuclease protection assay in basal and tumor necrosis factor (TNF)-alpha- stimulated epithelial cells. Of the eicosanoid products that enhance neutrophil survival, platelet-activating factor and leukotriene B(4) were not detected in the supernatants, whereas prostaglandin E(2) (PGE(2)) was produced in modest amounts. The levels of IL-6, GM-CSF, and PGE(2) in epithelial supernatants were significantly increased after transient TNF-alpha stimulation. This induction was suppressed if dexamethasone (Dex) was added during TNF-alpha stimulation. Only IL-6, GM-CSF, and PGE(2) promoted neutrophil survival over the range of concentrations detected in the supernatants, and a combination of neutralizing antibodies to GM-CSF and IL-6 completely inhibited the enhanced neutrophil survival in epithelial supernatants. Both the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling technique and morphologic scoring of apoptotic neutrophils confirmed that epithelial supernatants, as well as purified IL-6, GM-CSF, and PGE(2) all delayed neutrophil apoptosis. Finally, the effects of Dex on neutrophil survival and on epithelial cytokine production were investigated. Dex independently prolonged neutrophil survival but suppressed epithelial production of survival-enhancing factors in a dose-dependent manner. The net effect of Dex appeared to favor neutrophil survival.
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PMID:Multiple epithelial cell-derived factors enhance neutrophil survival. Regulation by glucocorticoids and tumor necrosis factor-alpha. 1042 10

The purpose of this study was to determine if aberrant apoptosis plays a role in pathologic wound healing as manifested by hypertrophic scarring and keloid formation. Apoptosis has recently been found to participate in the transition between granulation tissue and the development of definitive scar. The question that remains to be answered is what stimuli initiate apoptosis during wound healing. Hitherto, regulatory factors and pathways involved have been largely undefined. We investigated heterogeneity among fibroblasts derived from normal skin and keloid scar, by examining apoptotic profiles and pathways for these cells. Quantitative analysis of apoptotic cells using an Annexin-V-FITC binding assay showed that normal skin fibroblast cultures were found to have a two-fold higher percentage of apoptotic cells than did keloid fibroblast cultures. To study apoptotic pathways and related death-associated genes, a ribonuclease protection assay was performed for fibroblasts exposed to anti-Fas antibody and tumor necrosis factor-alpha to activate the Fas/TNF receptor apoptotic pathway. Compared with normal skin fibroblasts, keloid fibroblasts exhibited decreased expression of apoptosis-associated genes.
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PMID:Expression of apoptosis-associated genes by human dermal scar fibroblasts. 1063 11

beta-Amyloid plaque deposition observed in brains from Alzheimer patients, might function as immune stimulus for glial/macrophages activation, which is supported by observations of activated microglia expressing interleukin (IL)-1beta and elevated IL-6 immunoreactivity in close proximity to amyloid plaques. To elucidate the mechanisms involved in beta-amyloid-mediated inflammation, transgenic mice (Tg2576) expressing high levels of the Swedish double mutation of human amyloid precursor protein and progressively developing typical beta-amyloid plaques in cortical brain regions including gliosis and astrocytosis, were examined for the expression pattern of a number of cytokines. Using ribonuclease protection assay, interleukin (IL)-1alpha,-beta, IL-1 receptor antagonist, IL-6, IL-10, IL-12, IL-18, interferon-gamma, and macrophage migration inhibitory factor (MIF) mRNA were not induced in a number of cortical areas of Tg2576 mice regardless of the postnatal ages studied ranging between 2 and 13 months. Using immunocytochemistry for IL-1alpha,beta, IL-6, tumor necrosis factor (TNF)-alpha, and macrophage chemotactic protein (MCP)-1, only IL-1beta was found to be induced in reactive astrocytes surrounding beta-amyloid deposits detected in 14-month-old Tg2576 mice. Using non-radioactive in situ hybridization glial fibrillary acidic protein (GFAP) mRNA was detected to be expressed by reactive astrocytes in close proximity to beta-amyloid plaques. The local immune response detected around cortical beta-amyloid deposits in transgenic Tg2576 mouse brain is seemingly different to that observed in brains from Alzheimer patients but may represent an initial event of chronic neuroinflammation at later stages of the disease.
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PMID:Induction of cytokines in glial cells surrounding cortical beta-amyloid plaques in transgenic Tg2576 mice with Alzheimer pathology. 1081 26

1. Elevated proinflammatory cytokines within the central nervous system (CNS) of individuals infected with human immunodeficiency virus (HIV) may contribute to altered CNS processes prior to the onset of AIDS. Most studies of HIV-induced alterations in cytokine expression within the CNS have focused on interleukin (IL)-1 and tumor necrosis factor (TNF). 2. We used a ribonuclease protection assay (RPA) to elucidate further the pattern of cytokine mRNA expression in the rat CNS in response to HIV envelope glycoprotein 160 (gp160). Male Sprague-Dawley rats were surgically implanted with a guide cannula directed into a lateral cerebral ventricle. HIV gp160 was injected intracerebroventricularly and rats were sacrificed immediately (time = 0) or at 1, 2, or 4 hr postinjection. Discrete brain regions were dissected, and peripheral glands removed. All tissues were frozen in liquid nitrogen until RNA extraction and assay. 3. IL-1beta IL-1alpha, TNF-alpha, and TNFbeta mRNAs were constitutively expressed in brain tissues. Central administration of gp160 dramatically increased mRNA expression for IL-1beta and TNFalpha in the hypothalamus, hippocampus, brainstem, and cerebellum. Furthermore, although mRNA expression for IL-5, IL-6, and IL-10 was never detected under basal conditions, these mRNAs were increased in brain tissue after administration of gp160. Peak expression in each brain region was detected 2 hr after administration. Multiple cytokine mRNAs were detected in peripheral tissues, but their expression was not altered by central administration of gp160. 4. Our results indicate that gp160 induces mRNA expression in brain for cytokines other than IL-1 and TNF. Screening for multiple cytokine mRNA in this manner provides extensive information concerning the particular cytokines that may be involved in HIV-induced pathologies and alterations in CNS processes.
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PMID:Human immunodeficiency virus glycoprotein 160 induces cytokine mRNA expression in the rat central nervous system. 1090 Dec 64

Endothelial synthesis of the C-C chemokine monocyte chemotactic protein-1 (MCP-1) has been implicated in the regulation of monocyte recruitment for extravascular pools under both physiologic and inflammatory conditions. We designed and characterized five antisense phosphorothioate oligodeoxynucleotides (PS-ODN) targeting MCP-1 secretion by human pulmonary artery endothelial cells (HPAEC) and pulmonary microvascular endothelial cells (HMVEC-L). The most effective PS-ODN (MCP-1 AS 2) dose-dependently suppressed the secretion of MCP-1 but not the secretion of the C-X-C chemokine interleukin-8 (IL-8) in both HPAEC and HMVEC-L in the nanomolar concentration range. Mismatch controls bearing 2 or 4 bp substitutions showed markedly reduced inhibitory capacity. MCP-1 mRNA levels were not affected even at the highest PS-ODN doses employed (ribonuclease protection assay), suggesting a translational arrest of MCP-1 production. Accordingly, PS-ODN exhibited no nonspecific side effects on immediate-early gene regulation of the transcription factor nuclear factor-kappaB (NF-kappaB), as analyzed by gel shift assays. Antisense pretreatment of HPAEC reduced the monocyte chemotactic bioactivity liberated from tumor necrosis factor-alpha (TNF-alpha)-activated endothelial cells (EC) and reduced the TNF-alpha-induced transendothelial monocyte migration. We conclude that nanomolar concentrations of specific antisense oligodeoxynucleotides effectively inhibit human endothelial MCP-1 synthesis and may thus provide a rational approach to modulate monocyte recruitment under inflammatory conditions.
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PMID:Antisense oligomers for selective suppression of MCP-1 synthesis in human pulmonary endothelial cells. 1090 55

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

Although 3':5' cyclic adenosine monophosphate (cAMP) is known to modulate cytokine production in a number of cell types, little information exists regarding cAMP-mediated effects on this synthetic function of human airway smooth-muscle (HASM) cells. We examined the effect of increasing intracellular cAMP concentration ([cAMP](i)) on tumor necrosis factor (TNF)-alpha-induced regulated on activation, normal T cells expressed and secreted (RANTES) and interleukin (IL)-6 secretion from cultured HASM cells. Pretreatment of HASM with prostaglandin (PG) E(2), forskolin, or dibutyryl cAMP inhibited TNF-alpha-induced RANTES secretion but increased TNF-alpha-induced IL-6 secretion. Moreover, stimulation with PGE(2), forskolin, or dibutyryl cAMP alone increased basal IL-6 secretion in a concentration-dependent manner. SB 207499, a specific phosphodiesterase type 4 inhibitor, augmented the inhibitory effects of PGE(2) and forskolin on TNF-alpha-induced RANTES. Collectively, these data demonstrate that increasing [cAMP](i) in HASM effectively increases IL-6 secretion but reduces RANTES secretion promoted by TNF-alpha. Reverse transcriptase/polymerase chain reaction and ribonuclease protection assays suggested that these opposite effects of increased [cAMP](i) on TNF-alpha- induced IL-6 and RANTES secretion may occur at the transcriptional level. Accordingly, we examined the effects of TNF- alpha and cAMP on the regulation of nuclear factor (NF)-kappaB, a transcription factor known to modulate cytokine synthesis in numerous cell types. Stimulation of HASM cells with TNF-alpha increased NF-kappaB DNA-binding activity. However, increased [cAMP](i) in HASM neither activated NF-kappaB nor altered TNF-alpha- induced NF-kappaB DNA-binding activity. These results were confirmed using a NF-kappaB-luciferase reporter assay. Together, our data suggest that TNF-alpha-induced IL-6 and RANTES secretion may be associated with NF-kappaB activation, and that inhibition of TNF-alpha-stimulated RANTES secretion and augmentation of IL-6 secretion by increased [cAMP](i) in HASM cells occurs via an NF-kappaB-independent mechanism.
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PMID:Tumor necrosis factor-alpha-induced secretion of RANTES and interleukin-6 from human airway smooth-muscle cells. Modulation by cyclic adenosine monophosphate. 1110 33

To evaluate the immunomodulatory effects of histamine in vivo, we analyzed an experimental syngenic tumor model using a colon adenocarcinoma cell line, CT-26, in Balb/c mice. In this model, distinct tumor growth was observed around 6 days after inoculation. Daily administration of cimetidine (0.12 mg/kg/day) significantly suppressed the increases in tumor volume and weight. On day 6 and day 7, histidine decarboxylase (HDC) activity was markedly increased. To examine the alterations in the local immune system, the cytokine expressions in the tumor tissue were measured by ribonuclease protection assay. The cytokine expression levels such as lymphotoxin-beta, tumor necrosis factor-alpha, interferon-gamma, interleukin-10, and interleukin-15 were considerably lower in tissues on day 14 than those on day 6. These decreased expressions were all restored by cimetidine. These results indicated that the effects of cimetidine on tumor growth in this model might be mediated by restoration of the decreased local cytokine expression, which exerts antitumoral effects.
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PMID:Effect of cimetidine on intratumoral cytokine expression in an experimental tumor. 1124 50

Symptoms of nasal, pharyngeal and ocular discomfort have been reported among workers in the wood surface-coating industry. Symptoms were reported more often by workers using ultraviolet radiation-curable acrylate coatings (UV coatings), which contain potential chemical sensitizers, than by those using acid-curing coatings. Furthermore, increased levels of eosinophil cationic protein (ECP) and albumin, but not tryptase, in nasal lavage from workers exposed to UV coatings have been observed. To further examine whether air contaminants present in the UV-coating industry are causing the observed increase in symptoms, the inflammatory process in the nasal mucosa of workers exposed to UV coatings was investigated. Clinical and biochemical endpoints were selected to distinguish between specific and non-specific hypersensitivity and to test the hypothesis that the symptoms were consistent with Type IV hypersensitivity. The nasal lavage and nasal biopsy were performed under local anesthetic at the workplace during working hours after a minimum of 2 h of work in both the exposed and control groups. Albumin and ECP, and the cytokines tumor necrosis factor-alpha (TNF-alpha) and interleukin-8 (IL-8), were used as inflammatory markers. A multi-probe ribonuclease protection assay was used to attempt to detect cytokine variation in human nasal biopsies. The cytokine genes analyzed were TNF-alpha, GM-CSF, interferon-gamma, IL-2, IL-4 and IL-5. L32 and GAPDH were used as control genes for mRNA expression levels. Mucosal inflammation symptoms correlated with increased levels of albumin, but not with increased levels of ECP, secreted proinflammatory cytokines or cytokine gene mRNA expression. We conclude that the symptoms are non-specific and do not correlate with occupational exposure to UV coatings under the conditions of this investigation.
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PMID:Absence of proinflammatory cytokine gene expression in nasal biopsies from wood surface-coating industry workers. 1167 74


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