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P C McDonnell

Publications and source records attributed to P C McDonnell.

14 recordsLinked to original sources
PubMed2000

Identification and initial characterization of four novel members of the interleukin-1 family.

Interleukin-1 (IL-1), fibroblast growth factors (FGFs), and their homologues are secreted factors that share a common beta-barrel structure and act on target cells by binding to cell surface receptors with immunoglobulin-like folds in their extracellular domain. While numerous members of the FGF family have been discovered, the IL-1 family has remained small and outnumbered by IL-1 receptor homologues. From expressed sequence tag data base searches, we have now identified four additional IL-1 homologues, IL-1H1, IL-1H2, IL-1H3, and IL-1H4. Like most other IL-1/FGFs, these proteins do not contain a hydrophobic leader sequence. IL-1H4 has a propeptide sequence, while IL-1H1, IL-1H2, and IL-1H3 encode only the mature protein. Circular dichroism spectra and thermal stability analysis suggest that IL-1H1 folds similarly to IL-1ra. The novel homologues are not widely expressed in mammals. IL-1H1 is constitutively expressed only in placenta and the squamous epithelium of the esophagus. However, IL-1H1 could be induced in vitro in keratinocytes by interferon-gamma and tumor necrosis factor-alpha and in vivo via a contact hypersensitivity reaction or herpes simplex virus infection. This suggests that IL-1H1 may be involved in pathogenesis of immune mediated disease processes. The addition of four novel IL-1 homologues suggests that the IL-1 family is significantly larger than previously thought.

Amino Acid Sequence↗
PubMed1998

Herpesvirus entry mediator ligand (HVEM-L), a novel ligand for HVEM/TR2, stimulates proliferation of T cells and inhibits HT29 cell growth.

Herpesvirus entry mediator (HVEM), a member of the tumor necrosis factor (TNF) receptor family, mediates herpesvirus entry into cells during infection. Upon overexpression, HVEM activates NF-kappaB and AP-1 through a TNF receptor-associated factor (TRAF)-mediated mechanism. Using an HVEM-Fc fusion protein, we screened soluble forms of novel TNF-related proteins derived from an expressed sequence tag data base. One of these, which we designated HVEM-L, specifically bound to HVEM-Fc with an affinity of 44 nM. This association was confirmed with soluble and membrane forms of both receptor and ligand. HVEM-L mRNA is expressed in spleen, lymph nodes, macrophages, and T cells and encodes a 240-amino acid protein. A soluble, secreted form of the protein stimulates proliferation of T lymphocytes during allogeneic responses, inhibits HT-29 cell growth, and weakly stimulates NF-kappaB-dependent transcription.

Amino Acid Sequence↗
PubMed1997

Novel homologues of CSBP/p38 MAP kinase: activation, substrate specificity and sensitivity to inhibition by pyridinyl imidazoles.

A novel homologue of p38 MAP kinase, called SAPK4, has been cloned which shares 61% amino acid identity with p38 and is expressed predominantly in testes, pancreas and small intestine. We also cloned an alternative form of p38beta, termed p38beta2, which lacks the additional 8 amino acid insertion unique to p38beta. p38, p38beta, p38beta2, ERK6/p38gamma/SAPK3, and SAPK4 were characterized with respect to stimulus-dependent activation in transfected cells, substrate specificity, and sensitivity to inhibition by pyridinyl imidazoles. All homologues were stimulated, although to differing extents, by IL-1beta, TNF, sorbitol, and UV. Only SAPK3 and SAPK4 were stimulated significantly by PMA. p38beta showed the weakest activation overall. MBP, ATF-2, and both MAPKAP kinase-2 and kinase-3 were good substrates of p38 and p38beta in vitro. In contrast, only MBP, ATF2, and MAPKAP kinase-3 proved to be significant substrates of SAPK3 and SAPK4, and of these three, MAPKAP kinase-3 was by far the weakest. p38beta had very poor kinase activity for all substrates except MBP. While both p38 and p38beta2 were comparably inhibited by SB 203580 and SB 202190, neither SAPK3 nor SAPK4 were inhibited. p38beta was partially inhibited by both inhibitors. These data suggest that SAPK3 and SAPK4 form a distinct subset of the p38 MAP kinases with different expression pattern, response to stimuli, substrate specificity, and inhibitor sensitivity.

Activating Transcription Factor 2↗
PubMed1997

Pyridinyl imidazole inhibitors of p38 mitogen-activated protein kinase bind in the ATP site.

The site of action of a series of pyridinyl imidazole compounds that are selective inhibitors of p38 mitogen-activated protein kinase in vitro and block proinflammatory cytokine production in vivo has been determined. Using Edman sequencing, 125I-SB206718 was shown to cross-link to the nonphosphorylated Escherichia coli-expressed p38 kinase at Thr175, which is proximal to the ATP binding site. Titration calorimetric studies with E. coli-expressed p38 kinase showed that SB203580 bound with a stoichiometry of 1:1 and that binding was blocked by preincubation of p38 kinase with the ATP analogue, FSBA (5'-[p-(fluorosulfonyl)benzoyl]adenosine), which covalently modifies the ATP binding site. The intrinsic ATPase activity of the nonphosphorylated enzyme was inhibited by SB203580 with a Km of 9.6 mM. Kinetic studies of active, phosphorylated yeast-expressed p38 kinase using a peptide substrate showed that SB203580 was competitive with ATP with a Ki of 21 nM and that kinase inhibition correlated with binding and biological activity. Mutagenesis indicated that binding of 125I-SB206718 was dependent on the catalytic residues K53 and D168 in the ATP pocket. These findings indicate that the pyridinyl imidazoles act in vivo by inhibiting p38 kinase activity through competition with ATP and that their selectivity is probably determined by differences in nonconserved regions within or near the ATP binding pocket.

Adenosine↗
PubMed1997

Regulation of stress-induced cytokine production by pyridinylimidazoles; inhibition of CSBP kinase.

Members of three classes of pyridinylimidazoles bind with varying affinities to CSBP (p38) kinase which is a member of a stress-induced signal transduction pathway. Based upon SAR and protein homology modeling, the pharmacophore and three potential modes of binding to the enzyme are presented. For a subset of pyridinylimidazoles, binding is shown to correlate with inhibition of CSBP kinase activity, whereas no significant inhibition of PKA, PKC alpha and ERK kinase activity is observed.

Calcium-Calmodulin-Dependent Protein Kinases↗
PubMed1996

Activation of the HIV-1 long terminal repeat by cytokines and environmental stress requires an active CSBP/p38 MAP kinase.

The human immunodeficiency virus, type 1 (HIV-1) promoter is known to be activated by proinflammatory cytokines and UV light. These stimuli also activate various members of the mitogen-activated protein kinase family, including JNK/SAPK and CSBP/p38. In HeLa cells containing an integrated HIV-1 long terminal repeat (LTR) -driven reporter, we now show that the specific p38 inhibitor, SB203580, inhibits activation of the HIV-1 LTR by interleukin-1, tumor necrosis factor, UV light, and osmotic stress. Inhibition was 70-90% in all but the case of tumor necrosis factor stimulation, where inhibition was 50%. Each of these stimuli activated p38, which was inhibited by SB203580 in vitro and in vivo with an IC50 (between 0.1 and 1 microM) similar to that required to inhibit transcription. In contrast, SB203580 had no effect on JNK, which was also activated by these stimuli. The NFkappaB sites in the HIV-1 LTR were required for a response to cytokines but not to UV, and SB203580 remained capable of inhibiting UV activation in the absence of the NFkappaB sites. Studies in which SB203580 was added at different times relative to UV stimulation suggested that the critical p38-mediated phosphorylation event occurred between 2 and 4 h after UV treatment. These data indicate that p38 is required for HIV-1 LTR activation but that the action of p38 is delayed, presumably due to substrate unavailability or inaccessibility.

Calcium-Calmodulin-Dependent Protein Kinases↗
PubMed1996

Identification of mitogen-activated protein (MAP) kinase-activated protein kinase-3, a novel substrate of CSBP p38 MAP kinase.

CSBP p38 is a mitogen-activated protein kinase that is activated in response to stress, endotoxin, interleukin 1, and tumor necrosis factor. Using a catalytically inactive mutant (D168A) of human CSBP2 as the bait in a yeast two-hybrid screen, we have identified and cloned a novel kinase which shares approximately 70% amino acid identity to mitogen-activated protein kinase-activated protein kinase (MAPKAP kinase)-2, and thus was designated MAPKAP kinase-3. The binding of CSBP to MAPKAP kinase-3 was confirmed in vitro by the precipitation of epitope-tagged CSBP1, CSBP2, and CSBP2(D168A) and endogenous CSBP from mammalian cells by a bacterially expressed GST-MAPKAP kinase-3 fusion protein and in vivo by co-precipitation of the epitope-tagged proteins co-expressed in HeLa cells. MAPKAP kinase-3 was phosphorylated by both CSBP1 and CSBP2 and was then able to phosphorylate HSP27 in vitro. Treatment of HeLa cells with sorbitol or TNF resulted in activation of CSBP and MAPKAP kinase-3 and activation of MAPKAP kinase-3 could be blocked by preincubation of cells with SB203580, a specific inhibitor of CSBP kinase activity. These data suggest that MAPKAP kinase-3 is activated by stress and cytokines and is a novel substrate of CSBP both in vitro and in vivo.

Amino Acid Sequence↗
PubMed1995

Human mitogen-activated protein kinase CSBP1, but not CSBP2, complements a hog1 deletion in yeast.

CSBP1 and CSBP2 are human homologues of the Saccharomyces cerevisiae Hog1 mitogen-activated protein kinase which is required for growth in high osmolarity media. Expression of CSBP1, but not CSBP2, complemented a hog1 delta phenotype. A CSBP2 mutant (A34V) that complements hog1 delta was isolated and found to have approximately 3-fold lower kinase activity than the wild-type CSBP2. Further analysis revealed that both the kinase activity and tyrosine phosphorylation of CSBP1 and CSBP2 (A34V) is regulated by salt. In contrast, wild-type CSBP2 is constitutively active but dependent on the upstream kinase, Pbs2. Mutagenesis studies showed that reduction or elimination of CSBP2 kinase activity restores salt responsiveness as measured by tyrosine phosphorylation suggesting that too high a level of kinase activity can result in desensitization of the host cell and inability to grow in high salt.

Base Sequence↗
PubMed1994

Tumor necrosis factor-alpha expression in ischemic neurons.

BACKGROUND AND PURPOSE: Tumor necrosis factor-alpha (TNF-alpha) is a cytokine with diverse proinflammatory actions, including endothelial leukocyte adhesion molecule expression. Since leukocytes infiltrate into ischemic brain lesions, the present study was conducted to examine whether TNF-alpha messenger RNA (mRNA) and peptide are expressed in the brain after experimental focal stroke and before leukocyte accumulation. METHODS: TNF-alpha mRNA and protein expression were monitored in the ischemic and nonischemic cerebral cortex of rats after focal ischemia produced by permanent middle cerebral artery occlusion. The effect of TNF-alpha administered by microinjection into the brain cortex on leukocyte adherence to brain capillaries was also studied. RESULTS: Induction of TNF-alpha mRNA, normalized to a standard reference rat macrophage TNF-alpha mRNA, was detected as early as 1 hour after middle cerebral artery occlusion. TNF-alpha mRNA was elevated by 3 hours (29 +/- 6% versus 2 +/- 1% in sham-operated rats) only in the ischemic cortex, with peak expression at 12 hours (104 +/- 8%; P < .01). Five days after middle cerebral artery occlusion, TNF-alpha mRNA levels in ischemic cortex were still significantly elevated (38 +/- 5%; P < .05). Also, TNF-alpha mRNA expression was greater in the ischemic cortex of spontaneously hypertensive rats than in normotensive rats (P < .05). Double-labeling, immunohistochemical studies revealed the presence of TNF-alpha protein localized within nerve fibers in the evolving infarct at 6 and 12 hours after ischemia and further expression in the tissues immediately adjacent to the infarct 24 hours after ischemia. After 5 days, the neuronally localized peptide had diminished greatly, but macrophages located within the infarcted tissues were immunoreactive. Cortical microinjections of TNF-alpha (10 ng in 1 microL) produced a significant neutrophil adherence/accumulation in capillaries and small blood vessels 24 hours later. CONCLUSIONS: These results represent the first demonstration that focal cerebral ischemia in rats results in elevated TNF-alpha mRNA and protein in ischemic neurons. The neuronal expression of peptide appears to facilitate the infiltration of inflammatory cells that can further exacerbate tissue damage in cerebral ischemia and might contribute to increased sensitivity and risk in focal stroke.

Animals↗
PubMed1993

Cytokine-induced neutrophil chemoattractant mRNA expressed in cerebral ischemia.

Cytokine-induced neutrophil chemoattractant (CINC), originally identified as a chemoattractant in rat kidney epithelial cells, is related to human 'gro' and murine 'KC'. The proteins encoded by these genes belong to the chemokine alpha superfamily, most of which have neutrophil chemotactic activity. Since brain chemokines may play a significant role in neutrophil accumulation in cerebral ischemia which can contribute to the extent of tissue injury in stroke, we examined the expression of CINC mRNA in the cerebral cortex of rats subjected to focal cerebral ischemia induced by middle cerebral artery occlusion (MCAO). Significant CINC mRNA expression was observed in the ipsilateral (ischemic) cortex from 6 h (17.3 +/- 3.7%, n = 6, P < 0.05) to 24 h (32.1 +/- 3.7%, n = 5, P < 0.01) with a peak at 12 h (43.9 +/- 3.7%, n = 6, P < 0.01) after MCAO. Five days post-MCAO, CINC mRNA levels were no longer elevated. No significant CINC mRNA expression was observed in the contralateral (control) cortex. These studies suggest that message for the neutrophil chemoattractant CINC is induced early in brain tissue subjected to ischemia, and therefore supports the possibility that brain-derived chemokines support the infiltration of circulating inflammatory cells following focal stroke.

Animals↗
PubMed1993

Interleukin-1 beta mRNA expression in ischemic rat cortex.

BACKGROUND AND PURPOSE: Interleukin-1 beta is a proinflammatory cytokine produced by blood-borne and resident brain inflammatory cells. The present study was conducted to determine if interleukin-1 beta mRNA was produced in the brain of rats subjected to permanent focal ischemia. METHODS: Rat interleukin-1 beta cDNA, synthesized from stimulated rat peritoneal macrophage RNA by reverse transcription and polymerase chain reaction and cloned in plasmid Bluescript KS+, was used to evaluate the expression of interleukin-1 beta mRNA in cerebral cortex from spontaneously hypertensive rats and normotensive rats subjected to permanent middle cerebral artery occlusion. Interleukin-1 beta mRNA was quantified by Northern blot analysis and compared with rat macrophage RNA standard. To correct for gel loading, blots were also analyzed with cyclophilin cDNA, which encodes an abundant, conserved protein that was unchanged by the experimental conditions. RESULTS: Interleukin-1 beta mRNA produced in the ischemic zone was significantly increased from 6 hours to 120 hours, with a maximum of 211 +/- 24% of interleukin-1 beta reference standard, ie, 0.2 ng stimulated rat macrophage RNA, mRNA compared with the level in nonischemic cortices (4 +/- 2%) at 12 hours after ischemia (P < .01; n = 6). Interleukin-1 beta mRNA at 12 hours after ischemia was markedly elevated in hypertensive rats over levels found in two normotensive rat strains. Neurological deficits were also apparent only in the hypertensive rats. CONCLUSIONS: Brain interleukin-1 beta mRNA is elevated acutely after permanent focal ischemia and especially in hypertensive rats. These data suggest that this potent proinflammatory and procoagulant cytokine might have a role in brain damage following ischemia.

Animals↗
PubMed1992

Transcriptional activity of rel family proteins.

Our studies originally demonstrated that the v-rel oncoprotein repressed gene expression in chicken lymphoid cells, while it activated transcription in rodent fibroblasts. Here we report that the c-rel protein can activate expression of genes linked to kappa B motifs when low levels of endogenous kappa B-binding activity are present. In contrast v-rel, and to a lesser extent c-rel, inhibit NF-kappa B-mediated activation of the human immunodeficiency virus long terminal repeat (HIV LTR) in phorbol ester-stimulated HeLa cells. Competition assays show that v-rel competitively inhibits both NF-kappa B and c-rel-mediated transcriptional activation. Analysis of mutant HIV LTR-chloramphenicol acetyltransferase (CAT) constructs in which all Sp1 or both NF-kappa B elements have been deleted shows that NF-kappa B motifs are required for rel-mediated effects on gene expression. Transforming v-rel mutants compete efficiently with phorbol ester-activated kappa B factors, whereas a transformation-defective mutant of v-rel is impaired in this activity. Taken together, these results strengthen the hypothesis that v-rel functions as a dominant interfering member of rel family proteins. These results also suggest that the ability of v- and c-rel to activate or repress gene expression in specific cells may result from their capacity to compete with endogenous rel family proteins whose expression and/or activity are cell-specific.

Base Sequence↗
PubMedDate not supplied

A protein kinase involved in the regulation of inflammatory cytokine biosynthesis.

Production of interleukin-1 and tumour necrosis factor from stimulated human monocytes is inhibited by a new series of pyridinyl-imidazole compounds. Using radiolabelled and radio-photoaffinity-labelled chemical probes, the target of these compounds was identified as a pair of closely related mitogen-activated protein kinase homologues, termed CSBPs. Binding of the pyridinyl-imidazole compounds inhibited CSBP kinase activity and could be directly correlated with their ability to inhibit cytokine production, suggesting that the CSBPs are critical for cytokine production.

Amino Acid Sequence↗