Protein structure prediction: playing the fold.
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Computer Communication Networks↗
Biomedical subjects
J Park
Publications and source records attributed to J Park.
Effect of a nonsteroidal antiinflammatory drug on synovial fluid in osteoarthritis.
OBJECTIVE: The use of nonsteroidal antiinflammatory drug (NSAID) therapy in osteoarthritis (OA) is controversial because of suggestions that pure analgesics can be as effective as NSAID for pain relief. In addition, there is incomplete information whether antiinflammatory effects have any longterm benefit in OA. NSAID have been known to affect synovial fluid (SF) prostaglandins in rheumatoid arthritis. We describe the first examination of the effect of an NSAID, etodolac, on SF prostaglandins, cytokines, and cells in OA. METHODS: Joint fluids were studied before and 2 weeks after initiation of therapy with etodolac 400 mg tid. Leukocyte counts, prostaglandin, interleukin 6, and tumor necrosis factor were measured. RESULTS: Pretreatment features of SF did not predict clinical response. We found no change in the relatively low leukocyte counts. However, SF prostaglandin levels and interleukin 6 levels were significantly decreased and tumor necrosis factor alpha levels were increased after therapy with NSAID. CONCLUSION: This NSAID had potentially important local effects that could be either beneficial or deleterious. Further studies on effects of this and other NSAID on a broader variety of SF and synovial cytokines may help predict longterm effects of NSAID on progression of OA.
Anti-Inflammatory Agents, Non-Steroidal↗
Analysis of phosphorylation of tau with antibodies specific for phosphorylation sites.
Previously, we determined sites of tau protein phosphorylation by tau protein kinase (TPK) I/glycogen synthase kinase 3 beta (GSK-3 beta) and TPKII/(cyclin-dependent kinase 5 (CDK5) + p23). We prepared antibodies specific for these sites of tau phosphorylated by TPKI and TPKII, using chemically synthesized phosphopeptides as antigens. Each antibody specifically reacts with each phosphorylation site. With these antibodies, it was confirmed that TPKI and TPKII are responsible for these phosphorylation sites, as reported previously, except that Ser404 is also weakly phosphorylated by TPKI alone. It was also observed that TPKII-phosphorylation enhances TPKI-phosphorylation. These results indicate that these antibodies are useful tools for investigation of the phosphorylation of tau by TPKI and TPKII.
Amino Acid Sequence↗
Immunosuppressive drugs prevent a rapid dephosphorylation of transcription factor NFAT1 in stimulated immune cells.
The immunosuppressive drugs cyclosporin A and FK506 interfere with the inducible transcription of cytokine genes in T cells and in other immune cells, in part by preventing the activation of NF-AT (nuclear factor of activated T cells). We show that transcription factor NFAT1 in T cells is rapidly dephosphorylated on stimulation, that dephosphorylation occurs before translocation of NFAT1 into the cell nucleus, and that dephosphorylation increases the affinity of NFAT1 for its specific sites in DNA. Cyclosporin A prevents the dephosphorylation and the nuclear translocation of NFAT1 in T cells, B cells, macrophages, and mast cells, delineating at least one mechanism that contributes to the profound immunosuppressive effects of this compound.
Animals↗
Phosphorylation of the transcription factor NFATp inhibits its DNA binding activity in cyclosporin A-treated human B and T cells.
Cyclosporin A (CsA) exerts its immunosuppressive effect by inhibiting the activity of nuclear factor of activated T cells (NFAT), thus preventing transcriptional induction of several cytokine genes. This effect is thought to be largely mediated through inactivation of the phosphatase calcineurin, which in turn inhibits translocation of an NFAT component to the nucleus. Here we report that CsA treatment of Raji B and Jurkat T cell lines yields a phosphorylated form of NFATp that is inhibited in DNA-binding and in its ability to form an NFAT complex with Fos and Jun. Immunoblot analyses and metabolic labeling with [32P]orthophosphate show that CsA alters NFATp migration on SDS-polyacrylamide gel electrophoresis by increasing its phosphorylation level without affecting subcellular distribution. Dephosphorylation by in vitro treatment with calcineurin or alkaline phosphatase restores NFATp DNA binding activity and its ability to reconstitute an NFAT complex with Fos and Jun proteins. These data point to a new mechanism for CsA-sensitive regulation of NFATp in which dephosphorylation is critical for DNA binding.
Alkaline Phosphatase↗
Human immunodeficiency virus-type 1 replication can be increased in peripheral blood of seropositive patients after influenza vaccination.
Despite considerable evidence that cell activation enhances human immunodeficiency virus-type 1 (HIV-1) replication in vitro, there is very little data on the role of immune activation on in vivo HIV-1 replication. In this study, we examined the effect of influenza vaccination on HIV-1 replication in the peripheral blood of 20 study subjects, and in 14 control subjects who did not receive influenza vaccination. Blood was obtained from each subject on three occasions during the month before vaccination and again on three occasions during the following month. Over the study period, there was little change in levels of proviral DNA in peripheral blood mononuclear cells (PBMCs). However, peak PBMC viral RNA levels after influenza vaccination were significantly increased over the mean of prevaccination values. This change was not observed to the same extent in unvaccinated controls. Therefore, this is the first report showing that HIV-1 replication can increase in temporal association with influenza vaccination. Our results suggest that continued immunologic (antigenic) stimulation may result in increased virus load in vivo. To address the appropriateness of influenza vaccination in HIV-infected patients, expanded studies will be required to examine specific and generalized immune responses to vaccination, and differences in patient response based on disease stage.
Adult↗
Partial spectral weights of disordered CuxPd1-x alloys including the photoemission matrix-element effect.
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Journal Article↗
Cloning and functional expression of a rat kidney extracellular calcium/polyvalent cation-sensing receptor.
The maintenance of a stable extracellular concentration of ionized calcium depends on the integrated function of a number of specialized cells (e.g., parathyroid and certain kidney epithelial cells). We recently identified another G protein-coupled receptor (BoPCaRI) from bovine parathyroid that responds to changes in extracellular Ca2+ within the millimolar range and provides a key mechanism for regulating the secretion of parathyroid hormone. Using an homology-based strategy, we now report the isolation of a cDNA encoding an extracellular Ca2+/polyvalent cation-sensing receptor (RaKCaR) from rat kidney. The predicted RaKCaR protein shares 92% identity with BoPCaR1 receptor and features a seven membrane-spanning domain, characteristic of the G protein-coupled receptors, which is preceded by a large hydrophilic extracellular NH2 terminus believed to be involved in cation binding. RaKCaR cRNA-injected Xenopus oocytes responded to extracellular Ca2+, Mg2+, Gd3+, and neomycin with characteristic activation of inositol phospholipid-dependent, intracellular Ca(2+)-induced Cl- currents. In rat kidney, Northern analysis revealed RaKCaR transcripts of 4 and 7 kb, and in situ hybridization showed localization primarily in outer medulla and cortical medullary rays. Our results provide important insights into the molecular structure of an extracellular Ca2+/polyvalent cation-sensing receptor in rat kidney and provide another basis on which to understand the role of extracellular divalent cations in regulating kidney function in mineral metabolism.
Amino Acid Sequence↗
Fold recognition and ab initio structure predictions using hidden Markov models and beta-strand pair potentials.
Protein structure predictions were submitted for 9 of the target sequences in the competition that ran during 1994. Targets sequences were selected that had no known homology with any sequence of known structure and were members of a reasonably sized family of related but divergent sequences. The objective was either to recognize a compatible fold for the target sequence in the database of known structures or to predict ab initio its rough 3D topology. The main tools used were Hidden Markov models (HMM) for fold recognition, a beta-strand pair potential to predict beta-sheet topology, and the PHD server for secondary structure prediction. Compatible folds were correctly identified in a number of cases and the beta-strand pair potential was shown to be a useful tool for ab initio topology prediction.
Algorithms↗
Neurotoxicity of acute glutamate transport blockade depends on coactivation of both NMDA and AMPA/Kainate receptors in organotypic hippocampal cultures.
Excessive activation of glutamate receptors is neurotoxic, contributing to brain injury caused by cerebral ischemia. The pharmacology of glutamate neurotoxicity is difficult to study in animals because it is efficiently cleared from the extracellular space by a family of glutamate transporters. We have investigated the receptor specificity of endogenous glutamate's toxic effects in organotypic cultures of the hippocampus by acute blockade of these transporters. The organotypic cultures used in these transporters. The organotypic cultures used in these experiments preserve the intrinsic connections and regional differentiation of the hippocampus in long term culture and may more closely reproduce the pharmacology of the mature brain region. Membrane injury was measured with digital fluorescence imaging of the vital dye, propidium iodide, 24 h after a 30-min exposure to glutamate receptor agonists or to antagonists of glutamate transport. Confirming our previous results, bath-applied, exogenous glutamate caused dose-dependent neuronal injury. Glutamate was less potent than the selective agonists NMDA, AMPA, and quisqualate. Blockade of glutamate transport with the selective antagonists threo-hydroxy-aspartate and pyrrolidine-dicarboxylic acid also caused dose-dependent neuronal injury. Endogenous or exogenous glutamate toxicity was caused by a coactivation of both NMDA and AMPA/kainate receptors; blockade of either was sufficient to substantially prevent neuronal injury. Protective effects of combined application of antagonists were generally less than additive. We conclude that AMPA/kainate receptors play a more prominent role in glutamate neurotoxicity in organotypic cultures than in dissociated cortical or hippocampal cultures, acting together with NMDA receptors to cause neuronal injury.
ATP-Binding Cassette Transporters↗
Navigator: tools for informal structure-activity relationship discovery.
Navigator is a molecular database visualization system, designed to support exploratory data analysis and informal structure-activity relationship studies. In addition to the operations commonly found in chemical database systems, it provides new tools that facilitate substituent analysis and help elucidate the relationships among similar molecules and between related assays. Navigator's capabilities include two ways of displaying the relationships between analogs, mouse-sensitive charts of sets of molecules, mouse-sensitive plots of assay relationships, and access to a system for three-dimensional quantitative structure-activity relationship discovery. Navigator's mouse-based user interface provides a one-object/one-window paradigm that makes data manipulation easy even for inexperienced users. Navigator runs on Silicon Graphics workstations.
Computer Graphics↗
Detection of water proximity to tryptophan residues in proteins by single photon radioluminescence.
We recently developed a single photon radioluminescence (SPR) technique to measure submicroscopic distances in biological samples [Bicknese et al., and Shahrokh et al., Biophys. J., 63 (1992) 1256-1279]. SPR arises from the excitation of a fluorophore by the energy deposited from a slowing beta decay electron. The purpose of this study was to detect 3H2O molecules near tryptophan residues in proteins by tryptophan SPR. To detect small SPR signals, a sample compartment with reflective ellipsoidal optics was constructed, and amplified signals from a cooled photomultiplier were resolved by pulse-height analysis. A Monte Carlo calculation was carried out to quantify the relationship between SPR signal and 3H2O-tryptophan proximity. Measurements of tryptophan SPR were made on aqueous tryptophan; dissolved melittin (containing a single tryptophan); native and denatured aldolase; dissolved aldolase, monellin, and human serum albumin; and the integral membrane proteins CHIP28 (containing a putative aqueous pore) and MIP26 using 3H2O or the aqueous-phase probe 3H-3-O-methylglucose (OMG). After subtraction of a Bremsstrahlung background signal, the SPR signal from aqueous tryptophan (cps.microCi-1 mumol-1 +/- SE) was 8.6 +/- 0.2 with 3H2O and 7.8 +/- 0.3 with 3HOMG (n = 8). With 3H2O as donor, the SPR signal (cps.microCi-1 mumol-1) was 9.0 +/- 0.3 for monomeric melittin in low salt (trytophan exposed) and 4.6 +/- 0.8 (n = 9) for tetrameric melittin in high salt (tryptophans buried away from aqueous solution). The ratio of SPR signal obtained for aldolase under denaturing conditions of 8 M urea (fluorophores exposed) versus non-denaturing buffer (fluorophores buried) was 1.53 +/- 0.07 (n = 6). Ratios of SPR signals normalized to fluorescence intensities for monellin, aldolase, and human serum albumin, relative to that for d-tryptophan, were 1.42, 1.09, and 1.04, indicating that the cross-section for excitation of fluorophores in proteins is greater than that for tryptophan in solution. For the CHIP28 and MIP26 proteins in membranes, the ratio of SPR signal obtained with 3H2O versus 3HOMG was 1.35 +/- 0.13 (CHIP28, n = 5) and 0.99 +/- 0.02 (MIP26). These data are consistent with the existence of an aqueous channel through CHIP28 that excludes small solutes. We conclude that tryptophan radioluminescence in proteins is measurable and provides unique information about the presence of local aqueous compartments.
Evaluation Studies as Topic↗
Localization of the epitope in methamphetamine and its antibody use for the detection of methamphetamine and benzphetamine by polarization fluoroimmunoassay.
An antibody was prepared, using a four carbon-bridged methamphetamine molecule as an immunogen in order to develop a polarization fluoroimmunoassay for urine screening of methamphetamine and benzphetamine. Also, its binding characteristics were investigated to locate epitope sites of methamphetamine. The study showed that the antibody was highly capable of eliciting a polarization fluoroimmunoassay response. However, the detection limit was much greater for benzphetamine (0.05 ppm) than for methamphetamine (0.2 ppm) and weakly antibody binding was found with methamphetamine. This difference in sensitivity may reflect the similarity of benzphetamine to the immunogen used to produce the antibody. Both benzphetamine and the immunogen have a tertiary amine attached to a carbon bridges whereas methamphetamine has only a secondary amine and amphetamine has a primary amine group. The difference of cross-reactivity data between phenylethylamine drugs and beta-hydroxyl phenylethylamine drugs indicates that the beta-carbon position have a major influence on the antibody interaction. Thus, the substitution of hydroxyl group on beta-carbon resulted in virtually no antibody affinity, even if a tertiary amine or secondary amine group was present in the molecule. This suggests that the beta-carbon chain plays a primary role as the epitope site with cooperative binding site of tertiary amine or secondary amine in alpha-carbon position. A hydroxyl group at the beta-carbon position plays an important inhibitory role to the antibody binding.
Antibody Affinity↗
8-Methoxypsoralen photoadduct formation in complementary oligonucleotides containing a cross-linkable site.
The complete profile of 8-methoxypsoralen photoadduct formation in complementary oligonucleotides (5'-GAGTATGAG and 5'-CATAC) has been determined. Equimolar solutions of the oligonucleotides were irradiated at 4 degrees C in order to stabilize the mini-double helix. Photomodified oligonucleotides were separated by reversed phase chromatography on a Vydac C4 column. Photoadduct formation favored the 5'TAT site in the 9mer over the 5'ATA site in the 5mer by a factor of two. Split-dose studies showed that the monoadducts formed on GAGTATGAG were preferentially converted to cross-links by an additional UVA exposure.
Adenine↗
Role of Ca2+ and calmodulin in ehrlichial infection in macrophages.
Replication of Ehrlichia risticii was inhibited in P388D1 cells and murine peritoneal macrophages when a calmodulin antagonist (W-7, chlorpromazine, or trifluoperazine); a Ca2+ channel blocker (verapamil, diltiazem, nifedipine, or flunarizine); an extracellular Ca2+ chelator, EGTA [ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid]; an inhibitor of intracellular Ca2+ mobilization, TMB-8; or Ca2+ ionophore A23187 was added after internalization of the organism at 3 h postincubation. When intracellular ehrlichiae at their logarithmic stage of growth were treated with these reagents, not only was further proliferation prevented but also there was significant reduction in numbers of intracellular ehrlichiae. These reagents prevented spreading of E. risticii from P388D1 cells to THP-1 cells. None of these reagents prevented binding of [35S]methionine-labeled E. risticii to P388D1 cells, but all of these reagents prevented internalization of [35S]methionine-labeled E. risticii. Protein kinase C inhibitors, H-7 and staurosporin, had no effect. 14CO2 production from L-[14C]glutamine in Percoll-density-gradient-purified E. risticii was inhibited by A23187 but not by W-7 or verapamil, suggesting that Ca2+ but not calmodulin directly regulates ehrlichials glutamine oxidation. Pretreatment of E. risticii with W-7 or verapamil did not reduce its infectivity. These results indicate that calmodulin and Ca2+ are essential for ehrlichial internalization, replication, and spreading in macrophages but are not essential for binding.
Animals↗
Src activity increases and Yes activity decreases during mitosis of human colon carcinoma cells.
Src and Yes protein-tyrosine kinase activities are elevated in malignant and premalignant tumors of the colon. To determine whether Src activity is elevated throughout the human colon carcinoma cell cycle as it is in polyomavirus middle T antigen- or F527 Src-transformed cells, and whether Yes activity, which is lower than that of Src in the carcinoma cells, is regulated differently, we measured their activities in cycling cells. We observed that the activities of both kinases were higher throughout all phases of the HT-29 colon carcinoma cell cycle than in corresponding phases of the fibroblast cycle. In addition, during mitosis of HT-29 cells, Src specific activity increased two- to threefold more, while Yes activity and abundance decreased threefold. The decreased steady-state protein levels of Yes during mitosis appeared to be due to both decreased synthesis and increased degradation of the protein. Inhibition of tyrosine but not serine/threonine phosphatases abolished the mitotic activation of Src. Mitotic Src was phosphorylated at novel serine and threonine sites and dephosphorylated at Tyr-527. Two cellular proteins (p160 and p180) were phosphorylated on tyrosine only during mitosis. Tyrosine phosphorylation of several other proteins decreased during mitosis. Thus, Src in HT-29 colon carcinoma cells, similar to Src complexed to polyomavirus middle T antigen or activated by mutation at Tyr-527, is highly active in all phases of the cell cycle. Moreover, Src activity further increases during mitosis, whereas Yes activity and abundance decrease. Thus, Src and Yes appear to be regulated differently during mitosis of HT-29 colon carcinoma cells.
Aphidicolin↗
Muscarinic M3 receptor-mediated release of gastrin from canine antral G cells in primary culture.
The functional role of the cholinergic nervous system in regulating gastrin release was investigated using enriched canine antral G cells. Gastrin content was 30.1 +/- 2.9 pmol per well and basal gastrin release was 900 +/- 27 fmol per well (n = 45). Carbachol (10(-8) to 10(-5) M) dose-dependently stimulated gastrin release with a maximal stimulatory response achieved at a concentration of 10(-5) M (330% over basal). To characterize the muscarinic receptor which mediates gastrin release from antral G cells, we examined the effect of three muscarinic receptor antagonists on carbachol-stimulated gastrin release; atropine (nonselective muscarinic receptor antagonist), pirenzepine (M1 muscarinic receptor antagonist) and 4-DAMP (M3 muscarinic receptor antagonist). Atropine (10(-9) to 10(-6) M), pirenzepine (10(-8) to 10(-5) M) and 4-DAMP (10(-9) to 10(-6) M) had no effect on the basal gastrin release. However, carbachol (10(-5) M)-stimulated gastrin release was effectively inhibited by atropine and 4-DAMP with Ki values of 0.48 and 0.66 nM, respectively. Pirenzepine at a high concentration (10(-5) M) also inhibited carbachol-stimulated gastrin release with a Ki value of 46.3 nM. These results suggest that the cholinergic nervous system directly stimulates gastrin release via M3 muscarinic receptors located on antral G cells.
Animals↗
Influence of Helicobacter pylori colonization on histological grading of chronic gastritis in Korean patients with peptic ulcer.
OBJECTIVES: We conducted an analysis of correlation between histological grading of chronic gastritis and the presence of H. pylori infection to investigate if H. pylori influences histological severity of chronic gastritis in Korean patients with peptic ulcers. METHODS: Gastroscopic antral biopsy specimens and peripheral venous blood were taken from 80 patients with gastric or duodenal ulcers. H. pylori was identified microscopically in sections with Giemsa staining and quantitative grading of cultured H. pylori was reported on a scale 0 to 3. The histopathological features of biopsy specimens were reported according to the Sydney classification of chronic gastritis. Serum gastrin and pepsinogen concentrations were measured by radioimmunoassay. RESULTS: H. pylori was identified in 62.5%(20 of 32 GU, 30 of 48 DU) of the study group. Gastric clonization rate of H. pylori did not increased with age. Forty of 50 biopsy specimens with H. pylori and also 23 of 30 biopsy specimens without H. pylori showed active chronic gastritis. There was no significant correlation overall between the presence of H. pylori and histological grading of chronic gastritis, including activity, and also no association was found between the quantitative grading of H. pylori and the histological grading of chronic gastritis. With and without H. pylori, a mean of serum gastrin concentration(79.4 +/- 4.30 pg/ml and 80.2 +/- 31.9 pg/ml)showed no significant difference, but a mean of serum pepsinogen concentration(87.7 +/- 41.6 ng/ml and 119 +/- 34.4 ng/ml) showed significant difference between the populations with and without H. pylori(p = 0.001). CONCLUSIONS: The influence of H. pylori on histological grading of chronic gastritis in Korean is less than that in prior studies of Western countries, and further investigation of pathogenesis of H. pylori in chronic gastritis and peptic ulceration is necessary.
Adolescent↗