Histamine release from mast cells of EAE rats by Gi protein-dependent and IgE-dependent pathways.

We investigated both Gi protein-dependent and IgE-dependent pathways that control release of histamine by PMCs derived from EAE or Complete Freund's Adjuvant (CFA) immunized rats. The number and histamine content of MCs per rat were the same between normal and EAE rats. Activation of Gi pathway by substance P (SP), DSA, 48/80, and mastoparan resulted in a dose-dependent increase in release of histamine by PMCs in normal, EAE-, and CFA-immunized rats.

Cleavage of porcine high molecular weight kininogen by the action of porcine pancreatic kallikrein.

Pancreatic kallikrein (KK), converts the single chain high molecular weight kininogen (HK) into a two-chain molecule accompanied by kinin release. Further degradation was not observed. Molecular weights of the heavy (H)-chain and light (L)-chain were estimated to be 61 kDa and 56 kDa, respectively, by SDS-PAGE.

Determination of progesterone and 17-hydroxyprogesterone by high performance liquid chromatography after pre-column derivatization with 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionohydra zide.

Progesterone, 17-hydroxyprogesterone and four other 3-keto steroids were determined by high performance liquid chromatography with fluorescence detection. Each steroid was derivatized with 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene++ +-3- propionohydrazide (BODIPY FL hydrazide) and separated on a Wakosil 5C4 column with acetonitrile-water (7 + 3) as mobile phase. The limits of detection of progesterone, 17-hydroxyprogesterone, dehydroepiandrosterone, androstenedione, testosterone and 17-methyltestosterone were 550-3700 fmol per 10 microliters injection (signal-to-noise ratio = 5) serum.

Sensitivity of real time viral detection by an optical biosensor system using a crude home-made antiserum against measles virus as a ligand.

Virus identification in clinical materials during acute phase infections could give necessary information for the treatment of infections by human immunoglobulin (hIg) or interferon (IF). But because of a lack of information, most virus infections have not been treated. We have tried to develop a real time detection system for viruses in general using an optical biosensor and a model virus, Herpes simplex virus Type 1 (HSV-1), and have proved that the HSV-1 virus propagated in Vero cells and diluted in minimum essential medium (MEM) with 10% fetal bovine serum (FBS) could be detected in high sensitivity close to 10 infectious units (50% tissue culture infective dose [TCID50] units) using purified cellular receptor molecules as the ligand because the receptor could be the most specific ligand.

DNA sequencing of the gene encoding Salmonella typhimurium-derived T-cell inhibitor (STI) and characterization of the gene product, cloned STI.

In a previous study, we found a novel protein which inhibited T-cell responsiveness to interleukin-2 (IL-2) in Salmonella typhimurium and called it S. typhimurium-derived T-cell inhibitor (STI). In this study, we analyzed the DNA sequence of the gene encoding STI.

Ca2+-ATPase inhibitor induces IL-4 and MCP-1 production in RBL-2H3 cells.

The effects of a Ca2(+)-ATPase inhibitor, cyclopiazonic acid (CPA), and two hydroquinone-antioxidants, 2,5-di-(tert-butyl)-1,4-hydroquinone (DTBHQ) and 2,5-di-(tert-amyl)-1,4-hydroquinone (DTAHQ) on the release of IL-4 and MCP-1 from RBL-2H3 cells were investigated. CPA, DTBHQ and DTAHQ, all of which induce intracellular free Ca2+ concentration ([Ca2+]i) increase, induced IL-4 and MCP-1 release in a dose-dependent manner. The release of TNF-alpha required both a Ca2(+)-ATPase inhibitor and 12-O-tetradecanoylphorbol-13-acetate (TPA).

A labile sulfur in trisulfide affects cytochrome P-450 dependent lipid peroxidation in rat liver microsomes.

The effects of trisulfide derivatives were studied on cytochrome P-450-dependent lipid peroxidation using rat liver microsomal systems. Cytochrome P-450-dependent lipid peroxidation was induced by carbon tetrachloride or tert-butylhydroperoxide and was evident by an increase in thiobarbituric acid-reactive substances (TBA-RS) and oxygen consumption. In these cytochrome P-450-dependent lipid peroxidation systems, pretreatment of microsome with trisulfide derivatives (cystine trisulfide and thiocyclam) significantly inhibited TBA-RS formation and oxygen consumption compared with disulfide or thiol analogs (cystine, nereistoxin, or cysteine).

Studies on porcine high molecular weight kininogen. An improved method for the purification of porcine high molecular weight kininogen and cleavage of the kininogen by the action of porcine plasma kallikrein.

By introduction of stepwise DEAE Sephadex A-50 and copper-Chelating Sepharose 6B column chromatographies, about 18.5 mg of high molecular weight kininogen (HK) composed of a single polypeptide chain was obtained from 500 ml of porcine plasma. Molecular weights of reduced or non-reduced preparation were estimated to be 110 kDa and 116 kDa, respectively, by SDS-PAGE.

Optimum conditions for the 13C-phenylalanine breath test.

We have conducted optimization studies to develop a superior 13C-phenylalanine breath test for the diagnosis of liver disease. First, we examined the optimum 13C-labeling position in phenylalanine for use in a breath test based on infrared spectroscopic detection of 13CO2 in exhaled air. L-[1-13C]Phenylalanine gave the best result.

Dihydropyridine type calcium channel blocker-induced turbid dialysate in patients undergoing peritoneal dialysis.

We previously reported that manidipine, a new dihydropyridine type calcium channel blocker, produced chylous peritoneal dialysate being visually indistinguishable from infective peritonitis in 5 patients undergoing continuous ambulatory peritoneal dialysis (CAPD) [Yoshimoto et al. 1993]. To study whether such an adverse drug reaction would also be elicited by other commonly prescribed calcium channel blockers in CAPD patients, we have conducted postal inquiry to 15 collaborating hospitals and an institutional survey in International Medical Center of Japan as to the possible occurrence of calcium channel blocker-associated non-infective, turbid peritoneal dialysate in CAPD patients.

First report of Trichosporon ovoides isolated from the home of a summer-type hypersensitivity pneumonitis patient.

Trichosporon species have been known to cause summer-type hypersensitivity pneumonitis (SHP). During the isolation of yeasts from an SHP patient's house, we recovered a strain belonging to the genus Trichosporon. Morphologically, the isolate produced rectangular arthroconidia when grown on corn meal agar.

Enantioselective tissue distribution of the basic drugs disopyramide, flecainide and verapamil in rats: role of plasma protein and tissue phosphatidylserine binding.

Purpose: The stereoselective distribution of three basic drugs, disopyramide (DP), flecainide (FLC) and verapamil (VP), was studied to clarify the relationship between the tissue-to-unbound plasma concentration ratio (Kpf) and drug lipophilicity and binding to phosphatidylserine phs), which are possible factors determining the tissue distribution of these drug enantiomers.

Methods: The drug enantiomer or racemate was administered to rats by intravenous constant infusion. Their concentrations in plasma and tissues were determined using enantioselective high-performance liquid chromatography.

Identification of Trichosporon asahii by PCR based on sequences of the internal transcribed spacer regions.

Trichosporon asahii is a major causative agent of deep-seated trichosporonosis, which has a high mortality rate. To detect T. asahii, we have developed specific oligonucleotide primers based on the internal transcribed spacer regions of this organism's genome.

Quantitative determination of disopyramide, verapamil and flecainide enantiomers in rat plasma and tissues by high-performance liquid chromatography.

Enantiomers of disopyramide (DP), flecainide (FLC) and verapamil (VP) were extracted from rat plasma and tissues (brain, lung, heart, liver, kidney and muscle), followed by quantitative determination using enantioselective high-performance liquid chromatography with chiral stationary-phase columns. The recoveries of S-(+)- and R-(-)-DP from tissues were higher than 69%, and the within- and between-day coefficients of variation were very low (0.5 - 5.

Improved high-performance liquid chromatographic analysis of teniposide in human plasma.

A simple and practical high-performance liquid chromatographic analysis has been developed for measuring teniposide (VM26) in human plasma. The present analytical method has improved extraction efficiency from human plasma, therefore allowing determination of VM26 in a clinical setting using ultraviolet detection alone. Furthermore, sample preparation was simplified and shortened through use of a one-step extraction procedure.

Purification of haemorrhagic proteinase from the venom of Agkistrodon caliginosus (Kankoku-Mamushi).

A haemorrhagic proteinase was purified from A. caliginosus venom by ion-exchange chromatography on CM-Sephadex C-50, DEAE-Sephadex A-50, S-Sepharose Fast Flow and Q-Sepharose Fast Flow, and gel-filtration on a Sephadex G-100 column. By this procedure, about 17.

Inhibition of S-warfarin metabolism by nonsteroidal antiinflammatory drugs in human liver microsomes in vitro.

We studied the inhibition of S-warfarin metabolism by nonsteroidal antiinflammatory drugs (NSAIDs) in human liver microsomes in vitro. After screening for potential inhibitors among ten NSAIDs using human recombinant cytochrome P450, inhibition kinetic parameters were estimated using human liver microsomes. Phenylbutazone and bucolome were suggested to increase the unbound steady-state level of S-warfarin about four- and five-fold, respectively, as estimated from these metabolic parameters.

Isolation of a major metabolite (i-OHAP) of aprindine and its identification as N-[3-(N,N-diethylamino)propyl]-N-phenyl-2-aminoindan-5-ol.

i-OHAP, a major metabolite of aprindine (AP), was isolated by TLC from rat feces and identified as N-13-(N,N-diethylamino)propyl]-N-phenyl-2-aminoindan-5-ol, based on 1H-NMR, the H-H correlation spectroscopy (COSY) spectrum, MS and LC-MS. Its structure was also confirmed by comparison with the synthesized compound. The hydroxy group of i-OHAP was located at the 5-position of the indan ring.

Metabolism of warfarin enantiomers in Japanese patients with heart disease having different CYP2C9 and CYP2C19 genotypes.

Objective: To determine whether genetic polymorphism of cytochrome P450 (CYP) 2C9 affects the in vivo metabolism of warfarin enantiomers.

Methods: Eighty-six Japanese patients heart disease who were given warfarin participated in the study. Plasma unbound concentrations of warfarin enantiomers and urinary (S)-7-hydroxywarfarin concentrations were measured by means of a chiral HPLC and ultrafiltration technique to calculate the unbound oral clearance (CLpo,u) for the enantiomers and the formation clearance (CLm) for (S)-warfarin 7-hydroxylation.

Rhodocytin, a functional novel platelet agonist belonging to the heterodimeric C-type lectin family, induces platelet aggregation independently of glycoprotein Ib.

We isolated and characterized a functionally novel platelet agonist, designated as rhodocytin, from the Calloselasma rhodostoma venom. Rhodocytin was a disulfide-linked heterodimer consisting of 18- and 15-kDa subunits. The respective N-terminal amino acid sequences of both subunits were homologous to each other and to those of the carbohydrate-recognition domains (CRD) of C-type lectins.

[Real time observation of binding of measles virus to Vero cells and neutralization of measles virus by human immunoglobulin using optical biosensor--a new real time diagnosis system for viral infections].

An optical biosensor to monitor molecular intractions was developed. This system made possible to observe reactions with a few minutes. We tried to apply this system for rapid diagnosis of viral infections.

Stereoselective metabolism of benoxaprofen in rats. Biliary excretion of benoxaprofen taurine conjugate and glucuronide.

Benoxaprofen (BOP) was administered iv to bile duct-cannulated rats at a dose of 10 mg/kg. BOP and its metabolites in plasma, urine, and bile were quantified using HPLC. A previously unidentified BOP metabolite was found in HPLC chromatograms of rat bile, and the metabolite was isolated chromatographically.

Screening system for urease inhibitors using 13C-NMR.

Urease inhibitors are candidate drugs to treat infection with the human pathogen, Helicobacter pylori, which produces a potent urease [urea amidohydrolase; EC 3.5.1.

Rapid detection of species of the opportunistic yeast Trichosporon by PCR.

Trichosporon species are opportunistic pathogens, associated with a high mortality rate in immunocompromised patients. Oligonucleotide primers were used to amplify a 170-bp fragment of small-subunit ribosomal DNA of all species in the genus Trichosporon by PCR. The primers amplify DNAs of all species in the genus Trichosporon, including six causative agents of trichosporonosis.

Salmonella infection-induced non-responsiveness of murine splenic T-lymphocytes to interleukin-2 (IL-2) involves inhibition of IL-2 receptor gamma chain expression.

In a previous study we demonstrated that Salmonella typhimurium-induced immunosuppression involved T-cell non-responsiveness to interleukin-2 (IL-2). In this study we observed that Salmonella-induced T-cell non-responsiveness to IL-2 was not reversed completely by treatment with N(G)-monomethyl-L-arginine, which is known to inhibit nitric oxide (NO) secretion by macrophages in culture. Furthermore, when purified splenic T-lymphocytes from Salmonella-infected mice were activated with an anti-CD3 antibody, the responsiveness of these T-cells to IL-2 was suppressed significantly.