Biomedical subjects

T Watanabe

Publications and source records attributed to T Watanabe.

At least 19 recordsLinked to original sources

PubMed1992

Determination of a new bisphosphonate, YM175, in plasma, urine and bone by high-performance liquid chromatography with electrochemical detection.

A high-performance liquid chromatographic method for the determination of disodium dihydrogen(cycloheptylamino)methylene-bisphosphonate monohydrate (YM175) in plasma, urine and bone is described. Plasma obtained in high-dose animal studies is pretreated by Method A, a simple method using 1 ml of plasma, which is based on deproteinization of plasma followed by coprecipitation of the drug with calcium phosphate and removal of excess calcium ions by AG 50W-X8 resin. Plasma obtained in lower-dose clinical studies is treated by Method B, a more sensitive method using 10 ml of plasma, which is based on solid-phase extraction using a Sep-Pak C18 cartridge coupled with Method A. Urine and bone are treated similarly to Method B. The chromatographic system consists of a mobile phase at pH 11, an alkali-stable column and an electrochemical detector operating in the oxidation mode. The determination limit is 5 ng/ml for Method A and 0.5 ng/ml for Method B in plasma, 1 ng/ml in urine, and 25 ng/g in bone.

Animals↗

PubMed1992

The development of hypercalcemia in a patient with an ovarian tumor producing parathyroid hormone-related protein.

Hypercalcemia developed in a 34-year-old woman with a clear cell carcinoma of the ovary. Osseous involvement with the tumor cells was not present. Primary hyperparathyroidism was absent. Operative partial resection of the metastatic supraclavicular lymph node, followed by radiation therapy, decreased her serum calcium concentrations. This case belongs to the category of humoral hypercalcemia of malignancy (HHM). Detection of a significant quantity of immunoreactive parathyroid hormone-related protein (PTH-rP) in the metastatic lymph node suggested that the HHM of the patient was induced by PTH-rP produced by the tumor. From a review of 17 cases of ovarian tumors showing HHM-like morbidity, it was found that clear cell carcinoma and cystadenocarcinoma were the major types of ovarian tumors associated with HHM.

Adenocarcinoma↗

PubMed1992

Three types of Gi alpha protein of the guinea-pig lung: cDNA cloning and analysis of their tissue distribution.

cDNA clones encoding three types of Gi alpha, the alpha subunit of GTP-binding protein (Gi1 alpha, Gi2 alpha, and Gi3 alpha), were isolated from a cDNA library of the guinea-pig lung. Nucleotide sequence analysis revealed a high degree of homology with other mammalian Gi alpha cDNAs. By RNA blot analysis, the expression pattern of Gi1 alpha was more tissue-specific than those of other types of Gi alphas in the guinea-pig tissues examined. While Gi2 alpha and Gi3 alpha mRNAs were ubiquitously expressed in all tissues examined, Gi1 alpha mRNA was mainly expressed in the brain, lung and kidney. These results suggest that each Gi alpha protein may have a different role.

Amino Acid Sequence↗

PubMed1992

Expression of VLA-4 on thymocytes. Maturation stage-associated transition and its correlation with their capacity to adhere to thymic stromal cells.

The present study investigates the expression of VLA-4 on thymocytes at various stages of maturation and their capacity to adhere to thymic stromal cells. Whole thymocytes were stained with anti-CD4 and anti-CD8, as well as anti-VLA-4 antibodies. Flow microfluorometric analyses revealed that a) most of CD4-8- (double negative DN) and CD4-8intermediate thymocyte populations expressed large amounts of VLA-4, b) the levels of VLA-4 were considerably and markedly reduced on CD4+8+ (double positive DP) and single positive (SP) (CD4+8- or CD4-8+) populations, respectively. This contrasted with an increase in the levels of LFA-1 along with thymocyte maturation. DN, DP, and SP subsets were isolated and examined for their capacity to express VLA-4 and to adhere to fibronectin (FN) molecules as well as thymic stromal cells expressing FN. DN, DP, and SP subsets were confirmed to express the respective high, low, and very low levels of VLA-4, respectively. Approximately 70% of DN thymocytes became bound to FN-precoated culture plates, whereas 30 to 40% of DP and only 10 to 20% of SP cells adhered to FN. Similar patterns of adhesion were observed between these thymocyte subsets and thymic stromal monolayers. The binding of the DN subset to FN-plates or thymic stromal monolayers was inhibited only marginally by the RGDS peptide, but was efficiently inhibited by V10 peptide (cell-binding sequence that is located in the V region on FN and reacts with the VLA-4 integrin) or anti-VLA-4 antibody. Anti-VLA-4 antibody plus RGDS peptide strongly inhibited DN cell binding to FN-coated plates and thymic stromal monolayers. These results indicate that i) VLA-4 expressed on DN thymocytes functions as an important integrin for interacting with thymic stromal cells; ii) the expression level of this integrin decreases with the progress of thymocyte maturation, and iii) most of the mature thymocytes (SP) are rendered less adhesive to thymic stromal cells by reducing the level of VLA-4 expression.

Animals↗

PubMed1992

Adult coronary artery disease probably due to childhood Kawasaki disease.

We have surveyed adult survivors of childhood Kawasaki disease (KD) who had coronary artery disease that could be ascribed to KD. In response to questionnaires sent to cardiologists throughout Japan, 21 patients (17 men, 4 women, aged 20-63 years) with coronary lesions and a definite (2) or suspected (19) history of KD were reported. 5 patients had presented with acute myocardial infarction, 6 previous myocardial infarction, 9 angina pectoris, and 1 dilated cardiomyopathy. 16 patients had obstructions in two or more coronary arteries. 3 had died and 18 were alive with serious sequelae (mitral regurgitation, arrhythmias, congestive heart failure). Childhood KD should be included in the differential diagnosis of coronary artery disease in young adults.

Adult↗

PubMed1992

Opioid mu-deficient CXBK mouse and the role of mu 1-receptors in electrically induced convulsions.

Studies were made on the role of mu 1-receptors on electrically induced convulsions in mice. A relatively selective mu 1-agonist, DAGO ([D-Ala2-N-methyl-Phe4-Gly-ol]-enkephalin) decreased the durations of tonic and clonic convulsions in C57BL/6 mice, but not in opioid mu-deficient CXBK mice, indicating that the activation of mu 1-receptors had an anticonvulsive effect on these convulsions in mice. The selective mu 1-antagonists naloxonazine and naltrexonazine promoted the clonic phase of electrically induced convulsion in C57BL/6 mice. Moreover, the duration of the clonic phase was longer in mu-deficient CXBK mice than in C57BL/6 mice. These data for CXBK mice confirmed, at least some proconvulsant effects of mu 1-antagonists in C57BL/6 mice. Thus, blockade of mu 1-receptors has a proconvulsant effect, and mu 1-receptors have at least some protective role against electrically induced convulsions.

Animals↗

PubMed1992

Cell division arrest induced by phorbol ester in CHO cells overexpressing protein kinase C-delta subspecies.

Several lines of CHO cells stably overexpressing protein kinase C (PKC) subspecies to various extents were established by the DNA-mediated transfer. Upon treatment with phorbol 12-myristate 13-acetate, the growth of the cells expressing the PKC-delta subspecies was markedly inhibited, whereas cell lines expressing PKC-alpha, PKC-beta II, and PKC-zeta subspecies were not significantly affected. Flow cytometric analysis indicated that all cell lines overexpressing PKC-delta subspecies accumulated in G2/M phase in response to phorbol 12-myristate 13-acetate. In these arrested cells, dikaryons were predominant, implying that phorbol ester-induced inhibition of cell division is specific to telophase. These results suggest PKC-delta subspecies may play a role in the normal cell cycle progression.

Animals↗

PubMed1992

High affinity binding of the leucocyte adhesion molecule L-selectin to 3'-sulphated-Le(a) and -Le(x) oligosaccharides and the predominance of sulphate in this interaction demonstrated by binding studies with a series of lipid-linked oligosaccharides.

The binding of the leucocyte adhesion molecule L-selectin has been investigated toward several structurally defined lipid-linked oligosaccharides immobilized on silica gel chromatograms or plastic wells. In both assay systems the 3'-sulphated Le(a)/Le(x) type tetrasaccharides [formula: see text] were more strongly bound than 3'-sialyl analogues. A considerable binding was observed to the 3'-sulphated oligosaccharide backbone in the absence of fucose but not to a 3'-sialyl analogue or fuco-oligosaccharide analogues lacking sulphate or sialic acid. Affinity for other sulphated saccharides: 3'-sulphoglucuronyl neolactotetraosyl ceramide and glycolipids with sulphate 3'-linked to terminal or sub-terminal galactose or N-acetylgalactosamine was detected in the chromatogram assay only. These studies, together with earlier reports that L-selectin binding to endothelium is inhibited by sulphatide, highlight the relative importance of sulphate in the adhesive specificity of this protein.

Animals↗

PubMed1992

Functional analysis of alternatively spliced transcripts of the human histidine decarboxylase gene and its expression in human tissues and basophilic leukemia cells.

L-Histidine decarboxylase (HisDC) is the enzyme catalyzing the formation of histamine from L-histidine. HisDC activity is expressed specifically in mast cells/basophils, endocrine cells in stomach, and histaminergic neurons in brain. As a first step in the analysis of the regulation of HisDC gene expression, we have cloned the cDNA coding for HisDC from a cDNA library of a human basophilic leukemia cell line, KU-812-F. We identified two types of HisDC cDNA, representing the 2.4-kb and 3.4-kb HisDC mRNA constitutively expressed in these cells. Sequence analysis of these cDNA revealed that the 3.4-kb mRNA contains the insert sequence of 824 bases and suggests that both 2.4-kb and 3.4-kb mRNA may represent the alternatively spliced transcripts of the HisDC gene. Using expression plasmids containing a cDNA for each HisDC mRNA, we analyzed the function of possible HisDC isoforms. We show that only the 2.4-kb mRNA encodes functional HisDC and is expressed in human brain and lung. However, we were unable to detect the 3.4-kb mRNA in these tissues. Thus, the 3.4-kb mRNA may be generated by KU-812-F cell-specific splicing of the HisDC gene transcripts. Furthermore, we demonstrated the increase in the level of 2.4-kb HisDC mRNA and HisDC activity in KU-812-F cells following treatment with phorbol 12-myristate 13-acetate.

Alternative Splicing↗

PubMed1992

Purification and properties of Aspergillus niger beta-glucosidase.

Beta-glucosidase was purified from a crude cellulase preparation from Aspergillus niger by affinity chromatography on a methacrylamide-N-methylene-bis-methacrylamide copolymer bearing cellobiamine. The purified enzyme was a dimer with an isoelectric point of 4.0. The molecular mass of the enzyme was estimated to be 240 kDa by gel-permeation chromatography. The enzyme hydrolyzed specifically beta-glucosidic bonds and catalyzed transglucosylation of the beta-glucosyl group of cellobiose to yield 4-O-beta-gentiobiosylglucose in the presence of organic solvents or under neutral conditions.

Aspergillus niger↗

PubMed1992

Truncation of N-terminal extracellular or C-terminal intracellular domains of human ETA receptor abrogated the binding activity to ET-1.

We have investigated the function of N-terminal and C-terminal domains of the human ETA receptor by expressing truncated mutants in COS-7 cells. Three kinds of ETA receptors truncated in the N-terminal extracellular or C-terminal intracellular domains were produced. Deletion of the entire extracellular N-terminal or intracellular C-terminal domain completely inactivated the ET-1 binding activity. However, the deletion of one half of the N-terminal extracellular domain of the ETA receptor, missing one of two N-linked glycosylation sites, maintained complete binding activity. Specific monoclonal antibodies detected all the truncated ETA receptors in the cell membrane fraction of transfected COS-7 cells. The size of the ETA receptor was heterogeneous due to differential glycosylation and distributed in 48K, 45K and 42K dalton bands in Western blot analysis. These results demonstrated that a part of the N-terminal domain in close proximity to the first transmembrane region is required for the ligand binding activity of the ETA receptor, and the C-terminal domain is perhaps necessary as an anchor for maintenance of the binding site.

Amino Acid Sequence↗

PubMed1992

Calcium dependent conformational changes of surfactant protein A (SP-A) and its collagenase resistant fragment with or without dithiothreitol.

Calcium-dependent conformational changes of surfactant protein A (SP-A) and the collagenase resistant fragment (CRF) of SP-A were studied by measuring fluorescence spectra. The emission peaks of both SP-A and CRF in the absence of Ca2+ appeared at 343 nm when they were excited at 280 nm. In the presence of Ca2+, the peaks appeared at 340 nm and were accompanied by an increase in the fluorescence intensity. The magnitude of the fluorescence intensity change induced by Ca2+ was amplified by the addition of dithiothreitol (DTT) in both SP-A and CRF. The Ca2+ binding of CRF was measured by a flow dialysis method with 45CaCl2 in the Ca2+ concentration range where the Ca(2+)-induced fluorescence changes occurred. The maximum binding number of Ca2+ to CRF was about 2 mol per mol of CRF, and the value was independent of the presence of DTT.

Calcium↗

PubMed1992

Expression of platelet activating factor receptor in renal tubular cell line (LLC-PK1).

Northern blot analyses of poly(A)+ RNA extracted from cloned renal tubule cells, LLC-PK1, MDCK and JTC-12 cells, were performed using an isolated cDNA of guinea pig platelet activating factor (PAF) receptor as a probe. The results showed the expression of single entity of PAF receptor mRNA in LLC-PK1, but not in MDCK or JTC-12 cells. PAF also induced a dose-dependent elevation of intracellular calcium in LLC-PK1 cells as assessed by Fura-2 fluorescence signals, which was completely blocked by a PAF receptor antagonist, WEB 2170. These results indicate a PAF receptor-effector system in a renal tubular cell line LLC-PK1, providing a possible model system for studying physiological roles of PAF receptors in renal tubules.

Animals↗

PubMed1992

Alteration of phosphotyrosine-containing proteins at the early stage of erythroid differentiation of mouse erythroleukemia (MEL) cells.

Following treatment of mouse erythroleukemia (MEL) cells with dimethyl sulfoxide and other typical erythroid inducing agents, the profile of cellular phosphotyrosine-containing proteins was drastically altered. We found that the level of almost all of the phosphotyrosine-containing proteins was either decreased or disappeared at a very early stage of differentiation. Addition of sodium orthovanadate (Na3VO4), a specific inhibitor of phosphotyrosine phosphatases, prevented the alteration as well as erythroid differentiation. Mutant MEL cells, which are resistant to differentiation by dimethyl sulfoxide, were apparently insensitive to the alteration. These results indicate that dephosphorylation of phosphotyrosine residues in cellular proteins is coupled with a reaction(s) which is responsible for triggering differentiation of MEL cells.

Acetamides↗

PubMed1992

Uveitis associated with human T-cell lymphotropic virus type I.

Seroepidemiologic, clinical, and virologic studies were performed to determine whether human T-cell lymphotropic virus type I was closely associated with uveitis in two hospitals. One hospital was in an endemic area of the virus (Miyakonojo, Miyazaki) and the other hospital was in a less endemic area (Kurume). In the endemic area, the seroprevalence of the virus in patients with uveitis without defined causes (35.4%, 62 of 175 patients) was significantly higher than that in patients with nonuveitic ocular diseases (16.1%, 42 of 261 patients), or in patients with uveitis with defined causes (10.3%, eight of 78 patients). The seroprevalence in younger patients (20 to 49 years of age) with uveitis without defined causes in the area was 44.8% (30 of 67 patients), whereas it was only 9.3% (ten of 107 patients) in the other two groups. A similar observation was recorded even in the less endemic area (Kurume). Because the seroprevalence of the virus in the general population is known to be low in younger patients and to increase with age, these findings were interpreted to indicate that the association of human T-cell lymphotropic virus type I with uveitis was significant. Most patients, particularly those aged 20 through 49 years, had an intermediate uveitis characterized by a moderate inflammation in the vitreous body accompanied by an iritis and retinal vasculitis. The ocular symptoms in the patients differed from those of other types of uveitis common in Japan (Behçet's disease, Vogt-Koyanagi-Harada's disease, and toxoplasmosis, for example).(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗

PubMed1992

Consensus sequence for precursor processing at mono-arginyl sites. Evidence for the involvement of a Kex2-like endoprotease in precursor cleavages at both dibasic and mono-arginyl sites.

Many peptide hormones and neuropeptides are produced from larger, inactive precursors through endoproteolysis at sites usually marked by paired basic residues (primarily Lys-Arg and Arg-Arg), or occasionally by a monobasic residue (primarily Arg). Based upon data concerning processing of prorenin and its mutants around the native Lys-Arg cleavage site expressed in mouse pituitary AtT-20 cells, we present the following sequence rules that govern mono-arginyl cleavages: (a) a basic residue at the fourth (position -4) or the sixth (position -6) residue upstream of the cleavage site is required, (b) at position -4, Arg is more favorable than Lys, and (c) at position 1, a hydrophobic aliphatic residue is not suitable. These rules are compatible with those proposed by comparison of precursor sequences around mono-arginyl cleavage sites. We also provide evidence that precursor cleavages at mono-arginyl and dibasic sites can be catalyzed by the same Kex2-like processing endoprotease, PC1/PC3.

Amino Acid Sequence↗

PubMed1992

Sequence and expression of a rat cDNA for LECAM-1.

A rat cDNA clone encoding an adhesion molecule, LECAM-1, has been isolated from the SD rat and the partial nucleotide sequence was determined. It encodes a peptide of 372 amino acids (aa), including a signal peptide of 38 aa. The protein has three tandem domains: a lectin domain, an EGF-like domain and two repeats of complement regulatory proteins (CR domain). The lectin binding domain has 93.2% and 81.4% and the EGF-like domain has 85.3% and 76.5% aa identity with those of mouse and human, respectively. In the CR repeat domain, the amino acid identity was 72.6% between human and rat and 71.8% between mouse and rat. Northern blot analysis detects the main transcript of about 3 kb in peripheral blood mononuclear cells (PBMC), spleen and thymus. The expression was down-regulated by mitogen stimulation of PBMC and spleen T cells. The protein encoded by this cDNA interacted with PPME when it was expressed on gp90MEL-14 negative mouse EL-4 cells.

Amino Acid Sequence↗

PubMed1992

cDNA sequence analysis of CP94: rat lens fiber cell beaded-filament structural protein shows homology to cytokeratins.

To study the molecular structure of the gene responsible for a lens fiber cell beaded-filament structural protein of 94kDa (CP94), we isolated its specific cDNA from a rat lens cDNA library by use of anti-mouse CP94 antiserum. The expressed fusion protein kept the epitopes specific against anti-chick CP97 as well as anti-mouse CP94 antibody, and the size was estimated as 190-200kDa, indicating that the cDNA insert of the clone seemed to encode a polypeptide with 80-90kDa in appearance. Northern analysis indicated that CP94 mRNA is expressed only in the lens, and not in the brain, skin, heart, kidney, lung, and liver, and the size was estimated to 2.1-2.3kb. In a lens of inherited microphthalmic mouse, Elo, a trace amount of mRNA with the size closely similar to that of rat mRNA was observed. The entire compiled sequence (1,873bp) showed an open reading frame covering the sequence of 533 amino acids totalling 58,857Da. No sequence homologous to the entire CP94 was found among the entries of any nucleotide and amino acid sequence databases; but with respect to a limited amino acid sequence of N-side region of CP94, a significant homology with cytokeratins was found.

Amino Acid Sequence↗