Biomedical subjects
H Nishimura
Publications and source records attributed to H Nishimura.
Isolation and characterization of the THI6 gene encoding a bifunctional thiamin-phosphate pyrophosphorylase/hydroxyethylthiazole kinase from Saccharomyces cerevisiae.
Thiamin-phosphate pyrophosphorylase (TMP-PPase; EC 2.5.1.3) involved in de novo synthesis of thiamin in Saccharomyces cerevisiae is a bifunctional enzyme with 4-methyl-5-beta-hydroxyethylthiazole kinase (Th-kinase; EC 2.7.1.50) activity, which is an octamer of identical 60-kDa subunits (Kawasaki, Y. (1993) J. Bacteriol. 175, 5153-5158). Previous study demonstrated that the activities of both TMP-PPase and Th-kinase are reduced by the mutation of a single nuclear gene, designated THI6. We have cloned the THI6 gene from a yeast genomic library by functional complementation of the thi6 mutant and determined by DNA blot analysis that THI6 is located on chromosome XVI. The nucleotide sequence of the THI6 gene contained an open reading frame of 1,620 base pairs encoding a 540-amino acid polypeptide with a calculated molecular weight of 58,058, which is similar to the determined molecular mass of the purified bifunctional enzyme. Gene disruption demonstrated that the thi6 null strain is auxotrophic for thiamin, indicating that the THI6 protein is essential for thiamin synthesis in yeast. A recently isolated thi6 mutant, thi6-3, bearing a replacement of Glu370 by Lys370, showed a decrease in only Th-kinase activity, proving that the THI6 gene of S. cerevisiae encodes a structural gene of the thiamin biosynthetic bifunctional enzyme. Furthermore, complementation analysis of the thi6 null strain with the modified THI6 DNAs by a 12-nucleotide linker insertion suggested that a region from amino acids 138 to 187 and that from amino acids 370 to 453 are involved in functional domains of TMP-PPase and Th-kinase, respectively, whereas the COOH-terminal region is necessary for both enzyme activities. Strains conferring no Th-kinase but slight TMP-PPase activity could grow in medium without thiamin, suggesting that 4-methyl-5-beta-hydroxyethylthiazole is not involved in the pathway of de novo synthesis of thiamin via 4-methyl-5-beta-hydroxyethylthiazole monophosphate. Northern blot analysis demonstrated that THI6 gene expression is regulated at the mRNA level by intracellular thiamin pyrophosphate, a coenzyme form of thiamin, and that it requires the positive regulatory factors encoded by the THI2 and THI3 genes.
Purification and characterization of a novel substrate for plasma kallikrein (PK-120) in human plasma.
A 120 kDa plasma protein, which is susceptible to plasma kallikrein, was purified from human plasma by polyethylene glycol fractionation followed by ion exchange chromatography using Q-Sepharose, S-Sepharose, and hydroxyapatite and gel filtration on Sephacryl S-200. The 120 kDa protein, termed PK-120 in this paper, was a single polypeptide chain containing about 20% sugar by weight and its concentration in plasma was estimated to be 80 micrograms/ml by ELISA. At least three fragments, 100, 70, and 35 kDa, were produced from PK-120 by plasma kallikrein. The N-terminal sequence and Western blot demonstrated that PK-120 was first cleaved to yield the 100 and 35 kDa fragments, then the 100 kDa fragment was cleaved into the 70 kDa fragment. N-Terminal sequence analyses of PK-120 and its fragments demonstrated that it is a novel plasma protein, distinct from high molecular weight kininogen, a natural substrate for plasma kallikrein.
Activation of the alternative pathway of complement by apoptotic Jurkat cells.
Jurkat T cells die of apoptosis upon exposure to anti-Fas mAb. Here we show that although the alternative complement pathway generally does not attack homologous cells, anti-Fas-induced apoptotic Jurkat T cells were attacked antibody-independently by the alternative pathway of human complement and opsonized with iC3b, which is a ligand of the complement receptor type 3 (CR3) of phagocytes. These results suggest that apoptotic cells become the targets of the homologous alternative complement pathway, which facilitates the clearance of apoptotic cells by phagocytes.
Staurosporine inhibits phorbol 12-myristate 13-acetate- and insulin-stimulated translocation of GLUT1 and GLUT4 glucose transporters in rat adipose cells.
Staurosporine, a widely used protein kinase C inhibitor, completely inhibited both phorbol 12-myristate 13-acetate (PMA)- and insulin-stimulated glucose transport activity in isolated rat adipocytes. The inhibition was non-competitive and was attributed to a blockade of the PMA- and insulin-induced translocation of both GLUT1 and GLUT4 glucose transporters. The PMA-stimulated glucose transport activity was more sensitive to inhibition by staurosporine than was insulin-stimulated transport activity (PMA, IC50 = 1.1 +/- 0.1 microM; insulin, IC50 = 6.4 +/- 0.7 microM; P < 0.05, n = 3). At 1 microM staurosporine the insulin-sensitivity was decreased, i.e. EC50 increased from 0.12 nM to 5.4 nM, but the maximum response to insulin and the time course for stimulation were unaffected. At 6 microM staurosporine the insulin-sensitivity was further decreased, the maximal stimulation was decreased by 25%, and the apparent half-time for stimulation was extended from 2.5 min in control cells to 9.4 min. Staurosporine (30 microM) was able to block insulin's ability to stimulate glucose transport, whether added before or after insulin, by a mechanism that did not alter the rate of GLUT4 internalization. In intact adipose cells, staurosporine (30 microM) induced a slight (30%) decrease in the maximal insulin-induced receptor autophosphorylation and a similar decrease in the tyrosine phosphorylation of pp60 and pp160 (insulin-receptor substrate-1: 'IRS-1'), but was without effect on insulin binding to its receptor. Conversely, staurosporine induced a concentration-dependent inhibition of the constitutively tyrosine-phosphorylated (pp120) protein and of an insulin-stimulated protein pp53 in the cytosol. The locus of staurosporine's action appears to be distal from the initial insulin-receptor signalling, at a step that regulates the specific translocation of the glucose transporters to the plasma membranes.
Cocirculation of two distinct groups of influenza C virus in Yamagata City, Japan.
The antigenic and genetic relationships among 15 strains of influenza C virus isolated between August, 1988, and May, 1990, in Yamagata City, Japan, were investigated. Based on the results of antigenic analysis with monoclonal antibodies to the hemagglutinin-esterase (HE) glycoproteins and oligonucleotide mapping of total vRNA, the isolates were divided into two distinct groups closely related to either C/Yamagata/26/81 or C/Aichi/1/81. Antigenic differences between two groups could be detected clearly with heterogeneous antiviral sera. Comparison of the HE gene sequences between the representative Yamagata isolates and the previous isolates from other countries showed that C/Yamagata/26/81-like and C/Aichi/1/81-like viruses had high degrees of nucleotide sequence homology with C/pig/Beijing/115/81 and C/Johannesburg/1/66, respectively. These observations suggest that two lineages of influenza C virus, markedly different from each other in both antigenic and genetic structures, were simultaneously present in Yamagata City during 1988-1990 and that viruses belonging to each of the two lineages may also be prevalent in other areas of Japan and even in other countries.
Identification of regions of bovine factor VII essential for binding to tissue factor.
Initiation of the extrinsic blood coagulation pathway is mediated by a complex formed between plasma-derived factor VII/VIIa and cell-derived tissue factor (TF). To identify the site(s) of interaction, zymogen VII and VIIa were enzymatically and chemically modified, and their affinities for TF were estimated by measuring their inhibitory effects on the amidolytic activity enhanced after formation of the VIIa-TF complex. We found that the VIIa-light chain (Ki = 3.5 x 10(-7) M) and its fragment consisting of the gamma-carboxyglutamic acid (Gla)-domain and the first epidermal growth factor (EGF)-like domain (Gla-EGF1 peptide; Ki = 1.0 x 10(-6) M) have an affinity for TF. Therefore, one of the binding sites of VII with TF is probably located in the Gla-EGF1 region. On the other hand, a dansyl-Glu-Gly-Arg chloromethyl ketone-treated Gla-domainless VIIa (Ki = 0.7 x 10(-7) M) showed a high affinity for TF, whereas the corresponding Gla-domainless VII similarly treated showed no binding potential, thereby indicating that binding site(s) other than in the Gla-EGF1 region are present in VIIa but not in VII. Acetylation or carbamylation of the alpha-amino group of the NH2-terminal Ile-153 of VIIa resulted in the loss of binding affinity for TF; such modifications convert VIIa into a zymogen-like inactive form by destroying the salt bridge between Ile-153 and Asp-343 in VIIa. The rate of carbamylation of VIIa was reduced in the presence of TF. Protection of the alpha-amino group of Ile-153 from carbamylation after complex formation was consistent with salt bridge formation between Ile-153 and Asp-343 in the VIIa-TF complex. Therefore, binding of TF with the heavy chain of VIIa may induce a conformational change that brings the alpha-amino group of Ile-153 close to the beta-carboxyl group of Asp-343 to make a stable salt bridge.
Uniform multimegabar shock waves in solids driven by laser-generated thermal radiation.
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Gamma -X mixing effects on pseudodirect exciton transitions in GaAs/AlAs type-II superlattices.
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Activation peptide of human factor IX has oligosaccharides O-glycosidically linked to threonine residues at 159 and 169.
O-Linked oligosaccharide chains were identified in the activation peptide (AP) of human blood coagulation factor IX. The peptide obtained from human factor IX was separated into three molecular species (AP alpha, AP beta, and AP gamma) by reversed-phase high-performance liquid chromatography. Amino acid analysis showed that AP alpha, but not AP beta and AP gamma, contained galactosamine in addition to glucosamine, thereby suggesting the presence of an O-linked sugar chain(s) in the molecule of AP alpha. A nonapeptide (AP alpha-D4, residues 157-165) and an undecapeptide (AP alpha-D5, 166-176) derived from AP alpha contained Thr-159 and Thr-169, neither of which could be identified using a gas-phase protein sequencer. All other serine and threonine residues present in AP alpha were identified by peptide sequencing. Component sugar and sialic acid analyses of AP alpha-D4 and AP alpha-D5 revealed that they contained 1 mol each of N-acetyl-D-galactosamine (GalNAc), D-galactose (Gal), and sialic acid. Fast atom bombardment tandem mass spectrometric analysis of AP alpha-D4 suggested the existence of Gal-GalNAc-Thr, NeuNAc-(Gal-)GalNAc-Thr, and NeuNAc-Gal-GalNAc-Thr structures. On the basis of amino acid analysis after the isolation of AP alpha, it accounted for approximately 35% of the total activation peptide obtained. From these results, it was concluded that a part of the activation peptide of human factor IX in circulating blood has tri- and tetrasaccharides O-glycosidically linked to the threonine residues at 159 and 169.
A unique murine CD43 epitope Lp-3: distinct distribution from another CD43 epitope S7.
In foregoing studies, we found a unique B cell differentiation antigen Lp-3 which is expressed on pre-B and premature B cells in the bone marrow, but is negative on bone marrow mature B cells and peripheral resting B cells. Nonetheless, Lp-3 was clearly positive on the majority of CD5 B(B1) cells. When we examined the biochemical nature and partial amino acid sequences of purified 132-kDa Lp-3 molecules and the nucleotide sequence of the cDNA clones, we found that Lp-3 is an epitope of CD43. Thus, the monoclonal antibody (mAb) Lp-3 may be the first mAb to murine CD43 defined by primary target structure analysis. Comparison of tissue distribution of Lp-3 and S7, an epitope previously suggested to associate with murine CD43, showed that they were similarly distributed on thymocytes, peripheral B and T cells, granulocytes, and platelets. In the bone marrow, while both Lp-3 and S7 were negative on mature B cells, the former was positive on all B lineage cells at an early ontogeny and the latter was positive only on the minor population of pre-B cells and pro-B cells. Lp-3 and S7 epitopes also showed different distributions on basement membranes of renal glomerulus, bronchus, and endometrium, lining cells of choroid plexus and muscular cells of arterioles in a variety of tissues. As CD43 has various isoforms generated by different degrees of glycosylation of the common core peptide, it is likely that Lp-3 and S7 are associated with different CD43 isoforms.
The ability of influenza C virus to generate cord-like structures is influenced by the gene coding for M protein.
We observed previously that cord-like structures which had lengths up to 500 microns or greater were protruding from the surface of HMV-II cells infected with influenza C/Yamagata/1/88 virus (Nishimura et al., 1990, Virology 179, 179-188). Comparison of the cord-forming ability among a number of influenza C isolates revealed that the C/Taylor/1233/47 strain was unique in lacking the ability. It was also found that the major structural proteins, hemagglutinin-esterase (HE), nucleoprotein (NP), and matrix (M), could each be distinguished between C/Yamagata/1/88 and C/Taylor/1233/47 viruses by SDS-polyacrylamide gel electrophoresis. To determine the genes involved in the cord formation, a series of reassortant viruses were prepared between C/Yamagata/1/88 and C/Taylor/1233/47, and parental derivation of genes coding for HE, NP, and M was determined by gel electrophoresis. All reassortants which derived M gene from C/Yamagata/1/88, irrespective of derivation of genes coding for HE and NP, had the ability to generate cords, whereas none of reassortants which derived M gene from C/Taylor/1233/47 were capable of producing cords. With respect to several representative reassortants, the origins of genes encoding the polymerase proteins (PB2, PB1, P3) and the nonstructural proteins (NS1, NS2) were determined by T1-oligonucleotide fingerprinting of the isolated RNA segments. The results suggested that none of genes coding for these proteins are exclusively associated with the cord formation. Thus, it is likely that the M gene is the key determinant of the cord-forming ability of influenza C viruses. Nucleotide sequence analysis of the M genes revealed that compared to C/Yamagata/1/88, C/Taylor/1233/47 had two amino acid substitutions in the M molecule at positions 24 (Ala-->Thr) and 133 (Asp-->Asn).
The E-linked subregion of the major histocompatibility complex down-regulates autoimmunity in NZB x NZW F1 mice.
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Metallothionein mRNA in the testis and prostate of the rat detected by digoxigenin-labeled riboprobe.
Metallothionein (MT), a cysteine-rich heavy metal-binding protein, has been considered to play a role in the homeostatic control and detoxification of heavy metals, such as zinc, copper, and cadmium. In the present study, we have utilized a digoxigenin-labeled riboprobe to localize MT mRNA only by bright-field optics in the testis and prostate of the rat. In the rat testis, MT mRNA was found predominantly in primary spermatocytes and also in secondary spermatocytes and spermatids, but not in the spermatogonia, Sertoli cells, and Leydig cells. On the other hand, MT protein was present in these spermatogenic cells as well as in spermatozoa and Sertoli cells. In the prostate, MT mRNA was found predominantly in the epithelium of the dorsolateral lobes, but not in the ventral lobe, which is in agreement with the observed localization of MT protein. The utilization of both in situ hybridization and immunohistochemical staining on the same tissue specimens show MT gene expression in specific cell types in the male genital organs.
Modification of proteins with polyethylene glycol derivatives.
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Corticosteroid-responsive parkinsonism associated with primary Sjögren's syndrome.
A 74-year-old woman with primary Sjögren's syndrome confirmed by salivary gland biopsy presented with parkinsonism. Magnetic resonance imaging (MRI) of the brain revealed multiple small high intensity lesions in the deep white matter, basal ganglia and pons on T2-weighted images. Treatment with L-dopa failed to improve the parkinsonian features. After the initiation of prednisolone 30 mg/day, the parkinsonian signs and symptoms significantly improved. Some lesions on MRI were decreased in size after corticosteroid therapy. These findings suggest that parkinsonism associated with primary Sjögren's syndrome is at least in part attributable to small vessel vasculopathy such as focal inflammation or edema.
Amoxicillin concentration in pus from abscess caused by odontogenic infection.
1. Amoxicillin concentration in pus from odontogenic infection was assayed and the concentrations were compared with MIC (minimum inhibitory concentration) of alpha-hemolytic streptococci isolated from odontogenic infection. 2. Measurable amoxicillin concentrations in serum and pus were found in all instances (n = 16). 3. The mean peak concentrations in serum and pus were found at identical times, 1.5 hr after administration, which were 5.92 and 0.90 micrograms/ml, respectively. 4. The mean concentration ratio of pus/serum at the peak time was 0.15. 5. All amoxicillin concentrations in pus at the peak time exceeded the MIC for 90% of alpha-hemolytic streptococci (0.25 micrograms/ml).
Cephalexin concentrations in radicular granuloma following a single oral administration of 250- or 500-mg cephalexin.
1. Cephalexin concentrations in radicular granuloma and serum following a single oral administration of 250- or 500-mg cephalexin were measured by a paper disk method. 2. The highest concentration of cephalexin in radicular granuloma following administration of 250-mg cephalexin to nonfasting patients was observed at 2 hr, and was 1.62 micrograms/g. The mean cephalexin concentration ratio of radicular granuloma/serum at 2 hr was 0.35. 3. The highest concentrations of cephalexin in radicular granuloma following administration of 500-mg cephalexin to nonfasting and fasting patients occurred at 2 and 1.5 hr, and was 3.35 and 3.42 micrograms/g, respectively. Mean cephalexin concentration ratios of radicular granuloma/serum at 2 and 1.5 hr were 0.32 and 0.30, respectively. 4. All mean cephalexin concentrations in radicular granuloma following administration of 500-mg cephalexin to both fasting and nonfasting patients exceeded MIC for 90% (2 micrograms/ml) of clinically isolated strains of alpha-hemolytic streptococci. However, those concentrations obtained by 250-mg cephalexin did not exceed it.