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T Fujiwara

Publications and source records attributed to T Fujiwara.

At least 19 recordsLinked to original sources
PubMed1992

An active-site mutation (Gly633-->Arg) of dipeptidyl peptidase IV causes its retention and rapid degradation in the endoplasmic reticulum.

Dipeptidyl peptidase IV (DPPIV), a serine protease expressed on the cell surface, is deficient in a Fischer rat substrain. Northern blot analysis showed no difference in the size and amount of DPPIV mRNA between normal (344/NC) and deficient (344/CRJ) rats. Cloning and sequencing of DPPIV cDNAs revealed a G to A transition at nucleotide 1897 in the cDNA sequence of 344/CRJ, which leads to substitution of Gly633-->Arg in the active-site sequence Gly629-Trp-Ser-Tyr-Gly633 determined for the wild-type DPPIV [Ogata, S., Misumi, Y., Takami, N., Oda, K., & Ikehara, Y. (1992) Biochemistry 31, 2582-2587]. Pulse-chase experiments with hepatocytes showed that the wild-type DPPIV was initially synthesized as a 103-kDa form with high-mannose-type oligosaccharides, which was processed to a mature form of 109 kDa with the complex type during intracellular transport. In contrast, the mutant DPPIV, although being synthesized as the 103-kDa form, was rapidly degraded without being processed to the mature form. Site-directed mutagenesis of the wild-type and mutant cDNAs and their transfection/expression in COS-1 cells confirmed that the single substitution of Gly633-->Arg is sufficient to cause the rapid intracellular degradation of DPPIV. Immunoelectron-microscopic observations showed that the mutant DPPIV was detectable only in the endoplasmic reticulum (ER), in contrast to the distribution of the wild-type DPPIV in the Golgi complex and on the cell surface.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1992

Purification, characterization, cloning, and expression of a glutamic acid-specific protease from Bacillus licheniformis ATCC 14580.

A glutamic acid-specific protease has been purified to homogeneity from Bacillus licheniformis ATCC 14580 utilizing Phe-Leu-D-Glu-OMe-Sepharose affinity chromatography and crystallized. The molecular weight of the protease was estimated to be approximately 25,000 by SDS-polyacrylamide gel electrophoresis. This protease, which we propose to call BLase (glutamic acid-specific protease from B. licheniformis ATCC 14580), was characterized enzymatically. Using human parathyroid hormone (13-34) and p-nitroanilides of peptidyl glutamic acid and aspartic acid, we found a marked difference between BLase and V8 protease, EC 3.4.21.9, although both proteases showed higher reactivity for glutamyl bonds than for aspartyl bonds. Diisopropyl fluorophosphate and benzyloxycarbonyl Leu-Glu chloromethyl ketone completely inhibited BLase, whereas EDTA reversibly inactivated the enzyme. The findings clearly indicate that BLase can be classified as a serine protease. To elucidate the complete primary structure and precursor of BLase, its gene was cloned from the genomic DNA of B. licheniformis ATCC 14580, and the nucleotide sequence was determined. Taking the amino-terminal amino acid sequence of the purified BLase into consideration, the clones encode a mature peptide of 222 amino acids, which follows a prepropeptide of 94 residues. The recombinant BLase was expressed in Bacillus subtilis and purified to homogeneity. Its key physical and chemical characteristics were the same as those of the wild-type enzyme. BLase was confirmed to be a protease specific for glutamic acid, and the primary structure deduced from the cDNA sequence was found to be identical with that of a glutamic acid-specific endopeptidase isolated from Alcalase (Svendsen, I., and Breddam, K. (1992) Eur. J. Biochem. 204, 165-171), being different from V8 protease and the Glu-specific protease of Streptomyces griseus which consist of 268 and 188 amino acids, respectively.

Amino Acid Sequence↗
PubMed1992

Molecular characterization and expression of the cell-associated glucosyltransferase gene from Streptococcus mutans.

A gene encoding cell-associated glucosyltransferase (CA-GTase) was cloned from Streptococcus mutans MT8148 into Escherichia coli DH5 alpha by using a low-copy-number plasmid, pMW119. After screening of a gene library with the oligonucleotide probe designed on the basis of a partial amino acid sequence of CA-GTase, a recombinant plasmid, pSK6, that had a 5.6 kb insert carrying the CA-GTase gene was selected. The gene product (recombinant CA-GTase) of pSK6 was expressed by using a lac promoter in pMW119. Western blotting revealed that rCA-GTase reacted with antibody to CA-GTase. rCA-GTase was found to synthesize water-insoluble glucans. Southern blotting indicated that the MT8148 chromosome contained another gene which was homologous to pSK6. A plasmid harboring this gene (pSK16) was also isolated from the gene library, the gene product of pSK16 exhibited GTase activity but ten times lower than that of pSK6.

Base Sequence↗
PubMed1992

Specific inhibition of interleukin 1 beta gene expression by an antisense oligonucleotide: obligatory role of interleukin 1 in the generation of lymphokine-activated killer cells.

The purpose of the studies reported here is to determine whether interleukin 1 (IL-1) plays an important role in the regulation of lymphokine-activated killer (LAK) cell induction. The addition of exogenous IL-1 to peripheral blood lymphocyte culture containing suboptimal concentrations of interleukin 2 (IL-2) resulted in induction of cytoplasmic pore-forming protein expression. Polymerase chain reaction results revealed that the mRNAs of both IL-1 alpha and IL-1 beta were induced within 6 h when cultured in IL-2 alone or in a combination of IL-2 and IL-1; however, tumor necrosis factor alpha and beta mRNAs were expressed earlier in peripheral blood lymphocytes stimulated with the combination of IL-1 and IL-2. Furthermore, we have examined the functional role of endogenous IL-1 in LAK activity. The lytic potential was significantly inhibited by an IL-1 receptor antagonist, which could block IL-1-mediated effects, or by specific neutralizing antibodies for IL-1, suggesting that the extracellular autocrine/paracrine pathway of IL-1 is involved in LAK activation. However, a synthetic IL-1 beta antisense oligonucleotide, which could specifically inhibit intracellular IL-1 beta protein expression as detected by Western blot, was more effective in reducing LAK killing, but it could not suppress the cytotoxicity generated by exogenous IL-1 plus IL-2. These findings clearly indicate the existence of an intracellular IL-1 autocrine circuit. Taken together, our results strongly indicate that IL-1 should be considered an obligatory factor in the regulation of IL-2-mediated lymphocyte functions.

Base Sequence↗
PubMed1992

Nilvadipine as a neuroprotective calcium entry blocker in a rat model of global cerebral ischemia. A comparative study with nicardipine hydrochloride.

The effects of two dihydropyridine type calcium entry blockers, nilvadipine and nicardipine hydrochloride (nicardipine), on the liberation of free fatty acids (FFAs) were investigated using an experimental model of global cerebral ischemia in rats, and were compared with their pharmacokinetic properties. Nilvadipine, but not nicardipine, at a dose of 100 micrograms/kg i.v., significantly attenuated the liberation of FFAs, particularly docosahexaenoic and arachidonic acid. Furthermore, the brain concentration of nilvadipine was higher than that of nicardipine after equivalent dosing. The results of the present study demonstrate that pharmacokinetic differences between these two calcium entry blockers might explain the difference in their pharmacological efficacy.

Animals↗
PubMed1992

Selective cell-surface expression of dipeptidyl peptidase IV with mutations at the active site sequence.

The cell surface expression of dipeptidyl peptidase IV (DPPIV) was examined in COS-1 cells transfected with its cDNA with or without mutations at the active site sequence Gly-Trp-Ser-Tyr-Gly (positions 629-633). Mutants with substitution of Trp630----Glu or Ser631----Ala were expressed on the cell surface as normally as the wild-type DPPIV, although the mutant with Ala631 had no enzyme activity. In contrast, any single substitutions of Gly at positions 629 and 633 resulted in no surface expression of the mutants, which were, instead, detected within the cells. When Tyr632 was substituted, one mutant (Tyr----Phe) was expressed on the surface, whereas the others (Tyr----Gly or Leu) were intracellularly retained. These results indicate that the surface expression of DPPIV is critically influenced by mutations at the active site sequence.

Amino Acid Sequence↗
PubMed1992

Brefeldin A-resistant mutants of human epidermoid carcinoma cell line with structural changes of the Golgi apparatus.

We have isolated brefeldin A (BFA)-resistant cell lines, KB/BF-1 and KB/BF-2, from the human epidermoid carcinoma KB cell line. The BFA-resistant phenotypes have been stably maintained for more than 3 months in the absence of BFA. KB/BF-1 and KB/BF-2 showed 10-30-fold higher resistance to cytotoxicity of BFA but were 2-3-fold more sensitive to monensin and nigericin, than KB cells. KB/BF-1 showed aberrant structures of the Golgi complex with poorly developed cisternae surrounded by many small vesicles. Immunocytochemical studies were done with antibodies against a Golgi-specific antigen (chronic rheumatoid arthritis antigen) and a coatomer subunit (beta-subunit for coat proteins of non-clathrin-coated vesicles). Golgi-specific markers were distributed into the small vesicles which were localized diffusedly in cytoplasm of KB/BF-1 cells. Such Golgi markers were observed in a strictly confined perinuclear region of the parental KB cells, whereas in the mutant cells the markers were distributed more diffusedly in dot-like structures at perinuclear regions. In addition, when exposed to BFA, the mutant and parental cells showed a different distribution of these markers. Synthesis and maturation of low density lipoprotein receptor showed apparently slower rates in processing of low density lipoprotein receptor in KB/BF-1 and KB/BF-2 cells than those observed in their parental KB cells. Protein secretion in KB/BF-1 and KB/BF-2 cells was about 30% less than that in KB cells. Much less inhibition by BFA on the secretion was observed in KB/BF-1 and KB/BF-2 cells. A BFA-resistant mutation in BFA-resistant KB cell lines appears to affect assembly of the Golgi apparatus as well as some Golgi-specific functions.

Brefeldin A↗
PubMed1992

Purification, characterization and gene cloning of a novel glutamic acid-specific endopeptidase from Staphylococcus aureus ATCC 12600.

Twenty strains of Staphylococcus aureus from ATCC type cultures and strains found in clinical studies were cultivated, and their endopeptidase activity specific for glutamic acid was surveyed using benzyloxycarbonyl-Phe-Leu-Glu-p-nitroanilide (Z-Phe-Leu-Glu-pNA) as a substrate. The activity was found in two of the strains, ATCC 12600 and ATCC 25923. A glutamic acid-specific proteinase, which we propose to call SPase, was purified from the culture filtrate of S. aureus strain ATCC 12600 by a series of column chromatographies on DEAE-Sepharose twice and on Sephacryl S-200. A single band was observed on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of the purified SPase. The molecular weight of the proteinase was estimated to be 34000 by SDS-PAGE. When synthetic peptides and oxidized insulin B-chain were used as substrates, SPase showed the same substrate specificity as V8 proteinase, EC 3.4.21.9, which specifically cleaves peptide bonds on the C-terminal side of glutamic acid and aspartic acid. Examination with p-nitroanilides of glutamic acid and aspartic acid as substrates, however, revealed that both proteinases are highly specific for a glutamyl bond in comparison with an aspartyl bond. To elucidate the complete primary structure of SPase, its gene was cloned from genomic DNA of S. aureus ATCC 12600, and the nucleotide sequence was determined. Taking the amino acid sequence of SPase from the NH2-terminus to the 27th residue into consideration, the clones encode a mature peptide of 289 amino acids, which follows a prepropeptide of 68 residues. SPase was confirmed to be a novel endopeptidase specific for glutamic acid, being different from V8 proteinase which consists of 268 amino acids.

Amino Acid Sequence↗
PubMed1992

Regulation of lymphokine-activated killer cell induction by human recombinant IL-1 receptor antagonist. Obligate paracrine pathway of IL-1 during lymphokine-activated killer cell induction.

IL-2-stimulated human lymphocytes, referred to as lymphokine-activated killer (LAK) cells, can develop a broad range of lytic activity against fresh tumor cells and cultured tumor cell lines. IL-1, a pleiotropic cytokine shown to synergize with IL-2 on LAK induction, is endogenously synthesized and secreted by LAK cells. Immunoblot analysis demonstrated that IL-2-stimulated PBL produced the 31- to 34-kDa pro-molecules of IL-1 within 24 h and maintained their expression for at least 96 h. The role of secreted IL-1 has been examined using rIL-1R antagonist (IL-1ra). The addition of IL-1ra to LAK activation culture resulted in dose-dependent inhibited lytic activity, which was more apparent in LAK cells cultured with higher doses of IL-2. However, IL-1ra had no effect on proliferative responses elicited in LAK cells by IL-2. Moreover, when IL-1 binding was blocked by IL-1ra, the expression of the IL-2R p55 subunit was reduced compared with control LAK cells. The effect of IL-1 binding blockade on expression of other cytokine mRNA was further examined by polymerase chain reaction analysis, and, specifically, inhibition of both TNF-alpha and TNF-beta mRNA expression by IL-1ra was observed in PBL stimulated with IL-2. The reduced biologic activity of TNF in culture supernatants correlated well with the inhibition of mRNA expression. These findings suggest that autocrine/paracrine IL-1 is involved in the initial generation of LAK activity and, in particular, that TNF expression could be induced via an IL-1 autocrine pathway.

Base Sequence↗
PubMed1992

Molecular design of a eukaryotic messenger RNA and its chemical synthesis.

A designed mRNA consisting of 42 ribonucleotides having the cap structure was synthesized. The capped leader sequence of the brome mosaic virus (BMV) mRNA 4, m7G5'pppGUAUUAAUA (F-1), was synthesized by the phosphotriester method and followed by the capping reaction. A 32-mer consisting of an initiation codon (AUG), the coding region corresponding to a bacterial pheromone cAD1 and two stop codons, was constructed by the 18-mer (F-2) and 14-mer (F-3), which were synthesized by the phosphoramidite method. 2'-,3'-O-Methoxymethylene-guanosine 5'-phosphate was condensed with F-3 using P1-2',3'-O-methoxymethyleneguanosine-5'-yl P2-adenosine-5'-yl pyrophosphate (9) with T4 RNA ligase. The chemically synthesized RNA fragments were ligated successively with T4 RNa ligase to afford the whole RNA molecule.

Amino Acid Sequence↗
PubMed1992

Seed-specific repression of GUS activity in tobacco plants by antisense RNA.

beta-Conglycinin, the 7S storage protein of soybean, is expressed only in seeds and is regulated predominantly by gene transcription [5]. We applied an antisense strategy to modify expression of a beta-glucuronidase (uidA or gusA) gene in seeds using a promoter from a beta-conglycinin gene. Transgenic tobacco plants harboring the gusA gene under the control of the CaMV 35S promoter were retransformed with a gene construct comprising the beta-conglycinin promoter fused to the gusA gene in the antisense orientation. Double transformants were regenerated and transformation was confirmed by Southern blot hybridization. Seed-specific repression of GUS activity was observed in lines containing high copy numbers of the antisense gusA transgene. Suppression of GUS activity was correlated with the amounts of (-) sense gusA transcript detected and concomitantly with a decrease in gusA transcript levels. Furthermore, the amount of suppression of GUS activity was greatest during mid to late stages of seed development, when expression of the alpha' promoter is high. These results indicate that suppression of GUS activity is due to expression of the antisense gene.

Antigens, Plant↗
PubMed1992

Fluorescent dye rhodamine 6G as a molecular probe to study drug resistance of C6 rat glioma cells.

A study was made of the membrane transport of cytoplasm and mitochondria stained fluorescence dye Rhodamine 6G (R6G). In rat glioma C6 cells and 1-(4-amino-2-methyl-5-pyrymidinyl)-methyl-3-(2-chloroethyl) -3-nitrosourea hydrochloride (ACNU) and vincristine (VCR) resistant cell lines (C6/ACNU, C6/VCR), the rate of uptake of R6G decreased in C6/VCR cells, but verapamil increased the intracellular accumulation of R6G in C6/VCR. The intracellular accumulation of R6G of C6/ACNU cells was essentially the same as that of wild-type cells. C6/ACNU cells did not show cross resistance and were sensitive to VCR and cisplatin. C6/VCR cells showed cross resistance to ACNU and CDDP, but C6/VCR cells in the presence of verapamil were more sensitive to drugs than C6/VCR cells in the absence of verapamil. We conclude that the reduction of R6G fluorescence staining intensity in C6/VCR cells compared to wild-type cells may be associated with the mechanism of multidrug resistance (MDR) but does not reflect the mechanism of resistance to ACNU. Verapamil increased the accumulation of R6G in C6/VCR cells and overcame MDR, suggesting that there is a correlation between the MDR overcoming effect and enhancement of R6G accumulation, and that this correlation validates the use of the R6G staining test for clinical and laboratory investigation of MDR.

Animals↗
PubMed1992

The cytoarchitecture of the medial layer in rat thoracic aorta: a scanning electron-microscopic study.

The cytoarchitecture of the medial layer of rat thoracic aorta was examined by scanning electron microscopy after removal of the connective tissue. The outermost lamella showed a lattice-like structure of muscle bundles of closely apposed smooth muscle cells (SMCs), whereas the inner lamellae consisted of more-or-less continuous muscle sheets of vaguely defined subgroups of parallel SMCs. Longitudinal rows of ridges ran along the adventitial surface of these muscle bundles and sheets. The SMCs of the outermost lamella, were 5.1 microns wide, and varied in shape, whereas those of the inner lamellae, were 52.7 microns long, 2.6 microns wide and 4.1 microns thick, and were elongated, spindle-shaped cells with serrated outlines. These latter SMCs extended obliquely, and partially overlapped each other. The surface of the SMCs in the outermost lamella exhibited a rugged texture, with nodular protrusions and oblique and longitudinal laminar folds, while the inner lamellar cells showed longitudinal laminar folds and finger-like processes on both sides of the ridges, pointing in opposite directions to the ridges. The angle of deviation from the transverse axis of the vessel, of the muscle bundles and subgroups in the outermost lamella, was 33.6 degrees, in the second and third lamellae, 22.5 degrees, and in the innermost lamellae, 12.8 degrees. The mean angle of the muscle bundle and subgroup arrangement, with respect to the long axis of the vessel, however, was basically 90 degrees in all lamellae.

Animals↗
PubMed1992

Patent ductus venosus with hypoplastic right hepatoportal system in a young child born with asymmetric intra-uterine growth retardation.

We report a functioning ductus venosus with hypoplasia of the right hepatoportal system in a 2-year-old child born with asymmetric intra-uterine growth retardation. Postprandial galactosaemia and hyperammonaemia were clues to diagnosis of portal-systemic shunt through the patent ductus venosus, which was confirmed by ultrasonography and angiography.

Abnormalities, Multiple↗
PubMed1992

Latex agglutination test for detection of mutans streptococci in relation to dental caries in children.

A simple and rapid system based on a latex agglutination (LA) reaction was devised for the detection of mutans streptococci in dental plaque. Latex particles were sensitized with antibodies against whole cells of Streptococcus mutans strains MT8148 (serotype c), MT703R (e) and OMZ175 (f) and Strep. sobrinus strains B13 (d) and 6715 (g). These sensitized particles were agglutinated within a few minutes after addition of 1.0-10 ng serotype-specific antigen from the homologous organisms or the nitrous acid extract of whole cells at 10(5)-10(6) c.f.u. The LA test specifically differentiated not only mutans streptococci from the other oral streptococci but also Strep. sobrinus from Strep. mutans. The LA test was also applicable to extracts of plaque from 206 human subjects who harboured mutans streptococci. In clinical trials, the outcome of the LA test correlated significantly with the number of mutans streptococci found in plaque (p less than 0.0001), which was quantified by the selective cultivation of mutans streptococci. Furthermore, the LA test discriminated between Strep. mutans and Strep. sobrinus from human dental plaque. The sensitivity and the specificity of the LA test for detection of mutans streptococci were 78.9 and 100%. The degree of reactivity in the LA test correlated significantly with the number of decayed tooth surfaces (p less than 0.0001) and decayed and filled tooth surfaces (p less than 0.0001). These results suggest that the LA test could be useful clinically for the detection of mutans streptococci in dental plaque as well as serving as a caries-activity test.

Antibodies, Bacterial↗
PubMed1992

Ubiquinone biosynthesis by mitochondria, sonicated mitochondria, and mitoplasts of rat liver.

Ubiquinone was biosynthesized when rat liver mitochondria were incubated with S-adenosyl-L-methionine, solanesyl diphosphate, and [U-14C]p-hydroxybenzoate. The intermediates of ubiquinone biosynthesis but not ubiquinone were accumulated in mitochondria incubated without S-adenosyl-L-methionine and the accumulated intermediates were converted to ubiquinone by the addition of the methyl group donor and an excess of cold p-hydroxybenzoate. No solaneylated compounds except nonaprenyl p-hydroxybenzoate were found in sonicated mitochondria, while the biosynthesis of ubiquinone was observed in the sonicated preparation of mitochondria in which the intermediates accumulated. The results indicate that the initial decarboxylation reaction is completely abolished and the subsequent reactions of hydroxylation and methylation are not completely inhibited by the sonication treatment and therefore the decarboxylation reaction is the next step after nonaprenylation of p-hydroxybenzoate. Mitoplasts could biosynthesize ubiquinone with activity comparable to that of intact mitochondria, suggesting that components of the outer membrane and the intermembranous space of mitochondria are not involved in ubiquinone biosynthesis.

Animals↗
PubMed1992

A novel terminal oxidase, cytochrome baa3 purified from aerobically grown Pseudomonas aeruginosa: it shows a clear difference between resting state and pulsed state.

A novel type of cytochrome c oxidase was purified to homogeneity from Pseudomonas aeruginosa which was grown aerobically. The purified oxidase contained two molecules of heme a, two atoms of copper, and one molecule of protoheme per molecule. One of the two heme a molecules in the oxidase reacted with carbon monoxide, so that the enzyme was of baa3-type. The oxidase molecule was composed of three subunits with molecular weights of 38,000, 57,000, and 82,000. Although the oxidase oxidized ferrocytochrome c-550 obtained from the bacterial cells grown aerobically, the oxidizing activity was not high. The "resting form" and the "pulsed form" of the oxidase were observed clearly with this enzyme, and the transition from the resting form to the pulsed form was accompanied by a distinct change of the enzymatic activity. The difference in the kinetics of the catalytic reactions between the two forms is discussed.

Carbon Monoxide↗