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Biomedical subjects

S Fujisaki

Publications and source records attributed to S Fujisaki.

At least 55 records · Page 3Linked to original sources
PubMed1994

The identification of Escherichia coli ispB (cel) gene encoding the octaprenyl diphosphate synthase.

The gene located upstream of the nlp gene at min 69 on the chromosome of Escherichia coli was cloned from the plasmid pLC7-42 constructed by Clarke and Carbon. The extract of the strain harboring the plasmid containing this gene showed increased activity of the prenyltransferase. The product of the enzyme reaction was analyzed by two systems of the thin-layer chromatography and shown to be the phosphate ester of all-E-octaprenol. These results suggested that the gene was the structural gene for the octaprenyl diphosphate synthase which supplied the precursor of the side chain of the isoprenoid quinones.

Alkyl and Aryl Transferases↗
PubMed1994

Combined immunosuppressive therapy with low dose FK506 and antimetabolites in rat allogeneic heart transplantation.

Following rat heterotopic heart allotransplantation, low to lethal doses of the antimetabolites mizoribine (MIZ), RS-61443 (RS), and AZA were given alone or in combination with subtherapeutic doses of FK506 (0.04 mg/kg/day) for 14 days after transplantation. With the median effect analysis of Chou and Kahan for quantitative drug interactions, substantial therapeutic synergism was demonstrated between FK506 and non-toxic doses of MIZ (2.5, 5, and 10 mg/kg/day) or AZA (5, 30, and 45 mg/kg/day), which was particularly evident with the lowest dose MIZ (2.5 mg/kg/day). When FK506 was used in combination with MIZ or AZA but not with RS, the maximum effect (peak median graft survival) was enhanced significantly from 15 days (MIZ alone) to 26 days (P < 0.05), and from 19 days (AZA alone) to 32 days (P < 0.01). In contrast, RS interacted with FK506 no more than additively. Although RS was the most powerful single antimetabolite, the best overall survival was obtained by combining AZA and FK506. The addition of FK506 did not significantly increase the percent mortality and LD50 of the antimetabolites.

Animals↗
PubMed1994

Overexpression of the Saccharomyces cerevisiae MET17/MET25 gene in Escherichia coli and comparative characterization of the product with O-acetylserine.O-acetylhomoserine sulfhydrylase of the yeast.

The Saccharomyces cerevisiae MET17/MET25 gene encoding O-acetyl-L-serine (OAS).O-acetyl-L-homoserine (OAH) sulfhydrylase (EC 4.2.99.10) was overexpressed in Escherichia coli and the gene product was purified to homogeneity, using three steps, with a recovery of 28% from the total cell extract. The gene product has been compared with OAS.OAH sulfhydrylase purified from the yeast cells. These two protein preparations were indistinguishable with respect to their behavior in polyacrylamide gel electrophoresis, both with and without sodium dodecyl sulfate, their specificity for substrate amino acids, Michaelis constant (Km) value for OAH, sensitivity to carbonyl reagents, absorption spectrum, isoelectric point, behavior in HPLC (both ion-exchange chromatography and gel filtration), sensitivity to heat treatment, susceptibility to trypsin digestion, and their N-terminal amino acid sequence. The results obtained imply that the gene product is properly processed in E. coli, and the technique developed in this study to overexpress the gene in bacterial cells provides us with a large amount of the purified preparation of the enzyme. In contrast to a previous report we found that cystathionine gamma-lyase of S.

Amino Acid Sequence↗
PubMed1993

Suppression of cytotoxic T-lymphocyte activity during human pregnancy.

We have investigated alterations in Epstein-Barr virus antigen specific cytotoxic T-lymphocyte (EBV-CTL) activity during human pregnancy. EBV-CTL activity was determined by a modified EBV induced B-cell focus regression assay and was expressed in terms of a regression index (IR50), i.e. the initial cell concentration required to achieve a 50%-incidence of regression in EBV-infected cell culture. Increased values of IR50 indicate the suppression of EBV-CTL activity. In 113 human female T-cell leukemia type-I (HTLV-I) non-carriers, the IR50 values (mean +/- S.E.) in non-pregnant, pregnant (the first trimester, second trimester and third trimester of pregnancy) and puerperal women were 10.6 +/- 1.4, 16.1 +/- 1.1 (20.1 +/- 2.0, 14.8 +/- 2.0, 14.6 +/- 1.6), and 12.1 +/- 1.9 respectively. Among HTLV-I carriers, the IR50 values (mean +/- S.E.) were likewise 34.6 +/- 8.0, 87.4 +/- 5.2 (101.7 +/- 6.3, 88.3 +/- 8.4 and 79.5 +/- 9.2) and 39.2 +/- 7.1 respectively. This data demonstrate: 1) EBV-CTL activity was suppressed during pregnancy (P < 0.05), especially in the first trimester (P = 0.0003). 2). In HTLV-I carriers, this suppression was shown in the first trimester (P = 0.0002), in the second trimester (P = 0.0002) and in the third trimester of pregnancy (P = 0.0014) and 3). One month after delivery, this suppression had returned to the non-pregnant level in both HTLV-I non-carriers and HTLV-I carriers. Pregnancy therefore has a suppressive effect on antigen specific cytotoxic T-lymphocyte activity and this effect is amplified in HTLV-I carriers.

Adult↗
PubMed1993

Cloning and bacterial expression of the CYS3 gene encoding cystathionine gamma-lyase of Saccharomyces cerevisiae and the physicochemical and enzymatic properties of the protein.

By screening a yeast genomic library, we isolated and characterized a gene rescuing the cysteine requirement in a "cys1" strain of Saccharomyces cerevisiae. Except for four residues in the open reading frame composed of 1,182 nucleotides, the DNA sequence was the same as that for the CYS3 (CYI1) gene, encoding cystathionine gamma-lyase (EC 4.4.1.1), and isolated previously as a cycloheximide-induced gene (B. Ono, K. Tanaka, K. Naito, C. Heike, S. Shinoda, S. Yamamoto, S. Ohmori, T. Oshima, and A. Toh-e, J. Bacteriol. 174:pp.3339-3347, 1992). S. cerevisiae "cys1" strains carry two closely linked mutations; one (cys1) causes a defect in serine O-acetyltransferase (EC 2.3.1.30), and another, designated cys3, impairs cystathionine gamma-lyase activity. Rescue of the cysteine requirement by the gene encoding cystathionine gamma-lyase is consistent with both defects being responsible for the cysteine auxotrophy. In an effort to further determine the physicochemical and enzymatic properties of this enzyme, a coding fragment was cloned into an Escherichia coli expression plasmid, and the protein was produced in the bacteria. The induced protein was extracted by sonication and purified to homogeneity through one course of DEAE-cellulose column chromatography. The yield of the protein was approximately 150 mg from cells cultured in 1 liter of L broth. The protein showed molecular weights of approximately 194,000 and 48,000 (for the subunit), suggesting a tetrameric structure. An s20,w value of 8.8 was estimated by centrifugation in a sucrose concentration gradient. No sulfhydryl groups were detected, which is consistent with the absence of cysteine residues in the coding sequence. The isoelectric point was at pH 5.2. The protein showed a number of cystathionine-related activities, i.e., cystathionine beta-lyase (EC 4.4.1.8), cystathionine gamma-lyase, and cystathionine gamma-synthase (EC 4.2.99.9) with L-homoserine as substrate. In addition, we demonstrated L-homoserine sulfhydrylase (adding H2S) activity but could find no detectable serine O-acteyltransferease activity. In this paper, we compare the enzymatic properties of the protein with those of homologous enzymes previously reported and discuss the possibility that this enzyme has a physiological role as cystathionine Beta-lyase and cystathionine gamma-synthase in addition to its previously described role as cystathionine gamma-lyase.

Acetyltransferases↗
PubMed1992

An exploration of the binding site of aldolase using alkyl glycolamido phosphoric esters and alkyl monoglycolate phosphoric esters.

Alkyl glycolamido phosphoric esters (P-O-CH2-CO-NH-(CH2)n-CH3) and alkyl monoglycolate phosphoric esters (P-O-CH2-CO-O-(CH2)n-CH3), which are analogs of the aldolase substrate fructose-1-phosphate, were synthesized and use for probing the active site of rabbit muscle aldolase. The inhibition constants (Ki) were affected by the length of the alkyl groups of these compounds and a maximum value of Ki was observed between the number of methylene groups 2 and 4, depending on the type of compound. In the previous investigation, N-(omega-hydroxyalkyl)-glycolamido bisphosphoric esters (P-O-CH2-CO-NH-(CH2)n-O-P) and alkanediol monoglyclolate bisphosphoric esters (P-O-CH2-CO-O-(CH2)n-O-P) have a minimum Ki value between the number of methylene groups 1 and 4. The difference spectra of aldolase caused by binding of alkyl glycoamido phosphoric esters or alkyl monophosphates resembled that of their analogous bisphosphoric esters, but the intensity of absorbance was smaller than that of the bisphosphoric ester analogs. These results suggest that rabbit muscle aldolase has two binding sites for the phosphate groups on the entrance end of the active site cavity, the singly wound beta-barrel of the parallel alpha/beta class structure. The distance between the phosphate binding site Lys-107 in the beta-sheet structure (c) and Arg-148 in the beta-sheet structure (d) may possibly be expanded or contracted by the forms of the bending structure of the biphosphate compounds. Also, the change of distance between the beta-sheet structure (c) and (d) containing Trp-147, may have an effect on the environment of the tryptophan and cause a change of the absorbance of aldolase especially at 295-299 nm. On the other hand, the synthetic monophosphate compounds bind at only one of the two phosphate binding sites and have very little effect on the absorbance of Trp-147, in a similar manner as orthophosphate. The alkyl groups of monophosphate may be repelled by the ionic amino acid side chains, Asp-33, Lys-146, Glu-187 and/or Lys-229 in the middle of the active site cavity. However, the end of the long alkyl group of some monophosphates may possibly contact the hydrophobic bottom of the active site cavity without effect on the environment of Trp-147.

Binding Sites↗
PubMed1991

Effects of endometrial IgG on PHA-induced T cell mitogenesis.

To clarify the characterization and immunologic mechanisms of endometrial extract as a suppressive factor in tissues of the implantation site, the effects of endometrial extract and IgG on mitogen-stimulated cultures of lymphocytes from human peripheral blood were investigated. The inhibitory activity of endometrial extracts was observed to be augmented markedly in the secretory phase of the menstrual cycle as compared to the proliferative phase. Secretory endometrial extract, at a concentration of 0.6 mg protein/ml, caused 50% suppression of PHA-induced lymphocyte blastogenesis (PHA-BL). Column fractionation of endometrial extract on a Sephadex G-200 column showed a profile with three peak fractions and demonstrated that the 2nd peak fraction was mainly responsible for the suppression of PHA-BL. The 2nd peak fraction was shown to contain IgG by the method of immunodiffusion with anti-human IgG. The 2nd peak fraction from which IgG was removed with affinity chromatography caused significant depression of PHA-BL. Furthermore, the Fc fraction of IgG showed marked suppression compared to the F(ab')2 fraction. From these results, we suggest the possibility of an endogenous substance containing IgG as a suppressive factor which is implicated in the suppression of T cell function. The Fc fragment seemed to be the major fraction possessing such suppressive activity.

Cells, Cultured↗
PubMed1991

[The effect of extract from human tubercle bacilli (SSM) on HBeAg positive type B chronic hepatitis].

UNLABELLED: The effect on the HBeAg/anti-HBe system of SSM (specific substance Maruyama), an extract from human tubercle bacilli, was evaluated in patients with HBeAg positive type B chronic hepatitis. SUBJECTS AND METHODS: Twenty-five (25) HBeAg positive patients with biopsy-proven chronic hepatitis were injected subcutaneously with SSM solution twice a week for two years. Fifteen (15) comparable patients served as untreated controls for one year. HBeAg and anti-HBe were measured by the RIA method. RESULTS: (1) In the group receiving SSM injections, HBeAg disappeared in 8 out of 25 patients (32.0%) one year after the treatment and in 15 out of 23 (65.2%) within two years. Seroconversion from HBeAg to anti-HBe occurred in 5 of the 25 patients (20.0%) after one year and in 7 out of 23 (30.4%) within two years. Of the 15 untreated controls, HBeAg disappeared in 3 patients (20%), and seroconversion from HBeAg to anti-HBe occurred in 1 (6.7%) within one year. (2) In the SSM group, the HBeAg cut off index decreased significantly (p less than 0.01), from 5.2 +/- 1.9 during the pretreatment period to 2.7 +/- 2.3 twelve months after the treatment. (3) In the SSM group, a transient rise in sGOT and sGPT was observed in some patients three to nine months after starting the treatment. HBeAg disappeared in all these patients. (4) No notable side effects were observed in any of the patients treated with SSM. CONCLUSION: These results indicate that SSM treatment improves the HBeAg/anti-HBe system in patients with HBeAg positive chronic hepatitis.

Adolescent↗
PubMed1991

Angiosarcoma of the vagina. A light and electronmicroscopy study.

A 73-year-old woman, whose medical history reported radical hysterectomy and radiotherapy for squamous cell carcinoma of the cervix uteri 20 years earlier, was found to have an angiosarcoma of the vagina. The histological diagnosis was confirmed by immunohistochemical staining of tumor cells for factor VIII-related antigen and ultrastructurally defined vasoformative structures. Primary angiosarcoma of the vagina is quite rare and reported in only two published cases. The present case is a third in general, and the first report confirmed by light microscopy, immunohistochemical, and electronmicroscopy studies.

Aged↗
PubMed1990

An exploration of the binding site of aldolase using N-(omega-hydroxyalkyl) glycolamidobisphosphoric esters.

N-(omega-Hydroxyalkyl)glycolamidobisphosphoric esters (P-O-CH2-CO-NH-(CH2)n -O-P), which are analogues of the aldolase (D-fructose-1,6-bisphosphate D-glyceraldehyde-3-phosphate-lyase, EC 4.1.2.13) substrate fructose 1,6-bisphosphate, were synthesized and used for probing its active site. These phosphate compounds competitively inhibited aldolase activity. The Ki value was lowest when the maximum distance between the phosphorus atoms of the bisphosphate was brought close to that of fructose 1,6-bisphosphate. The inhibitor constants, Ki, were compared to those of alkanediol monoglycolate bisphosphoric esters and alkanediol bisphosphate compounds, which were reported previously by Ogata et al. The values of Ki for the bisphosphate compounds containing an amide group, the amide bisphosphate compounds, were smaller than those for the bisphosphate compounds containing an ester group, the ester bisphosphate compounds, and those for alkanediol bisphosphates were the largest for the same distance between phosphorus atoms in these bisphosphates. The difference spectra of aldolase caused by binding of a saturating concentration of N-(omega-hydroxypropyl)glycolamidobisphosphoric ester resembled that of butanediol monoglycolate bisphosphoric ester. However, the effects of the amide bisphosphate compounds on the absorption spectrum of aldolase were smaller than those of the ester bisphosphate compounds for the same distance between phosphorus atoms in these bisphosphate compounds. These results suggest that the synthesized phosphate compounds bind to aldolase at the active site and the -CO-NH- group of the compounds might be held more tightly than the -CO-O- group by hydrogen bonds, presumably with the amino acid residues in the active site, such as Lys-146 or -229 and Asp-33 or Glu-187. On the other hand, the -CO-O- group might be more effective in changing the environment of the Trp-147 residue in the active site of this enzyme.

Animals↗