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Biomedical subjects

S Fujiwara

Publications and source records attributed to S Fujiwara.

At least 109 records · Page 6Linked to original sources
PubMed1999

Gene analysis and enzymatic properties of thermostable beta-glycosidase from Pyrococcus kodakaraensis KOD1.

A beta-glycosidase with broad substrate specificity was identified from a hyperthermophilic archaeon, Pyrococcus kodakaraensis KOD1. The gene encoding beta-glycosidase (Pk-gly) consists of 1449 nucleotides corresponding to a polypeptide of 483 amino acids. The protein showed similarity with other beta-glycosidases from family-1 glycosyl hydrolases, in particular, it showed high identity to beta-mannosidase from P. furiosus (55.7%), beta-glycosidase from Sulfolobus solfataricus (42.7%) and beta-glucosidase from P. furiosus (41.9%). The cloned gene was expressed in Escherichia coli and the recombinant protein was purified. The beta-glycosidase showed optimal activity at pH 6.5 and at an extremely high temperature of 100 degrees C, and had a half-life of 18 h at 90 degrees C. The beta-glycosidase hydrolyzed various pNp-beta-glycopyranosides, with kcat K(m) values in the order of pNp-beta-glucopyranoside = pNp-beta-mannopyranoside > pNp-beta-galactopyranoside > pNp-beta-xylopyranoside. pNp-beta-mannopyranoside was the substrate exhibiting the lowest K(m) value [0.254 mM] with a kcat K(m) ratio comparable to that of pNp-beta-glucopyranoside. This substrate specificity was distinct from previously reported beta-glycosidases. We observed that the region in PK-Gly corresponding to the fifth alpha-helix and beta-strand region of beta-glycosidase from S. solfataricus, which constitutes a large portion of the channel for substrate incorporation, displayed a chimeric structure, with the N-terminal region similar to beta-glycosidases and the C-terminal region similar to beta-mannosidases. An exo-type hydrolytic activity and transglycosylation activity were also observed towards cellooligomers.

Journal Article↗
PubMed1999

Differences in health characteristics between native Japanese and Japanese-Americans.

Health characteristics were compared among 1193 Japanese in Hiroshima, and 2306 Japanese-Americans in Hawaii. Japanese women experienced later menarche, earlier menopause, and a shorter interval between menarche and menopause than Japanese-Americans. Japanese men and women were shorter and lighter and the men bad lower body mass index than Japanese-Americans. Differences between populations were also found for the prevalence of artificial menopause, number of live births, lactation period, smoking, and alcohol consumption. Within one or both populations, significant trends with birth year were found for age at menarche, age at menopause, interval between menarche and menopause, lactation period, height, weight, and body mass index, suggesting the existence of cohort effects. Some of these trends may also reflect age-related changes. Environmental factors likely contribute to the differences in physique and reproductive factors, which may explain differences in frequency of osteoporosis, cardiovascular diseases, and cancer between the two populations.

Journal Article↗
PubMed1999

Fas ligand-induced apoptosis of hepatocytes in natural killer cell leukaemia.

Neoplastic natural killer (NK) cells overexpress Fas ligand (FasL), which may cause damage of Fas-bearing tissues. We report a patient with NK cell leukaemia who developed liver injury after pharyngitis. The NK leukaemic cells expressed functional FasL. In addition to soluble FasL, serum levels of interleukin-6 and interferon-gamma were increased dramatically when liver injury was aggravated. Moreover, hepatocytes expressed Fas and apoptotic hepatocytes were detected in the portal areas. These findings are consistent with the notion that inflammatory cytokines enhance the sensitivity to FasL and trigger apoptosis of hepatocytes in NK cell malignancies.

Adult↗
PubMed1999

Purification and characterization of a novel protein produced by Bifidobacterium longum SBT2928 that inhibits the binding of enterotoxigenic Escherichia coli Pb176 (CFA/II) to gangliotetraosylceramide.

A novel protein (BIF) which shows inhibitory activity on the binding of enterotoxigenic Escherichia coli Pb176 (ETEC with colonization factor antigen (CFA) II, which consists of coli surface-associated antigens CS1 and CS3) to gangliotetraosylceramide (asialo GM1 or GA1) was isolated from the culture supernatant fluid of Bifidobacterium longum SBT2928 (BL2928) at its stationary phase. The homogeneity of the final preparation of BIF was demonstrated by SDS-PAGE, polyacrylamide gel electrofocusing and N-terminal amino acid sequencing. The BIF was characterized as (i) a protein with an M(r) of approximately 104 kDa when chromatographed on a gel filtration column, and 52 kDa when separated on SDS-PAGE, and (ii) having an isoelectric point of 5.9. No change in size was produced by thiol reduction. These results suggest that BIF is a homodimer consisting of identical 52 kDa monomers. The purified BIF at the concentration of 25 micrograms protein ml-1 caused a 50% reduction in binding of the ETEC strain to GA1.

Amino Acid Sequence↗
PubMed1999

Invasion of the lacrimal system by basal cell carcinoma.

BACKGROUND: The rate of recurrence of basal cell carcinoma (BCC) in the periorbital region is higher than that in other areas because of the spread of the tumor along barrier structures. OBJECTIVE: A better understanding of the biological behavior of BCC in this area, in particular as it relates to the lacrimal system, should improve the outcome of surgery. METHODS: A study was made of two cases of BCC that developed in the periorbital region and invaded the lacrimal system. RESULTS: The tumors were found to have invaded the lacrimal system along the mucosal epithelium. Magnetic resonance imaging (MRI) did not suggest any abnormalities in this area. In one patient, the tumor had infiltrated the nasal cavity without destruction of the periorbital bone and nasal cartilage. A preoperative fiberscopic examination clearly demonstrated the involvement of the nasal cavity in this case. CONCLUSION: The lacrimal system is often invaded by BCC that originates from the periorbital region. Physicians and surgeons need to be well aware of the possibility of such aggressive infiltration by BCC.

Aged↗
PubMed1999

UDP-GlcNAc:Galbeta1-->3GalNAc (GlcNAc to GalNAc) beta1-->6N-acetylglucosaminyltransferase holds a key role on the control of CD15s expression in human pre-B lymphoid cell lines.

Expression mechanism of CD15s (sialyl-Lex, sLex) antigen has been investigated using human B lymphoid cell lines. sLexstructures were not expressed in mature B lymphoids but highly expressed in pre-B leukemia and pre-B lymphoma cell lines. The expression site was mainly on the O -linked oligosaccharide chains and E-selectin mediated-cell adhesion capability of sLex-positive cells were significantly suppressed by benzyl-alpha-GalNAc treatment. Subsequently, the bases of the sLexexpression control mechanism were examined at the levels of enzymatic activities and transcripts of glycosyltransferases. (1) The activities of alpha1-->3fucosyltransferase, alpha2-->3sialyltransferase, beta1-->4Gal-transferase, and elongation beta1-->3GlcNAc-transferase, did not correlate with sLexexpression levels. (2) The transcripts of Fuc-TVII were not parallel with sLexexpression, and those of ST3Gal IV and beta1-->4Gal-transferase were constitutively detected in all cell lines tested. (3) There was no detectable enzyme activity for core 3 and 4 backbone structure synthesis in human B cell lines. (4) By contrast, the enzyme activities and transcripts of UDP-GlcNAc:Galbeta1-->3GalNAc (GlcNAc to GalNAc) beta1-->6 N -acetylglucosaminyltransferase (Core2GnT) had significant correlation with the cell surface expression of sLexantigen. (5) Moreover, Western blot analysis revealed the presence of a major approximately 150 kDa glycoprotein that carries O -linked oligosaccharides recognized by anti-sLexmonoclonal antibody in sLex-positive pre-B leukemia cell lines. This correlation of Core2GnT with CD15s expression suggests that Core2GnT is a regulator of the cell surface expression of sLexin human pre-B lymphoid cells.

Antigens, CD↗
PubMed1999

Crystallographic studies on a family B DNA polymerase from hyperthermophilic archaeon Pyrococcus kodakaraensis strain KOD1.

A hyperthermostable family B DNA polymerase from the hyperthermophilic archaeon, Pyrococcus kodakaraensis strain KOD1, has been crystallized by the hanging-drop vapor diffusion method at 293 K with 2-methyl-2,4-pentanediol as the precipitant. The diffraction pattern of a crystal extends to 3.0 A resolution, and two full sets of 3.0 A resolution diffraction data for native crystals were successfully collected at 290 K and 100 K upon exposure to synchrotron radiation at KEK-PF, Japan. The crystals belong to the space group, P212121, with unit-cell dimensions of a = 112.8, b = 115.4, and c = 75.4 A at 290 K, and a = 111.9, b = 112.4, and c = 73.9 at 100 K. Structural analysis by means of the multiple isomorphous replacement method is now in progress.

Crystallography, X-Ray↗
PubMed1999

Characterization and application to hot start PCR of neutralizing monoclonal antibodies against KOD DNA polymerase.

DNA polymerase from Pyrococcus kodakaraensis KOD1 (KOD DNA polymerase) is one of the most efficient thermostable PCR enzymes exhibiting higher accuracy and elongation velocity than any other commercially available DNA polymerase [M. Takagi et al. (1997) Appl. Environ. Microbiol. 63, 4504-4510]. However, even when KOD DNA polymerase was used for PCR, troubles with nonspecific DNA amplification and primer dimer formation still remain because of undesirable DNA polymerase activity during the first denaturing step of PCR. In order to inhibit this undesirable DNA polymerase activity (hot start PCR), two neutralizing monoclonal antibodies (mAbs), 3G8 and betaG1, to KOD DNA polymerase were obtained. Both of these antibodies belong to subclass IgG(1), k. K(d) values were 7.3 x 10(-8) for 3G8 and 1.1 x 10(-6) for betaG1. Nucleotide sequencing of cDNAs of these monoclonal antibodies revealed their sequences to differ in their CDRs (complementarity determining region). Exonuclease activity measurement and epitope mapping revealed that the epitope for 3G8 is located in conserved regions among alpha-like (family B) DNA polymerases (Region II), and the epitope for betaG1 is located in the 3'-5' exonuclease domain. When hot start PCR with each of these mAbs was performed, the specificity of target gene amplification became much higher than in reactions without monoclonal antibody. Furthermore, this method can easily be applied to long distance PCR (>17.5 kbp).

Amino Acid Sequence↗
PubMed1999

A unique chitinase with dual active sites and triple substrate binding sites from the hyperthermophilic archaeon Pyrococcus kodakaraensis KOD1.

We have found that the hyperthermophilic archaeon Pyrococcus kodakaraensis KOD1 produces an extracellular chitinase. The gene encoding the chitinase (chiA) was cloned and sequenced. The chiA gene was found to be composed of 3,645 nucleotides, encoding a protein (1,215 amino acids) with a molecular mass of 134,259 Da, which is the largest among known chitinases. Sequence analysis indicates that ChiA is divided into two distinct regions with respective active sites. The N-terminal and C-terminal regions show sequence similarity with chitinase A1 from Bacillus circulans WL-12 and chitinase from Streptomyces erythraeus (ATCC 11635), respectively. Furthermore, ChiA possesses unique chitin binding domains (CBDs) (CBD1, CBD2, and CBD3) which show sequence similarity with cellulose binding domains of various cellulases. CBD1 was classified into the group of family V type cellulose binding domains. In contrast, CBD2 and CBD3 were classified into that of the family II type. chiA was expressed in Escherichia coli cells, and the recombinant protein was purified to homogeneity. The optimal temperature and pH for chitinase activity were found to be 85 degrees C and 5.0, respectively. Results of thin-layer chromatography analysis and activity measurements with fluorescent substrates suggest that the enzyme is an endo-type enzyme which produces a chitobiose as a major end product. Various deletion mutants were constructed, and analyses of their enzyme characteristics revealed that both the N-terminal and C-terminal halves are independently functional as chitinases and that CBDs play an important role in insoluble chitin binding and hydrolysis. Deletion mutants which contain the C-terminal half showed higher thermostability than did N-terminal-half mutants and wild-type ChiA.

Amino Acid Sequence↗
PubMed1999

Isolation and characterization of a second subunit of molecular chaperonin from Pyrococcus kodakaraensis KOD1: analysis of an ATPase-deficient mutant enzyme.

The cpkA gene encoding a second (alpha) subunit of archaeal chaperonin from Pyrococcus kodakaraensis KOD1 was cloned, sequenced, and expressed in Escherichia coli. Recombinant CpkA was studied for chaperonin functions in comparison with CpkB (beta subunit). The effect on decreasing the insoluble form of proteins was examined by coexpressing CpkA or CpkB with CobQ (cobyric acid synthase from P. kodakaraensis) in E. coli. The results indicate that both CpkA and CpkB effectively decrease the amount of the insoluble form of CobQ. Both CpkA and CpkB possessed the same ATPase activity as other bacterial and eukaryal chaperonins. The ATPase-deficient mutant proteins CpkA-D95K and CpkB-D95K were constructed by changing conserved Asp95 to Lys. Effect of the mutation on the ATPase activity and CobQ solubilization was examined. Neither mutant exhibited ATPase activity in vitro. Nevertheless, they decreased the amount of the insoluble form of CobQ by coexpression as did wild-type CpkA and CpkB. These results implied that both CpkA and CpkB could assist protein folding for nascent protein in E. coli without requiring energy from ATP hydrolysis.

Adenosine Triphosphatases↗
PubMed1999

Purification and characterization of an extremely thermostable cyclomaltodextrin glucanotransferase from a newly isolated hyperthermophilic archaeon, a Thermococcus sp.

The extremely thermophilic anaerobic archaeon strain B1001 was isolated from a hot-spring environment in Japan. The cells were irregular cocci, 0.5 to 1.0 micrometers in diameter. The new isolate grew at temperatures between 60 and 95 degrees C (optimum, 85 degrees C), from pH 5.0 to 9.0 (optimum, pH 7.0), and from 1.0 to 6.0% NaCl (optimum, 2.0%). The G+C content of the genomic DNA was 43.0 mol%. The 16S rRNA gene sequencing of strain B1001 indicated that it belongs to the genus Thermococcus. During growth on starch, the strain produced a thermostable cyclomaltodextrin glucanotransferase (CGTase). The enzyme was purified 1,750-fold, and the molecular mass was determined to be 83 kDa by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. Incubation at 120 degrees C with SDS and 2-mercaptoethanol was required for complete unfolding. The optimum temperatures for starch-degrading activity and cyclodextrin synthesis activity were 110 and 90 to 100 degrees C, respectively. The optimum pH for enzyme activity was pH 5.0 to 5.5. At pH 5.0, the half-life of the enzyme was 40 min at 110 degrees C. The enzyme formed mainly alpha-cyclodextrin with small amounts of beta- and gamma-cyclodextrins from starch. This is the first report on the presence of the extremely thermostable CGTase from hyperthermophilic archaea.

Amino Acid Sequence↗
PubMed1999

Epstein-barr virus (EBV) nuclear protein 2-induced disruption of EBV latency in the Burkitt's lymphoma cell line Akata: analysis by tetracycline-regulated expression.

The Burkitt's lymphoma (BL) cell line Akata retains the latency I program of Epstein-Barr virus (EBV) gene expression and cross-linking of its surface immunoglobulin G (IgG) by antibodies results in activation of viral replication. When EBV nuclear antigen 2 (EBNA2) was artificially expressed by a constitutive expression vector, the Cp EBNA promoter remained inactive and accordingly the latency III program was not induced. In contrast, expression of LMP2A and activity of the Fp lytic promoter were activated. Consistent with this Fp activity, the rate of spontaneous activation of the EBV replicative cycle was increased significantly, suggesting the possibility that EBNA2 can induce EBV replication. The efficiency of anti-IgG-induced activation of the viral replication was reduced in Akata cells expressing EBNA2. To obtain more direct evidence for EBNA2-induced activation of the EBV replicative cycle, this protein was next expressed by a tetracycline-regulated expression system. EBNA2 was undetectable with low doses (<0.5 microgram/ml) of tetracycline, while its expression was rapidly induced after removal of the antibiotic. This induced expression of EBNA2 was immediately followed by expression of EBV replicative cycle proteins in up to 50% of the cells, as shown by indirect immunofluorescence and immunoblot analysis. These results suggest an unexpected potential of EBNA2 to disrupt EBV latency and to activate viral replication.

Burkitt Lymphoma↗
PubMed1999

Biclonal lymphoplasmacytic immunocytoma associated with Crohn's disease.

A 33-year-old man with a 4-year history of Crohn's disease presented with marked ascites and an abdominal tumor. Two M-protein peaks, immunoglobulin (Ig) G-kappa and IgA-kappa, were detected in the serum. Neoplastic lymphoplasmacytic cells were infiltrated in the bone marrow and ascites. Histological examination of the abdominal tumor showed marked proliferation of lymphoplasmacytic cells that were positive for either IgG or IgA. Moreover, DNA sequences of the expressed IgG and IgA genes were different in the complementarity-determining region 3. These results suggest that chronic inflammation in Crohn's disease contributes to the simultaneous development of biclonal lymphoplasmacytic immunocytoma of the small intestine.

Abdominal Neoplasms↗
PubMed1999

Inhibition of Ca(2+) entry caused by depolarization in acetylcholine-stimulated antral mucous cells of guinea pig: G protein regulation of Ca(2+) permeable channels.

The effects of depolarizing conditions resulting from increasing extracellular K(+) concentration or nystatin treatment on intracellular Ca(2+) concentration ([Ca(2+)](i)) were studied in guinea pig antral mucous cells following acetylcholine (ACh) stimulation. ACh stimulation evoked a biphasic increase in [Ca(2+)](i), that is, an initial transient increase followed by a plateau. Depolarizing conditions reduced the [Ca(2+)](i) in the plateau phase during ACh stimulation. However, pertussis toxin (PTX, a G protein inhibitor) treatment caused [Ca(2+)](i) in the ACh-evoked plateau phase to increase under depolarizing conditions, while it had no effect on [Ca(2+)](i) under hyperpolarized conditions. Based on these observations, Ca(2+) permeable channels are regulated by a G protein which is activated by depolarized conditions and inhibited by hyperpolarized conditions and PTX; activation of the G protein (depolarization) causes Ca(2+) permeable channels to inhibit, and in turn, inhibition of the G protein (hyperpolarization) causes them to activate.

Acetylcholine↗
PubMed1999

99mTc-bicisate and 99mTc-HMPAO SPECT imaging in early spontaneous reperfusion of cerebral embolism.

Two patients with a cerebral embolism were evaluated by using both 99mTc-ethyl cysteinate dimer (ECD, or Bicisate) and 99mTc-hexamethylpropyleneamine oxime (HMPAO) single-photon emission computed tomography (SPECT). In one patient, 99mTc-ECD SPECT images revealed hypoactivity in a reflow hyperemic area where an infarct was seen later on CT scans. In another patient, a reperfused area showed hyperactivity on 99mTc-ECD SPECT without any abnormality on follow-up CT. 99mTc-ECD represents a potential agent with which to evaluate cerebral tissue viability in early reperfusion after ischemia.

Aged↗
PubMed1999

[Emergency coronary artery bypass grafting for acute coronary syndrome].

Between 1987 and 1998, 22 patients underwent emergency coronary artery bypass grafting for the treatment of acute coronary syndrome. Overall hospital mortality was 22.7% (5/22) versus 1.8% (9/501) in patients operated on electively (p < 0.01). There were no hospital deaths in the unstable angina group. Three patients (37.5%) in the myocardial infarction group and two patients (40%) in the failed catheter intervention group died due to postoperative low cardiac output syndrome (LOS) or mediastinitis. As a complication of mechanical supports, lower extremity ischemia was a risk factor for mortality. Postoperatively six patients (27.3%) had LOS. Univariate analyses showed that age greater than 70 years, old myocardial infarction, poor ejection fraction, preoperative shock, and left main trunk or three vessel disease were significantly associated with increased LOS. To improve the surgical results, it is considered that improvement of intraoperative myocardial protection, effective combination with catheter interventional therapy and careful use of mechanical supports are needed for these compromised patients.

Adult↗