Biomedical subjects

N Hamada

Publications and source records attributed to N Hamada.

At least 73 records · Page 4Linked to original sources

PubMed1999

Isolation and transcriptional analysis of a cellulase gene (cell) from the basidiomycete Irpex lacteus.

A gene (named cell) homologous to the cellobiohydrolase I gene (cbhl) of Trichoderma reesei was isolated and sequenced from the white rot basidiomycete Irpex lacteus MC-2. The cell open reading frame consists of 1551 bp, which is interrupted by two introns, encoding a polypeptide of 517 amino acid residues with a calculated molecular mass of 54,522 Da. The deduced amino acid sequence showed that CEL1 (the protein encoded by cell) has a modular structure consisting of a catalytic domain of 449 amino acids and a C-terminal cellulose-binding domain (CBD) of 36 amino acids separated by a proline-, serine-, threonine-rich linker region of 32 amino acids. The CEL1 catalytic domain is homologous with fungal cellobiohydrolases (CBHs) belonging to family 7 of the glycosyl hydrolases. The transcription of cell was induced in the presence of various cellulosic substrates and repressed by glucose. It was therefore concluded that the reported sequence represents the first cellulase gene isolated from the basidiomycete Irpex.

Journal Article↗

PubMed1999

Purification, characterization and gene analysis of exo-cellulase II (Ex-2) from the white rot basidiomycete Irpex lacteus.

A new exo-type cellulase, named exo-cellulase II (Ex-2), was purified from the crude enzyme preparation of Irpex lacteus. Ex-2 was very similar to the previously characterized exo-cellulase I (Ex-1) with respect to enzymatic features such as optimal pH, temperature, heat stability, and catalytic activity. However, Ex-2 exhibited greater pH stability than Ex-1. The molecular mass and carbohydrate content of Ex-2 (56,000, 4.0%) were different from those of Ex-1 (53,000, 2.0%). A cellulase gene (named cel2) encoding both Ex-2 and Ex-1 was isolated from an I. lacteus genomic library. The cel2 gene was found to consist of 1569 bp with an open reading frame encoding 523 amino acids, interrupted by two introns. The deduced amino acid sequences revealed that cel2 ORF has a modular structure consisting of a catalytic domain and a fungal-type cellulose-binding domain (CBD) separated by a serine-rich linker region. The catalytic domain was homologous to those of fungal cellobiohydrolases belonging to family 7 of the glycosyl hydrolases. Northern blot analysis showed that expression of the cel2 gene was induced by various cellulosic substrates and repressed by glucose, fructose, and lactose.

Journal Article↗

PubMed1999

Role of the amino-terminal region of Porphyromonas gingivalis fimbriae in adherence to epithelial cells.

Porphyromonas gingivalis fimbriae elicit many responses in eukaryotic cells, including mitogenicity, cytokine production, epithelial cell invasion, and cellular immune response. Specific domains of the major fimbrial protein (FimA) have been shown to be important in triggering some of these functions. The goal of the present study was to identify the domain(s) of P. gingivalis FimA responsible for specific interaction with human mucosal epithelial cells. Fimbriated P. gingivalis strains have been shown to bind to buccal epithelial cells, whereas nonfimbriated strains bind at low levels or not at all. This and other studies provide evidence that FimA mediates the adherence of P. gingivalis to oral epithelial cells. To determine the specific region(s) of P. gingivalis FimA involved in epithelial cell binding, specific antipeptide antibodies were used to inhibit the binding of iodinated purified fimbriae as well as the binding of P. gingivalis cells to epithelial cells. Antibodies directed against peptides 49 to 68 (VVMANTAGAMELVGKTLAEVK) and 69 to 90 (ALTTELTAENQEAAGLIMTAEP) were found to highly inhibit both the binding of fimbriae and the binding of P. gingivalis cells to epithelial cells. The antibody against FimA peptides 69 to 90 also reacted with P. gingivalis fimbriae in immunogold labeling and immunoblot analysis, thereby indicating that this peptide domain is exposed on the surface of fimbriae. Our results suggest that the amino-terminal domain corresponding to amino acid residues 49 to 90 of the fimbrillin protein is a major epithelial cell binding domain of P. gingivalis fimbriae.

Amino Acid Sequence↗

PubMed1999

Transformation and expression of a cloned fimA gene in Porphyromonas gingivalis.

The Porphyromonas gingivalis fimbria is an important virulence factor involved in the adherence and colonization of the organism in the oral cavity. In this study, we transformed this organism with a gene, fimA381, encoding the fimbrial subunit of P. gingivalis 381 (fimbrillin) by using the host-vector system that we developed previously and examined expression of the cloned fimA381 gene. The recombinant plasmid pYHF2 was constructed by ligating a fragment containing the fimA381 gene into the plasmid vector pYH420 and transformed into the restriction-deficient P. gingivalis host YH522. pYHF2 was autonomously maintained in YH522 cells, and the fimbrillin polypeptide (recombinant fimbrillin) was fully expressed. The molecular mass of the recombinant fimbrillin was evaluated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis as 41 kDa, which was identical to that of the native fimbrillin of strain 381. The amino acid sequences of the 20 amino-terminal residues of the recombinant fimbrillin and the native fimbrillin of the strain 381 were identical. In addition, characteristic long and thin fimbrial structures (recombinant fimbriae) that were distinguishable from the host's native fimbriae when examined by immunogold electron microscopy were observed around the cell surface of the transformants containing the fimA381 gene. These results suggested that transformation of fimA gene from a different strain of P. gingivalis followed by accumulation of the mature fimbrial subunit protein was sufficient for production of fimbrial structures that were observable by electron microscopy.

Bacterial Proteins↗

PubMed1999

An augmentation of Fas (CD95/APO-1) antigen induced by radiation: flow cytometry analysis of lymphoma and leukemia cell lines.

An augmentation of Fas antigen induced by radiation was examined using flow cytometry. Six cell lines established from lymphomas or leukemias (HD-70, FLAM-76, CML-C-1, CML-C-2, DL-40 and DL-95) were used in this study. Each cell line was distributed to two dishes. The cells in one dish were irradiated at 10 Gy with cobalt-60 gamma rays. The control cells were not irradiated. At 6 h, 24 h, and 48 h after treatment, irradiated and non-irradiated cells of each cell line were stained with fluorescein isocyanate (FITC)-conjugated anti-human Fas antibody, and analyzed with flow cytometry. Mean fluorescence intensity (MFI) values of irradiated or non-irradiated cells were examined. MFI rates (MFI value of irradiated cells/MFI value of non-irradiated cells) of each cell line were calculated at each investigated time point, and an augmentation of the Fas antigen expression with radiation was evaluated. FLAM-76 did not express Fas antigen at any time. An augmented expression of Fas antigen due to irradiation was observed in the other five cell lines. These findings strongly suggest that radiation can augment Fas antigen expression in certain tumor cells. Further studies using Fas ligand or specific anti-Fas antibody are needed.

Antibodies↗

PubMed1999

Lung metastasis suppression of the bifunctional new radiosensitizer KIN-806.

KIN-806 is one of the newly developed 2-nitroimidazole derivative hypoxic cell radiosensitizers. The tumor growth control and the suppression of lung metastasis induced by KIN-806 were evaluated. Female C3H/He mice and SCCVII tumor cells were used. 30 Gy or 40 Gy was delivered as local irradiation. 0.2 g/kg or 0.4 g/kg KIN-806 was administered 30 min before this treatment to the KIN-806 administered groups. The enhancement ratio of KIN-806 evaluated using the growth delay method was 1.8. KIN-806 showed an excellent effect as a radiosensitizer. Furthermore, KIN-806 suppressed lung metastasis regardless of radiation, and the control of the metastatic lung nodules did not depend on the irradiation dose but rather on the KIN-806 dose (0.2 g/kg < 0.4 g/kg). KIN-806 is a promising bifunctional radiosensitizer which promotes anti-tumor activity in addition to having a radiosensitizing effect.

Animals↗

PubMed1998

Structures involved in the interaction of Porphyromonas gingivalis fimbriae and human lactoferrin.

The ability of laboratory and clinical strains of Porphyromonas gingivalis to bind lactoferrin has been assessed (FEMS Immunology and Medical Microbiology, 1996, 14, 135-143). Relative binding for P. gingivalis to lactoferrin varies among strains from 3.78 to 26.62%. We also observed that fimbriated strains of P. gingivalis bind more strongly to lactoferrin as compared to nonfimbriated strains of P. gingivalis. This observation led us to study fimbrial interaction with human lactoferrin and the fine structure of these interactions. Binding of iodinated purified fimbriae was studied using an overlay assay. Iodinated fimbriae bind specifically and strongly to human lactoferrin. When various sugars were used to inhibit binding, only N-acetylgalactosamine and fucose were inhibitory. To confirm further that oligosaccharide of lactoferrin is involved in the interaction, lactoferrin was chemically deglycosylated, and fimbriae failed to bind deglycosylated lactoferrin. Antifimbriae, as well as four antipeptide antibodies against different regions of the P. gingivalis fimbrillin, were used to inhibit the interaction. Antipeptide E, directed against amino acids 81-98 (AAGLIMTAEPKTIVLKAG-C), was found to be the most effective inhibitor for the lactoferrin-fimbriae interaction. These results suggest that the binding of P. gingivalis cells to lactoferrin is lectin like, directed to a oligosaccharide of lactoferrin. Furthermore, these studies suggest that the region of fimbriae that binds to lactoferrin is the N-terminus of the molecule. It is likely that binding of lactoferrin to P. gingivalis cells results in antimicrobial activity directed against these cells by virtue of its ability to deprive the bacterial cell of needed iron.

Amino Acid Sequence↗

PubMed1998

A long-term surviving patient with cancer of papilla of Vater after local resection.

We report successful local resection for cancer of papilla of Vater in an 86-year-old woman. She was referred to our hospital because of right hypochondralgia. Abdominal ultrasonography and computed tomography showed marked dilatation of the common bile duct (CBD). Endoscopic retrograde cholangiography disclosed a small shadow defect in the terminal of the dilated CBD. Biopsy of the papilla revealed well-to-moderately differentiated adenocarcinoma. Considering her extreme old age and keeping in mind her quality of life after the operation, and the finding that the tumor was localized within the papilla and highly differentiated, we performed local resection. In addition, the intrapancreatic portion of the CBD and part of the main pancreatic duct (MPD) were further resected to secure a negative margin, confirmed by frozen section. The MPD was reapproximated to the duodenal mucosa and a choledocho-duodenostomy was performed for CBD reconstruction. Histopathological examination showed the tumor was papillary adenocarcinoma, 10 x 15 mm in size; there was no invasion beyond the sphincter of Oddi, it had partly infiltrated the CBD, but had not invaded to the pancreas or duodenum. The patient's postoperative course was not eventful and she has had good quality of life for the past 6 years since the operation, without any evidence of recurrence. Although radical pancreaticoduodenectomy is now the standard procedure in patients with malignant tumor of the papilla of Vater, local resection is a reasonable alternative for high-risk patients with highly differentiated, apparently localized carcinomas.

Adenocarcinoma↗

PubMed1998

Hepatocellular carcinoma with obstructive jaundice successfully treated with a self-expandable metallic stent.

We report on a 71-year old man with hepatocellular carcinoma (HCC) whose obstructive jaundice was successfully treated with external irradiation and a self-expandable metallic stent (EMS); Wallstent; Schneider (Europe) AG, Bülach, Switzerland. He was admitted to our hospital because of jaundice. HCC was found in S8; the tumor had invaded the bile duct with growth in the common hepatic duct. Endoscopic nasobiliary drainage was performed with difficulty. Radiation therapy to the stenosis enabled us to place a Wallstent endoscopically. He survived without icterus for 1 year.

Aged↗

PubMed1998

Allograft rejection of small bowel transplantation in pigs.

Small bowel allograft rejection in large animals has yet to be well defined. There are no specific early signs of graft rejection. The present experiments were undertaken to compare acute small bowel allograft rejection in pigs with and without FK506 and also to examine the usefulness of mucosal biopsies. Thirty-six outbred Large-White pigs were divided into (1) group 1 (n = 9): nonimmunosuppressed recipients; (2) group 2 (n = 8): FK506-immunosuppressed recipients; (3) group 3 (n = 2): autotransplant controls; and (4) donors (n = 17). Orthotopic small bowel transplantations were performed with Thiry-Vella loops for daily biopsies. The survival rate of group 2 was significantly longer than that of group 1 (P < 0.05). One best survivor in group 2 was killed at postoperative day (POD) 365. Treatment by FK506 prevented rejection, but most of the pigs died of pneumonia. In group 1, rejection began on POD 3 and progressed to severe rejection rapidly within 7 days. In group 2, rejection began from POD 6 to POD 8, but either remained mild or spontaneously improved. The differences in the routine laboratory data and the tumor necrosis factor-alpha level were not evident between the groups. Histological studies of repeated graft biopsies are thus considered to be essential for detecting signs of graft rejection.

Animals↗

PubMed1998

Nucleotide sequence of the gene encoding the RNA polymerase and the 3' non-coding region of a bovine enterovirus Japanese isolate: rapid synonymous substitutions between European and Japanese strains.

The nucleotide sequences of the genome RNA encoding the RNA polymerase and the 3' non-coding region (NCR) of bovine enterovirus (BEV) serotype I Japanese isolate, MZ468, were determined. The genetic distance between the two BEV serotype I strains, MZ468 and VG-5-27, was calculated by pairwise comparison of nucleotide sequences. The synonymous substitution rate was high (1.40 x 10(-2)/site/year), and of the same order as those of influenza virus HA, HIV-1 gag and env, and enterovirus 70 VP1 genes.

Amino Acid Sequence↗

PubMed1998

Biliary tract cancer accompanied by anomalous junction of pancreaticobiliary ductal system in adults.

BACKGROUND: Anomalous junction of the pancreaticobiliary ductal system (AJPBDS) is a congenital anomaly in which the junction is located outside the duodenal wall. Recently, attention has been focused on the high incidence of malignancy in this anomaly. The purpose of this study was to clarify the clinicopathological features of this anomaly and to determine the appropriate surgical approach for biliary tract cancer associated with AJPBDS. METHODS: The data for 38 patients with AJPBDS, including 14 who had been treated for biliary tract cancer (2 with bile duct cancer and 12 with gallbladder cancer), were retrospectively reviewed. We assessed the clinical features, characteristics of the tumor, operative procedure, and outcome for each patient. RESULTS: The incidence of malignancy in AJPBDS was 17.8% (2 patients with bile duct cancer and 3 with gallbladder cancer) in the bile duct dilatation group (n = 28) and 90% (9 patients with gallbladder cancer) in the no-dilatation group (n = 10) . The mean length of the common channel was 24.7 mm (range 20 to 35 mm) . Resection with lymphadenectomy was performed in 9 (64.3%) of 14 patients, and curative resection in 5 of these 9 patients. Ten (71%) of the 14 patients had lymph node involvement noted either at the time of initial diagnosis or at surgery. The incidence of lymph node metastasis was closely related to the depth of tumor involvement. Ten patients died of recurrence or primary cancer, from 3 to 30 months after operation. Four patients are still alive without recurrent disease from 2.5 to 13 years after operation. CONCLUSION: For patients with AJPBDS without bile duct dilatation, prophylactic cholecystectomy is recommended even if no malignant lesion is found in the gallbladder because of the high incidence of gallbladder cancer and the poor prognosis. Both early detection and curative resection of the tumor are essential for successful treatment of biliary tract cancer.

Adenocarcinoma↗

PubMed1998

Purification and some properties of 2-hydroxychromene-2-carboxylate isomerase from naphthalenesulfonate-assimilating Pseudomonas sp. TA-2.

A 2-hydroxychromene-2-carboxylate isomerase was purified from a cell-free extract of naphthalenesulfonate-assimilating Pseudomonas sp. TA-2 to an electrophoretically homogeneous state by successive column chromatography on DEAE-cellulose, DEAE-Toyopearl 650M, Sephadex G-75, Hydroxyapatite, and Mono Q. The enzyme had a molecular mass of 25 and 27 kDa as estimated by SDS-PAGE and Superdex 200, respectively. Its N-terminal 30 amino acid sequence had high homology with the deduced amino acid sequences of the 2HC2CA isomerase of nahD (a gene of naphthalene metabolism), pahD (a gene of naphthalene and phenanthrene metabolism), and doxJ (a gene of dibenzothiophene metabolism). The enzymatic product was a trans isomer. The isomerase activity was inhibited in the presence of monoiodoacetate or Hg2+, but not by preincubation with monoiodoacetate or N-ethylmaleimide. GSH functioned as a cofactor and activated the enzyme at above 0.15 mM.

Amino Acid Sequence↗

PubMed1998

Role of Arg-gingipain A in virulence of Porphyromonas gingivalis.

In order to access the role of the Porphyromonas gingivalis Arg-gingipain proteases in the virulence of this organism, a mutant defective in the rgpA gene was constructed in strain 381. This mutant, MT10, displayed only 40% of the Arg-specific cysteine protease activity of the wild-type strain. In addition, MT10, as well as the recently characterized protease mutant G-102, which is defective in the rgpB gene, displayed reduced self-aggregation, hemagglutination, and the ability to bind to immobilized type I collagen compared to levels of the wild-type parent. However, unlike mutant G-102, the rgpA mutant displayed increased binding to epithelial cells relative to that of the parental organism. Mutant MT10 also did not express detectable levels of the FimA protein as assessed by both Western and Northern blotting or fimbriae visible by electron microscopy of the cells. Furthermore, the ability of MT10 to degrade rat tail collagen fibers when it was cultured at 37 degrees C was markedly attenuated compared to that of strain 381. These results suggest that Arg-gingipain A may play a significant role in the pathogenicity of P. gingivalis by altering the colonization and toxic properties of the organism.

Adhesins, Bacterial↗

PubMed1998

Induction of DNA fragmentation by total-body irradiation in murine liver.

Total-body irradiation (TBI) is an accepted modality to treat patients with disseminated tumors. The influence of the treatment on normal tissues is evaluated using mice by measuring the rate of the induction and distribution of apoptosis, as well as DNA fragmentation which occurs in the murine liver within hours of irradiation. Unanesthetized female C3H/He mice were exposed to gamma-ray TBI of 2, 7, and 20 gray (Gy) delivered from 60Co at a dose rate of 114 cGy/min. Frozen sections of livers which were excised from the animals at various times after irradiation were stained by hematoxylin-eosin (H-E) to count numbers of apoptotic cells, or were examined to detect DNA fragmentation. The percentages of apoptotic cells and length of the period during which the maximum levels of the percentages were exhibited showed a dose-dependent increase in the sections stained with H-E. No positive cells for 3'-OH ends of fragmented DNA were found in the liver before TBI, whereas positive cells were observed immediately after irradiation without dose-dependency, these positive cells returned to nearly basal levels after several hours. Positive cells were observed prior to showing apoptosis, suggesting that DNA fragmentation occurs immediately after TBI independent of apoptosis. The difference in the time courses between induction of DNA fragmentation and of apoptosis was not observed in other organs or in the samples treated with the detergent. These results suggested that the 3'-OH ends newly generated by TBI were masked by a detergent-soluble DNA-binding molecule which might be preferentially present in the murine liver.

Animals↗

PubMed1998

Hematuria in patients with renal hypouricemia.

The characteristics of urate metabolism in renal hypouricemic patients with hematuria were studied to clarify the risk factors for hematuria in patients with renal hypouricemia. In 16 Japanese patients with isolated renal hypouricemia, urate metabolism was measured using the urate clearance study and the subtype of renal hypouricemia [defective presecretory reabsorption (Pre), defective postsecretory reabsorption (Post), enhanced tubular secretion (Secretion) and defective presecretory and postsecretory reabsorption (Pre&Post)] were determined by the pharmacological tests. Hematuria was seen in 7 out of the 16 patients (44%), all of whom were females (58%). Serum urate and urinary urate concentrations were significantly higher in the group with hematuria (Sur = 1.76 +/- 0.31 mg/dl and Uur/Ucr = 0.75 +/- 0.12: p<0.05) than in the group without hematuria (Sur = 1.44 +/- 0.46 mg/dl and Uur/Ucr = 0.56 +/- 0.04), although there was no difference in the urate excretion rate between the two groups. Hematuria was more likely to be accompanied by Post (75%) and Secretion (75%), which showed significantly higher urinary urate concentration (Uur/Ucr = 0.75 +/- 0.1 and 0.69 +/- 0.13, respectively) than by Pre (25%) and Pre&Post (0%), which showed lower urinary urate concentration (0.61 +/- 0.06 and 0.62 +/- 0.05, respectively). The risk factors for hematuria in patients with renal hypouricemia are the elevation of urinary urate concentration and the subtypes of Post and Secretion.

Adult↗

PubMed1998

Source of reactive oxygen species in anti-Thy1 nephritis.

In proliferative glomerulonephritis, both macrophages and mesangial cells generate reactive oxygen species (ROS), contributing to the development of glomerular injury. We have attempted to determine which cell produces ROS during anti-Thy1 nephritis (ATN) in rats. The generation of ROS was studied using luminol amplified chemiluminescence (GCL) on isolated glomeruli. Immunohistochemical studies used avidin-biotin complex (ABC) to label macrophages and mesangial cells. Immediately after ATN induction, mesangiolysis and infiltration with ED-1 positive cells (referred to as macrophage) was noted with a peak at day 1. After day 4, mesangial proliferation appeared with a decrease of the ED-1 positive cells and a prominent increase of PCNA positive cells (regarded as mesangial cells). In the early phase of ATN, GCL, reflecting ROS generation, increased along with the appearance of ED-1 positive cells. GCL subsequently decreased as mesangial cells increased. This suggested that macrophage were the principal participants in ROS generation in the early phase of ATN although mesangial cells cannot be completely disregarded in the generation of ROS and development of glomerular injury.

Animals↗

PubMed1998

Changes in telomerase activity of advanced cancers of oral cavity and oropharynx during radiation therapy: correlation with clinical outcome.

Telomerase activity has been evaluated for many kinds of malignancies, and it has been clarified that the activity reflected the malignant potential of the tumor. With regard to radiation therapy for cancer, simple and reliable assays for the prediction of therapeutic effect have not been fully developed yet. Therefore, we aimed to determine whether telomerase activity is changed by radiotherapy for advanced head and neck malignancies, and to clarify the possible correlation of telomerase activity with the therapeutic effect of radiation therapy and patients' clinical outcomes. Twenty-five patients with advanced cancers of the oral cavity and oropharynx were examined. All the cancers were confirmed histopathologically as squamous cell carcinomas. Biopsies were performed before treatment and at doses of 4 and/or 10 Gy, and 20 Gy of radiotherapy. To semi-quantitate telomerase activity, a highly sensitive PCR-based telomeric repeat amplification protocol assay was performed. Nineteen of the 21 cancers (90.1%) showed telomerase activity of varied extents prior to radiation therapy. Nine of the 25 patients showed continuous elevations of telomerase activity (> 10 TPG units/microg protein) in their tumors during radiation therapy, and these nine showed 13.8-216.9 TPG units/microg protein at 20 Gy of radiotherapy. In eight of the nine patients, tumors did not respond well to radiotherapy and relapsed locally in a short period after the treatment. On the other hand, in another group of 11 patients, who showed low (< 10) and/or no activity of telomerase in their tumors at 20 Gy of radiotherapy, there were nine patients whose tumors responded well to radiotherapy (p < 0.025 by the chi2 test). None of them relapsed locally in the follow-up period of approximately 21 months, and the difference was statistically significant (p < 0.025 by the chi2 test). It is concluded that the high activity of telomerase in tumor tissue at 20 Gy of radiotherapy can predict a poor therapeutic effect of radiation therapy and unfavorable patients' outcomes for advanced cancers of oral cavity and oropharynx.

Aged↗