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Biomedical subjects

M Hirose

Publications and source records attributed to M Hirose.

At least 325 records · Page 18Linked to original sources
PubMed1996

Structural and functional features of bile canaliculi in adult rat hepatocyte spheroids.

Spheroids of adult rat hepatocytes are spherical cell aggregates which retain three-dimensional architecture and hepatocyte specific functions. In this study, we investigated the detailed structure and function of bile canaliculi in spheroids. Hepatocytes were prepared from adult rat liver and cultured with epidermal growth factor (50 ng/ml). Hepatocytes formed floating spheroids 4 days after inoculation. The morphology of hepatocyte spheroids was investigated after fluorescent staining for actin using confocal laser scanning microscopy and electron microscopy. To study the function of bile canaliculi, the transcellular transport of fluorescein diacetate was observed. These experiments were performed in a control group and in a group treated with the actin inhibitor cytochalasin B. In a control group, spheroids contained bile canalicular structures which were surrounded by actin filaments. Added fluorescent dye was secreted and pooled in bile canaliculi. Cytochalasin B caused marked distention of bile canaliculi and prominent accumulation of secreted fluorescent dye in dilated bile canaliculi. This phenomenon was based on the impairment of contractile movement of bile canaliculi. These results demonstrate that hepatocyte spheroids maintain functional and morphological peculiarity, and therefore this model may be useful in investigation of the mechanism of bile formation and intrahepatic cholestasis.

Actins↗
PubMed1996

Studies on 13C-phenacetin metabolism. II. A combination of breath test and urine test of in vivo metabolites in the diagnosis of liver disease.

We determined the optimum 13C-labeling position in phenacetin for use in a breath test to diagnose liver disease based on infrared spectroscopy detection of 13CO2 in exhaled air. ([1-13C]Ethoxy)phenacetin gave the best result. This compound was also employed in a urine test using 13C-NMR spectroscopy. In the urine test, healthy subjects gave a higher signal of phenacetin than of its metabolite, phenetidine, whereas in patients with liver disease the situation was the reverse. The combination of the breath and urine tests may be a valuable new tool for the diagnosis of liver disease.

Acute Disease↗
PubMed1996

Negative chronotropic and inotropic effects of U-92032, a novel T-type Ca2+ channel blocker, on the isolated, blood-perfused dog atrium.

We investigated the effects of U-92032 ((7-((bis-4-fluorophenyl) methyl)-1-piperazinyl)-2-2(2-hydroxyethylamino)-4-(1-methylethyl)- 2,4, 6-cycloheptatrien-1-one), a novel T-type Ca2+ channel blocker, on sinus rate and atrial contractile force in the isolated, blood-perfused atrium of the dog. U-92032 (1 to 300 nmol) induced negative chronotropic and inotropic responses in a dose-dependent manner, and the percentage decrease in sinus rate was less than that in atrial contractile force. Atropine did not affect the negative responses to U-92032. These results suggest that U-92032, a T-type Ca2+ channel blocker, simultaneously decreases the sinus rate and atrial force as do L-type Ca2+ channel blockers in the isolated dog atrium.

Analysis of Variance↗
PubMed1996

Role of the intrachain disulfide bond of ovalbumin during conversion into S-ovalbumin.

Disulfide-reduced and carboxymethylated ovalbumin was treated at pH 9.9 and 55 degrees C for 24 h as a specific condition for preparation of S-ovalbumin. The stability and conformation of the product were investigated. Such alkaline treatment converted native protein to S-ovalbumin, but this modified ovalbumin was not stabilized, according to results of calorimetric analysis. Instead, it had lost its native like conformation; the magnitude of CD spectra decreased. The conformation after alkaline treatment was not clear, but the possibility of aggregation was excluded by electrophoretic analysis. These observations showed that the transformation of native ovalbumin into S-ovalbumin requires the presence of the disulfide bond.

Alkalies↗
PubMed1996

Effects of wortmannin, a novel myosin light-chain kinase inhibitor, on bile canalicular contraction in vitro and in vivo.

BACKGROUND: The cytoskeletal system is believed to play an important role in normal bile formation. The effects of wortmannin, a new myosin light-chain kinase inhibitor, on bile canalicular contraction and bile flow have been observed. METHODS: The bile canalicular contraction of cultured hepatocyte doublets was investigated, using an image analyzer with a phase contrast microscope, and the intracellular Ca2+ concentration was measured, using microscopic fluorometry. We also investigated bile flow by in vivo intraportal infusion of the drug in rats. RESULTS: Treatment with wortmannin inhibited norepinephrine-induced canalicular contraction and caused a decrease in bile flow without changing systematic and portal blood pressure. Morphologic examination of the electron microscopic study showed that most bile canaliculi were dilated, with loss of microvilli, but no other apparent damage was seen in parenchymal hepatocytes. CONCLUSIONS: These data suggest that the integrity of the phosphorylation system of myosin is essential for normal bile flow.

Androstadienes↗
PubMed1996

[Clinicopathologic assessment of primary thymic carcinoma based on eighteen cases].

In order to study clinicopathologic characteristics of thymic carcinomas, 18 patients with thymic carcinoma and 56 patients with invasive thymoma were reviewed. All of them were underwent thoracotomy. Both 5-year and 10-year survival rates were 44% in the thymic carcinoma patients, while 5-year survival and 10-year survival rates were respectively 62% and 52% in the invasive thymoma patients. The survival rate of the thymic carcinoma cases was significantly lower than that of invasive thymoma cases (p < 0.05). Both surgical technique and anticancer chemotherapy. However, have been improved in these years, such as artificial vessels for great veins, panpleuro-pneumonectomy, cisplatin and so on. Those new techniques have brought the significantly higher survival rate in recent 10 cases of thymic carcinoma patients. Three-year survival rate of 10 cases after 1990 was 80% and it was 13% in 8 cases before 1989 (p < 0.01). We conclude that surgical procedure are necessary for the treatment of thymic carcinoma patients, and both chemotherapy and radiotherapy should be combined with the surgical procedures to completely remove the tumors.

Adult↗
PubMed1996

[Assessment of prognostic factors in surgical treatment for the pulmonary metastasis of osteosarcoma based on results of 17 patients].

Seventeen cases with surgical treatment for pulmonary metastases of osteosarcomas have been experienced for past 20 years in our institute. Disease free intervals, tumor doubling time, the number of metastatic lesions, the size of metastatic lesions, pathological features on nuclear findings were assessed in relation to their prognosis. These parameters were concluded to have no significant relations to the survival outcomes after the surgery for pulmonary lesions except for the number of metastatic lesions less than 20 in total which is likely to be associated with long survivals, supporting previous reports on the resectability of pulmonary metastatic lesions as an only reliable prognostic indicator. It was also stressed from our experience that the meticulous follow-up after each surgery for both primary and metastatic lesions is absolutely necessary for the early detection of recurrent pulmonary lesions in order not to overlook curable cases with aggressive treatment.

Adolescent↗
PubMed1996

[Fundamental study of lung-cancer screening by helical CT: second report: evaluation of CT images using normal volunteers].

In order to examine the use of helical computed tomography (CT) in lung cancer screening, we evaluated the effects of tube current and table feed speed on image quality. CT images of normal volunteers, which were scanned under various tube currents (150mA, 100mA, 50mA) and various table feed speeds (15mm/sec, 20mm/sec, 25mm/sec, 30mm/sec), were visually evaluated by experienced diagnostic radiologists. The images were not affected by decreasing the milliamperage. However, as table feed speed increased, images were evaluated as significantly worse. Particularly, since the degradation of the images scanned at greater than 25mm/sec was remarkable, they were judged to be unacceptable for detection of the abnormality. In conclusion, a tube current of less than 50mA and table feed speed of less than 20mm/sec are suitable in the application of helical CT for lung cancer screening.

Adult↗
PubMed1996

[Present international status and basic approach of lung and heart-lung transplantation].

Lung transplantation has been established as an optional treatment for variable irreversible diffuse lung diseases. To date, more than 5,000 patients have underwent lung transplantation, and nearly 1,000 procedures a year are performed recently. Although heart-lung transplantation has also been technically established, this procedure is confined to quite limited conditions due to the severe shortage of donors and many difficulties in operative procedure and the patient management. Preservation, post-transplantation edema, diagnosis of rejection, chronic rejection, shortage of donor organs, are principal problems in clinical lung transplantation, 24-hour preservation proved to be possible in several recent experimental studies, and the reperfusion injury has been revealed to be one of causes of post-transplantation edema. Establishment of methods for long-term pulmonary preservation and for the treatment of post-transplantation edema may be promising in the near future. Shortage of donor lung is one of major limiting factors. Research works on xenotransplantation and cadaver lung transplantation are on going, and these may help in solving this problem.

Heart-Lung Transplantation↗
PubMed1996

Interleukin-2 activated lymphocytes show improved sensitivity for class 1 HLA typing.

The use of phytohemagglutinin (PHA)/interleukin-2 (IL-2) activated lymphocytes for HLA typing and the comparison with those of fresh peripheral blood lymphocytes are described. For detecting class 1 HLA antigens, PHA/IL-2 activated lymphocytes showed higher sensitivity than fresh lymphocytes of the peripheral blood in serological microcytotoxicity testing method. Their reaction specificity for the panel of alloantisera for class 1 HLA antigens was the same. On the contrary, for detecting class 2 HLA antigens, PHA/IL-2 activated lymphocytes showed lower sensitivity than those with fresh B-cells. For class 2 HLA typing, the typing with DNA methods showed the best results. Thus, we conclude that a combination of a serological typing method using PHA/IL-2 activated lymphocytes for class 1 HLA antigens with a DNA method for typing class 2 HLA antigens currently offers the best approach for the detection of HLA antigens.

Adult↗
PubMed1995

Protein disulfide isomerase mutant lacking its isomerase activity accelerates protein folding in the cell.

We investigated the effect of protein disulfide isomerase (PDI) on in vivo protein folding of human lysozyme (h-LZM) in a specially constructed yeast coexpression system. Coexpression with PDI increased the amounts of intracellular h-LZM with the native conformation, leading to an increase in h-LZM secretion. The results indicated that PDI is a real catalyst of protein folding in the cell. The secretion of h-LZM increased even when both active sites of PDI were disrupted, suggesting that the effect of PDI resulted from a function other than the formation of disulfide bonds. This is the first finding that PDI without isomerase activity accelerates protein folding in vivo.

Base Sequence↗
PubMed1995

Structural and functional characteristics of partially disulfide-reduced intermediates of ovotransferrin N lobe. Cystine localization by indirect end-labeling approach and implications for the reduction pathway.

Ovotransferrin N lobe contains six intrachain disulfides (SS-I/Cys10-Cys45; SS-II/Cys20-Cys36; SS-III/Cys115-Cys197; SS-IV/Cys160-Cys174; SS-V/Cys171-Cys182; SS-VI/Cys228-Cys242) in a single polypeptide chain of 332 amino acid residues. Upon the protein disulfide reduction with dithiothreitol under nondenaturing conditions, the intermediate species with four, three, and two disulfides were generated. The partially disulfide-reduced intermediates were isolated, and the localization of intact disulfides in the intermediates was determined by an indirect end-labeling method. This method included the S-cyanocysteine-specific protein fragmentation, followed by gel electrophoresis and the immunochemical visualization of the C terminus-intact fragments using antiserum raised against a non-cysteine C-terminal fragment (Ser280-Arg332). Results clearly showed that first SS-IV and SS-V, second SS-III, and then SS-VI are cleaved. No reduction was observed for SS-I and SS-II under the employed reducing conditions. The conclusion was confirmed by peptide mapping analyses for the same disulfide intermediates using reverse phase high performance liquid chromatography. Transverse urea gradient gel electrophoresis and visible absorption spectra revealed that the four-disulfide intermediate, but not the three- or two-disulfide intermediate, retains essentially the same iron-binding function as the native protein. By far-UV CD analyses, the residual native conformation of the partially disulfide-reduced intermediates was found to decrease with increased number of the reduced disulfides. Implications of the partially disulfide-reduced intermediates for the disulfide-reductive unfolding pathway in ovotransferrin N lobe are discussed.

Amino Acid Sequence↗
PubMed1995

Crystal structure of diferric hen ovotransferrin at 2.4 A resolution.

The three-dimensional structure of diferric hen ovotransferrin has been determined by X-ray crystallography at 2.4 A resolution. The structure was solved by molecular replacement, using the coordinates of diferric human lactoferrin as a search model. Several rounds of simulated annealing and restrained least-squares refinement have resulted in a model structure with an R-factor of 0.171 for the data between 11.0 and 2.4 A resolution. The model comprises 5284 protein atoms (residues 5 to 686), 2 Fe3+, 2 CO3(2)- and 132 water molecules. The overall structure of ovotransferrin is similar to those of human lactoferrin and rabbit serum transferrin, being folded into two homologous lobes, each containing two dissimilar domains with one Fe3+ and one CO3(2)- bound at a specific site in each interdomain cleft. However, the relative orientation of the two lobes, which may be related to the class specificity of transferrins to receptors, is different from either human lactoferrin or rabbit serum transferrin. The angle of the relative orientation in ovotransferrin is increased by 6.8 degrees and 15.7 degrees as compared with to those in rabbit serum transferrin and human lactoferrin, respectively. Interdomain Lys209-Lys301 and Gln541-Lys638 interactions are found near the metal binding site of each lobe. The interlobe interactions and their role in the stabilization of iron binding are discussed.

Animals↗
PubMed1995

Chemoprevention by dehydroepiandrosterone and indomethacin in a rat multiorgan carcinogenesis model.

The chemopreventive efficacy of dehydroepiandrosterone (DHEA) and indomethacin (IM) alone or in combination was investigated in a rat multiorgan carcinogenesis model. These two chemicals were selected as chemopreventive agents with different functions. Animals were sequentially given five carcinogens with different organ target sites in the first 4-week initiation period. One week after its completion, the rats received 0.3% DHEA in the diet, 20 ppm IM in the drinking water, or 0.3% DHEA + 20 ppm IM until experimental week 28. DHEA enhanced hepatocarcinogenesis, but concurrent treatment with IM suppressed tumor development as compared to the DHEA group. DHEA inhibited tumor development in the thyroid, with a similar tendency observed for the small intestine. In addition, treatment with this hormone decreased occurrences of preneoplasias in the urinary bladder and seminal vesicles. Treatment with IM clearly suppressed development of preneoplasias or neoplasias in the lung and small and large intestines. In the urinary bladder, treatment with IM tended to decrease preneoplastic lesion development. Analysis of multiplicity of total tumors of any category revealed comparable values for DHEA and control groups, while the IM group showed a significant reduction. IM in combination with DHEA caused suppression as compared to DHEA alone. In a separate 8-week experiment, DHEA or IM were administered for 4 weeks after prior carcinogen application, and biochemical responses in the target organs were investigated. DHEA increased glucose-6-phosphate dehydrogenase levels in the liver but caused a decrease in the small intestine. In addition, DHEA decreased serum T4 but not T3. IM decreased prostaglandin E2 content in the small intestine. In conclusion, although DHEA or IM exert significant chemopreventive effects in multiorgans with the exception of the DHEA-treated liver case, treatment in combination did not result in amplification of their beneficial influence. Our results suggest the possible application of IM for chemoprevention in high-risk individuals, but the question of effects of DHEA in the liver must be answered before this hormone can be considered for use in humans.

Animals↗
PubMed1995

Hepatocyte growth factor inhibits intercellular communication via gap junctions in rat hepatocytes.

We demonstrated that HGF reduces gap junctional intercellular communication of rat hepatocyte doublets and increases cell spreading. Intercellular communication via gap junctions was markedly reduced in hepatocyte doublets with HGF (20 ng/ml) 3-12 hr after inoculation. Immunocytochemistry revealed spotty localization of connexin 32 on the plasma membrane of 3-hr cultured control hepatocyte doublets, whereas HGF-treated doublets showed significantly less connexin 32 staining. Genistein maintained intercellular communication in the presence of HGF, indicating that HGF reduces intercellular communication by down regulation of connexin 32 through the c-met/HGF receptor-tyrosine-kinase-mediated pathway. Furthermore, TGF-beta 1 maintained intercellular communication in the presence of HGF. Regulation of intercellular communication in hepatocytes by HGF and TGF-beta 1 might play an important role during liver regeneration.

Analysis of Variance↗
PubMed1995

Production of chicken ovalbumin in Escherichia coli.

For better understanding of the structure-function relationship in serine proteinase inhibitors, a protein engineering approach for converting non-inhibitory chicken ovalbumin (Ova) to the inhibitory form would be a highly useful model system. A prerequisite expression system for the Ova-encoding gene (Ova) was established in this study. The Ova gene was expressed in Escherichia coli with high yield using the T7 phage promoter; the amount of the recombinant Ova (re-Ova) was 29.4% of cellular proteins. SDS-PAGE and Western blotting analysis revealed that re-Ova immunoreacting with the egg ovalbumin antibody is not glycosylated. The re-Ova was purified by anion exchange chromatography into homogeneity, as evaluated by SDS-PAGE. Amino-acid and N-terminal sequence analyses confirmed that the purified product had the correct sequence designed for Ova production. As for secondary structure, re-Ova showed a far-UV circular dichroism spectrum indistinguishable from natural egg Ova. Furthermore, the proteolytic fragmentation pattern that should reflect protein conformation was exactly the same for the natural egg and re-Ova. Using the proteolytic fragments, the identity of the internal sequences for the natural and re- proteins was confirmed.

Amino Acid Sequence↗
PubMed1995

Modulating effects of ellagic acid, vanillin and quercetin in a rat medium term multi-organ carcinogenesis model.

Effects of dietary supplementation with the antioxidants ellagic acid, quercetin and vanillin were examined using a medium term multi-organ carcinogenesis model in rats. Groups of 10-15 male F344 rats were given i.p. injections of diethylnitrosamine (DEN, 100 mg/kg body wt.) and N-methylnitrosourea (MNU, 20 mg/kg body wt), s.c. injections of 1,2-dimethylhydrazine (DMH, 40 mg/kg body wt.), together with 0.05% N-butyl-N-(4- hydroxybutyl)nitrosamine (BBN) and 0.1% 2,2'-dihydroxy-di-n-propylnitrosamine (DHPN), both in the drinking water, for a total multiple initiation period of 4 weeks (DMBDD) treatment). Ellagic acid, quercetin or vanillin, each at a dose of 1% each in the diet were administered from 1 day before and throughout the carcinogen exposure period, or after completion of the initiation regimen. All surviving animals were sacrificed at the end of week 36, and major organs were examined histopathologically. In the small intestine, significant reductions in the incidence and number of tumors (adenomas and carcinomas) were observed in the groups administered ellagic acid during (8%, 0.08 +/- 0.29) or after (8%, 0.08 +/- 0.29) DMBDD treatment, and those receiving quercetin after DMBDD treatment (0%) compared to the control value (57%, 1.07 +/- 1.21). Although the incidences were not statistically significant, slightly decreased numbers of small intestinal tumors were found in the groups receiving vanillin during (0.33 +/- 0.72), or after (0.40 +/- 0.83) DMBDD treatment. The incidence of large intestinal carcinomas in the group treated with vanillin during DMBDD treatment was significantly higher (73%) than the control value (21%). These results indicated that while ellagic acid and quercetin exerted potent chemopreventive action in both the initiation and promotion stages in the present experimental system, their beneficial effects were restricted to the small intestine. Since small intestinal carcinomas are very infrequent in humans, the advantages of these phenolic compounds for human application as chemopreventors should not be overestimated.

1,2-Dimethylhydrazine↗