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Biomedical subjects

T Satoh

Publications and source records attributed to T Satoh.

At least 271 records · Page 15Linked to original sources

A chimeric inorganic pyrophosphatase derived from Escherichia coli and Thermus thermophilus has an increased thermostability.

Factors contributing to the thermostability of inorganic pyrophosphatase (PPase) were investigated by examining chimeric PPases from Escherichia coli and Thermus thermophilus (Tth). Two chimeric PPase genes, T1-135E (residues 1-135 from the N terminus are comprised of Tth PPase and residues 136-173 are derived from the C terminus of E. coli PPase) and T1-149E [residues 1-149 from the N terminus are from Tth PPase and the rest (150-175) are from E. coli PPase], were constructed by random chimeragenesis. After the genes were overexpressed in the E. coli BL21(DE3) strain and the expression products were purified, we compared the characteristics of these chimeric PPases with those of the parental PPases. We found that the two chimeras had higher activity than either parent PPase at the optimum temperature. We also examined thermal stability in terms of CD spectra, fluorescence spectra, and thermal changes in enzyme activity. The results revealed that the thermal stability of T1-149E is similar to that of Tth PPase, but T1-135E is much more stable. This suggests that the four residues that are different between T1-135E and T1-149E may be critical for thermostability between the two chimeras. By comparing the three-dimensional structures of Tth and E. coli PPases, we deduced that the following two factors may contribute to differences in thermostability. (1) Two residues (Thr138 and Ala141 in the Tth PPase and His140 and Asp143 in the E. coli PPase) in the vicinity of the trimer-trimer interface were different. (2) The Ala144-Lys145 loop in the Tth PPase was deleted in the E. coli PPase and also in the T1-135E chimera. Therefore, we conclude that T1-135E was thermostabilized by these two factors, and also, the Tth PPase moiety may contribute to the structural integrity of the chimeric enzymes.

Amino Acid Sequence↗

Anti-apoptotic function of Rac in hematopoietic cells.

The small GTP-binding protein Rac plays a pivotal role in the regulation of diverse physiological events including reorganization of the actin cytoskeleton, cell cycle progression, and transformation. Here we show an anti-apoptotic effect of Rac in interleukin-3-dependent murine hematopoietic BaF3 cells. Activated Rac(G12V), when ectopically expressed in BaF3 cells, rendered the cells resistant to apoptosis upon interleukin-3 deprivation, while activated mutants of Rho and Cdc42 displayed no significant anti-apoptotic effect. In contrast to activated Ras, which also supports cell survival in the absence of interleukin-3, Rac required fetal bovine serum for the prevention of cell death. The involvement of phosphatidylinositol 3-kinase downstream of Rac was demonstrated by the inhibition of Rac-induced cell survival by wortmannin and LY294002 and the presence of phosphatidylinositol kinase activity in the Rac immunoprecipitate. Furthermore, the serine/threonine kinase Akt was stimulated by activated Rac and fetal bovine serum in a synergistic manner. Rac-induced Akt activation was mediated by phosphorylation of threonine-308 and serine-473. In addition to the phosphatidylinositol 3-kinase/Akt pathway, the p38 mitogen-activated protein kinase pathway was crucial for Rac-dependent survival, whereas p38 mitogen-activated protein kinase nas not implicated in Ras-induced anti-apoptotic signaling. These findings provide evidence for the involvement of Rac in survival signaling of hematopoietic cells.

Animals↗

A study of novel antiallergic agents with eosinophilic infiltration inhibiting action.

The antiallergic action of a series of novel mono-O-substituted trimethylhydroquinones was investigated. Among this series of the compounds, 4-[4-[4-(diphenylmethyl)-1-piperazinyl]butoxy]-2,3,6- trimethylphenol (compound 3) showed a potent antihistaminic action (pA2 = 7.11) and an antiasthmatic action (100 mg/kg. p.o) on sensitized guinea pigs. Moreover, this compound exhibited a strong eosinophilic infiltration inhibiting action on sensitized mice (100 mg/kg p.o.).

Albumins↗

Advantage of the use of human liver S9 in the Ames test.

The mutagenicity of 13 chemicals was compared using human liver S9 or liver S9 prepared from male Sprague-Dawley rats either non-treated (R-n) or pretreated with phenobarbital/5,6-benzoflavone (R-i). The test compounds used in this study were well recognized procarcinogens requiring cytochrome P450 for metabolic activation. These included polycyclic aromatic hydrocarbons, aromatic amines, heterocyclic aromatic amines, nitrosoamines, and nitropyrene. We used four human liver S9 fractions, one of which was prepared from the liver sample having higher levels of the P450-catalyzed drug metabolizing enzyme activities, a possible explanation for which was enzyme induction by anti-asthma agents for 10 years. The results of the present study are as follows: (1) there were individual differences in the magnitude of the mutagenic activity of the procarcinogens by each S9 fraction used, (2) equivalent mutagenicity of chemicals was seen with three human S9 fractions (H3, H8, and H12), while a human H14 S9 fraction showed higher P450 enzyme activity, leading to much higher mutagenicity than the other three human S9 specimens, (3) the order of magnitude of the mutagenicity of the procarcinogens using human and rat liver S9 fractions was R-i>/=H14>/=R-n>/=H3, H8, and H12, while with 2-aminoanthrathene, N-nitrosodimethylamine, and 1-nitropyrene, this relationship was H3, H8, H12, and H14>/=R-n>/=R-i. The experimental data in the present study strongly suggest that the complementary use of human liver S9 fraction in the Ames test is a much more useful tool than rat S9 for evaluation of genotoxicity to humans.

Animals↗

The three copies of the ATP1 gene are arranged in tandem on chromosome II of Saccharomyces cerevisiae S288C.

In the yeast Saccharomyces cerevisiae there are three copies of the F(1)F(0)-ATPase alpha-subunit gene ATP1 on chromosome II (Takeda et al., 1995). However, after genome analysis using S. cerevisiae strain S288C, only one ATP1 gene sequence was observed (Feldman et al., 1994; Obermaier et al., 1995). To check whether the number of copies of ATP1 is strain-dependent or not, we carried out three different experiments: (a) long-PCR analyses of total DNAs isolated from several reference strains, carried out by preparing 29-mer oligonucleotides based on the 5'- and 3'- up- and downstream regions of the ATP1 nucleotide sequence using the data from the genome project to synthesize primers; (b) restriction analyses of chromosome II from the reference strains with SplI; and (c) long-PCR analyses of prime clones 70113 and 70804, both of which contained two ATP1 gene copies, ATP1a and ATP1b, and ATP1b and ATP1c, respectively, using 30 nucleotides just inside the 3'-end (sense) and 5'-end (antisense) of the ATP1-coding region as primers. In the case of the long-PCR experiments, the reference strains DC5, SEY2102, W303-1A, W303-1B, LL20 and DBY746, as well as strain S288C, generated a DNA fragment of approximately 32 kb, which hybridized with ATP1. During SplI digestion, a DNA fragment of more than 50 kb which hybridized with ATP1, was obtained from all reference strains. In the case of prime clone analyses using the long-PCR experiments, the distance between ATP1a and ATP1b or ATP1b and ATP1c was approximately 10 kb or 7 kb, respectively. The S288C strain generated these two DNA fragments, as do the other strains. These results showed that all these strains contained three copies of ATP1 on chromosome II.

Base Sequence↗

Ultrastructure of bovine embryos developed from in vitro-matured and -fertilized oocytes: comparative morphological evaluation of embryos cultured either in serum-free medium or in serum-supplemented medium.

The ultrastructure of bovine embryos developed from in vitro-matured and -fertilized oocytes, cocultured with bovine cumulus/granulosa cells either in a serum-free medium (IVMD101) or in a serum-containing medium (TCM199+CS) was compared. Embryos up to the eight-cell stage had many cellular organelles and cytoplasmic components that were randomly distributed in the cytoplasm. Mitochondria were spherical or ovoid and had only a few peripheral cristae. There were no obvious differences in the ultrastructure between embryos developed in IVMD101 and TCM199+CS up to the eight-cell stage. However, conspicuous differences in the ultrastructural features between the embryos cultured in IVMD101 and TCM199+CS were observed at the morula and blastocyst stages. At the morula stage, embryos cultured in IVMD101 had cells containing elongated mitochondria, well-developed Golgi apparatus, lipid droplets, and large vesicles resembling lysosomes. The lysosome-like vesicles were partially filled with electron-dense materials and were frequently fused with lipid droplets. The blastomeres of morulae cultured in TCM199+CS contained numerous large lipid droplets and fewer lysosome-like vesicles than those cultured in IVMD101. In blastocysts cultured in IVMD101, lysosome-like vesicles were frequently observed in the trophoblast cells and lipid droplets were present in the cytoplasm of trophoblast and inner cell mass (ICM)-cells, but they were not abundant. On the other hand, the blastocysts developed in TCM199+CS contained fewer lysosome-like vesicles and large numbers of lipid droplets. This accumulation of lipid droplets was higher in the trophoblast cells than in the ICM-cells. This study showed major differences in the ultrastructural features between the morulae and blastocysts from serum-free and serum-supplemented cultures, suggesting that the ultrastructural differences may reflect physiological characteristics of embryos.

Animals↗

Evolution of visual pigments and related molecules.

The molecular phylogenetic tree of vertebrate visual pigments, constructed on the basis of amino acid sequence identity, suggests that the visual pigments can be classified into five groups (L, ML, MS, S and Rh) and that their genes have evolved along these five gene lines. Goldfish has a UV-sensitive visual pigment (S group) localized in miniature single cone cells. Medaka has one type of rod cell containing rhodopsin (Rh group) and four types of cone cells, each of which contains a specific visual pigment with an absorption maximum that differs from those of the others. Frogs have a violet-sensitive visual pigment (S group) in small single cone cells and a blue-sensitive visual pigment (MS group) in green rod cells. Although nocturnal and diurnal geckos have rod- and cone-based retinas, respectively, they have phylogenetically closely related visual pigments. The pigments in each line may have restricted absorption maxima. We have cloned cDNAs encoding molecules involved in the phototransduction system of visual cells, such as phosphodiesterase, opsin kinase and arrestin. We then constructed phylogenetic trees of these molecules with the deduced amino acid sequences. The resulting phylogenetic trees show that these molecules are classified into two groups; one is expressed in cones and another in rods, suggesting that rods and cones contain homologous molecules with different amino acid sequences. These differences may result in the different light responses of rods and cones.

Animals↗

Apoptosis during iron chelator-induced differentiation in F9 embryonal carcinoma cells.

We have previously demonstrated that three potent iron chelators, hinokitiol, dithizone and deferoxamine, induce differentiation of F9 embryonal carcinoma cells, as do other well-known morphogens such as retinoic acid (RA) and sodium butyrate (NaB). In this study, we compared the patterns of cell proliferation, cell death and cell cycle arrest during the process of differentiation induced by these five agents. When F9 cells were cultured with the agents at their individual differentiation-inducing concentrations, cell proliferation was rapidly inhibited by treatment with the iron chelators and NaB. In contrast, RA did not influence the rate of increase of cell number at the concentration of 1 microm. The three chelators also caused a marked reduction in cell viability, and the treated cells exhibited internucleosomal DNA fragmentation, whereas cells treated with NaB showed no apoptotic characteristics. RA induced apoptosis weakly at 1 microm and strongly at higher concentrations. In addition, all the iron chelators hindered cell cycle progression, resulting in an arrest at the G1-S interface or S phase. The phenomena observed in chelator-treated cells were considerably different from those in RA- or NaB-treated cells. It is concluded that the three iron chelators cause both severe apoptotic cell death and cell cycle arrest of proliferating F9 cells via cellular iron deprivation, and that this apoptotic change may be independent of the process of differentiation.

Animals↗

Effects of glutamate receptor antagonists on lower urinary tract function in conscious unanesthetized rats.

OBJECTIVES: To study the effects of the intrathecal administered glutamate receptor antagonists on the bladder and urethral activities during isovolumetric bladder contraction in conscious normal and chronic spinal rats. METHODS: Twenty-eight female Wistar rats with and without previous spinal cord transection were used. Before and after intrathecal administration of glutamate receptor antagonist, urodynamic parameters under isovolmetric condition of the bladder were analyzed. RESULTS: In normal rats, MK-801 (noncompetitive N-methyl-D-aspartate [NMDA] receptor antagonist) and LY 293558 (competitive AMPA receptor antagonist) produced a decrease in bladder contraction pressure and urethral activity with dose dependent manner. In chronic spinal rats, detrusor-sphincter dyssynergia (DSD) was developed before drug administration. MK-801 and LY 293558 partially inhibited bladder contraction pressure, and markedly depressed urethral contraction concomitant with bladder contraction. LY 293558 produced urethral relaxation concomitant with bladder contraction. CONCLUSIONS: In both normal rats and chronic spinal rats, two subtypes of glutamate receptors (NMDA and AMPA receptors) in the spinal cord were involved in the control of bladder and urethral activities. The AMPA receptor in the spinal cord seems to take an important role in the development of DSD.

Animals↗

Three new mutations in the hepatocyte nuclear factor-1alpha gene in Japanese subjects with diabetes mellitus: clinical features and functional characterization.

AIMS/HYPOTHESIS: Mutations in the hepatocyte nuclear factor-1alpha gene are a common cause of the type 3 form of maturity-onset diabetes of the young. We examined the clinical features and molecular basis of hepatocyte nuclear factor-1alpha (HNF-1alpha) diabetes. METHODS: Thirty-seven Japanese subjects with early onset Type II (non-insulin-dependent) diabetes mellitus and 45 with Type I (insulin-dependent) diabetes mellitus were screened for mutations in this gene. Functional properties of mutant HNF-1alpha were also investigated. RESULTS: Three new mutations [G415R, R272C and A site of the promoter (+ 102G-to-C)] were found. Insulin secretion was impaired in the three subjects. Insulin and glucagon secretory responses to arginine in the subject with the R272C mutation were also diminished. Molecular biological studies indicated that the G415R mutation generated a protein with about 50% of the activity of wild-type HNF-1alpha. The R272C mutation had no transactivating or DNA binding activity and acted in a dominant negative manner. The + 102 G-to-C mutation in the A site of the promoter activity was associated with an increase in promoter activity and it had 42-75% more activity than the wild-type sequence. CONCLUSION/INTERPRETATION: Mutations in the HNF-1alpha gene may affect the normal islet function by different molecular mechanisms.

Adult↗

Fine structure of bovine morulae and blastocysts in vivo and in vitro.

The ultrastructure of bovine morulae and blastocysts developed from in vitro-matured and -fertilized oocytes in a serum-supplemented medium was compared with that of morulae and blastocysts collected non-surgically from superovulated cows. In the in vivo-derived morulae, two characteristic cells types could be identified by the electron-density of their cytoplasm and by their ultrastructural features. One type appeared light in color with low electron-dense cytoplasm. These cells were located in the peripheral layer of the cluster of blastomeres, possessed numerous cellular organelles such as mitochondria and Golgi apparatus and had microvilli projecting into the perivitelline space. The other cell type was distinguished by cytoplasm that stained more densely than that of the lighter-appearing cells. The darker-appearing cells generally possessed fewer organelles than the lighter cells, but many lysosome-like structures were present in the cytoplasm. The in vitro-developed morulae also contained two types of cells similar to those observed in the in vivo morulae. However, most of the in vitro-developed cells possessed numerous lipid droplets and contained fewer lysosome-like structures than the cells of the in vivo-derived morulae. The blastocysts, both in vivo and in vitro, showed a clear differentiation of trophoblast cells and inner cell mass (ICM)-cells. In the in vivo-derived blastocyst, the apical membrane of trophoblast cells was covered with large, numerous microvilli and well-developed junctional complexes were observed. Lipid droplets were present in the cytoplasm of trophoblast and ICM-cells but were not abundant. In vitro-developed blastocysts showed less well-developed junctional complexes between trophoblast cells, less well-developed apical microvilli on the trophoblast cells, and contained large numbers of lipid droplets. This accumulation of lipid droplets was higher in the trophoblast cells than in the ICM-cells. The zonae pellucidae of in vitro-developed embryos were thinner than that of the in vivo-derived embryos. This study demonstrates conspicuous differences in the ultrastructural features between the in vivo-derived and in vitro-developed embryos, suggesting that the ultrastructure may reflect the various physiological anomalies observed in previous studies.

Animals↗

Transformation of bile acids and sterols by clostridia (fusiform bacteria) in Wistar rats.

The effects on bile acid and sterol transformation of clostridia (fusiform bacteria), the dominant intestinal bacteria in rodents (ca. 10(10) counts per g wet feces) were examined in Wistar rats. After inoculation of clostridia into germ-free rats and into rats previously inoculated solely with Escherichia coli, most of the endogenous bile acids were deconjugated, and cholic acid and chenodeoxycholic acid were 7alpha-dehydroxylated to deoxycholic acid and lithocholic acid, respectively. Tauro-beta-muricholic acid, another major bile acid in rats, was deconjugated, but only part of it (ca. 30%) was transformed into hyodeoxycholic acid. Cholesterol and sitosterol were also reduced to coprostanol and sitostanol, respectively. Escherichia coli transformed neither bile acids nor sterols. These data suggest that clostridia play an important role in the formation of secondary bile acids and coprostanol in rats.

Animals↗

Brain-derived neurotropic factor prevents superoxide anion-induced death of PC12h cells stably expressing TrkB receptor via modulation of reactive oxygen species.

In our previous report (Satoh et al., 1999. Regulation of reactive oxygen species by nerve growth factor but not by Bcl-2 as a novel mechanism of protection of PC12 cells from superoxide anion-induced death. J. Biochem. 125, 952-959), we reported that nerve growth factor (NGF) protected PC12 cells from superoxide anion (O2-)-induced cell death through a novel regulation of reactive oxygen species (ROS) which increased O2- and decreased hydrogen peroxide (H2O2), indicating that decreasing conversion from O2- to H2O2 is a critical process for the protection by NGF. In the present study, we performed a comparative study on protective mechanisms between NGF and brain-derived neurotrophic factor (BDNF) using TrkB-expressing PC12h cells. When compared with NGF, BDNF induced a weaker but significant protective effect on the cells from O2- induced death. BDNF did not seem to change the total amount of ROS in the cells treated with xanthine and xanthine oxidase. On the other hand, BDNF increased O2- and decreased H2O2- levels in the same cells, although not so strongly as NGF. These results suggest that decreasing conversion from O2- to H2O2 is also critical for the protection by BDNF, which is considered to play a central role in survival and differentiation of CNS neurons.

Animals↗

Retinal photoreceptor and related gene expression in normal and clinostat-treated fish embryos.

Medaka fish had performed mating behavior successfully in space for the first time among vertebrate, and the eggs which were laid in space developed normally, and hatched during the space travel of 15 days aboard the space shuttle in the second International Microgravity Laboratory (IML-2) mission in 1994 (Ijiri 1994). But there has been few studies whether microgravity affects the development of rather complex tissues in this fish. Investigating this problem, we focused on the organogenetic events in the retina in developing Medaka under normal and simulated microgravity conditions (by a three-dimensional clinostat, 3D-clinostat). Our results showed that both normal and 3D-clinostat-treated Medaka embryos developed on almost equal time course. Moreover, we investigated the development of the retina in normal and 3D-clinostat-treated embryos, but there were no differences in organogenesis of their retina. Lamination of retina occurred almost at equal timing and the expressions of opsin genes in the 3D-clinostat-treated group also began almost at the same time as control. Our observations suggest that there were no definite effects of simulated microgravity on the organizations of a complex tissue such as retina in developing fish embryos.

Animals↗

Effect of orally active prostacyclin analogue on survival of outpatients with primary pulmonary hypertension.

OBJECTIVES: This study sought to investigate the effect of beraprost sodium (BPS), an orally active prostacyclin analogue, on the survival of outpatients with primary pulmonary hypertension (PPH). BACKGROUND: Continuous intravenous administration of epoprostenol (prostacyclin) has been shown to improve survival in PPH. However, the effect of oral BPS on survival in PPH remains unknown. METHODS: Fifty-eight consecutive patients with PPH who could be discharged after the first diagnostic catheterization for PPH were retrospectively divided into two groups: patients treated with BPS (BPS group, n = 24) and those without BPS (conventional group, n = 34). The baseline demographic and hemodynamic data did not significantly differ between the two. RESULTS: Twenty-seven patients died of cardiopulmonary causes in the conventional group during a mean follow-up period of 44 +/- 45 months. In contrast, only 4 patients died of cardiopulmonary causes in the BPS group during a mean follow-up period of 30 +/- 20 months. In a subsample (n = 15) of patients in the BPS group, mean pulmonary arterial pressure and total pulmonary resistance significantly decreased, respectively, by 13% and 25% during a mean follow-up period of 53 days. Among the variables previously known to be associated with the mortality in PPH, the absence of BPS therapy and the reduced cardiac output were independently related to the mortality by a multivariate Cox proportional hazards regression analysis (both p < 0.05). The Kaplan-Meier survival curves demonstrated that the one-, two- and three-year survival rates for the BPS group were 96%, 86% and 76%, respectively, as compared with 77%, 47% and 44%, respectively, in the conventional group (log-rank test, p < 0.05). CONCLUSIONS: The oral administration of BPS may have beneficial effects on the survival of outpatients with PPH as compared with conventional therapy alone.

Administration, Oral↗

Clinical and laboratory features of lupus patients with complicating pulmonary disease.

The objective of this study was to determine the incidence of pulmonary involvement in patients with systemic lupus erythematosus (SLE) and to clarify the clinical and laboratory characteristics in SLE patients with various pulmonary involvements. A retrospective study (n = 137) revealed that the types of pulmonary involvement found in SLE patients were: pleuritis (9%), interstitial pneumonia (8%), pulmonary infarction (7%), pulmonary infection (4%), pulmonary hypertension (2%), restrictive dysfunction (28%) and decreased diffusion capacity (43%). The incidences of pericarditis (P < 0.01), arthralgia (P < 0.05) and hypoalbuminemia (P < 0.05) were significantly greater in patients with pleuritis than in those without, while in patients with interstitial pneumonia, the incidence of anti-SS-A antibody (P < 0.05) and sicca syndrome (P < 0.05) were significantly greater than in those without. A longitudinal follow-up study of patient groups with various pulmonary involvements revealed: 1. significant changes of erythrocyte sedimentation rate (ESR), C-reactive protein (CRP), lactate dehydrogenase (LDH) and thrombomodulin (TM) in patients with pleuritis, and 2. significant changes of WBC and LDH in patients with interstitial pneumonia. The increased ESR, CRP and TM levels during disease episodes suggest that the involvement of inflammatory processes is related to vasculitic events in the pathogenesis of lupus pleuritis. A higher incidence of anti-SS-A antibody in lupus patients with interstitial pneumonia suggests a potential role for this autoantibody in the pathogenesis of this complication.

Adolescent↗

The interaction of mitogen-activated protein kinases to Epstein-Barr virus activation in Akata cells.

To understand the mechanism by which Epstein-Barr virus (EBV) is activated in Akata cells by cross-linking of surface immunoglobulin, the interaction between mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) and EBV activation was investigated. Immunoblotting using an anti-phosphoMAPK antibody (Ab) revealed that anti-IgG Ab induced rapid phosphorylation of MAPK in the cells. The phosphorylation was inhibited by MAPK/ERK kinase specific inhibitor, PD98059. The expressions of the EBV immediate early BZLF1 mRNA and its protein product ZEBRA, and early antigen were also inhibited by the inhibitor. These results indicate that MAPK is involved in the pathways of EBV activation.

B-Lymphocytes↗

Identification of an alternative form of caspase-9 in human gastric cancer cell lines: a role of a caspase-9 variant in apoptosis resistance.

Overcoming apoptosis resistance to chemotherapy and radiation may lead to a reduction in gastric cancer death. We hypothesize that the apoptotic machinery in gastric cancer cells is dependent upon specific cellular conditions. In the course of our study of the expression of apoptosis-related genes in human gastric cancer cell lines, we have identified a cDNA clone which predicts an alternative form of caspase-9. The caspase-9 variant, which we designated as caspase-9 beta, retained a truncated structure of native caspase-9 without its catalytic domain and was expressed in seven cell lines from human gastric cancer. Among the cell lines examined, MKN-28 cells, which exhibited the most resistance against apoptotic stimuli, expressed the highest level of caspase-9 beta. The induction of apoptosis by staurosporine or actinomycin D was markedly suppressed in caspase-9 beta-transfected HeLa cells. These results are consistent with our hypothesis that the caspase-9 beta may be an endogenous dominant-negative molecule which attenuates apoptotic activity in human gastric cancer cells.

Journal Article↗