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H Takagi

Publications and source records attributed to H Takagi.

At least 163 records · Page 9Linked to original sources
PubMed2000

Enhanced thermostability of the single-Cys mutant subtilisin E under oxidizing conditions.

We obtained enhanced thermostability by replacing Ser161 with Cys in subtilisin E from Bacillus subtilis, a cysteine-free alkaline serine protease. The Ser161Cys mutant subtilisin E was purified from the culture supernatant of the recombinant B. subtilis in an oxidizing environment. SDS-polyacrylamide gel electrophoresis and mass spectrometry under oxidizing conditions indicated that the mutant enzyme in part formed an oligomeric protein, which may contain an intermolecular disulfide bond between two surface Cys residues at position 161. Further, no free sulfhydryl groups were detected in the mutant enzyme, suggesting the sulfhydryl modification in a monomeric form under oxidizing conditions. The Ser161Cys mutant enzyme showed a catalytic efficiency equivalent to that of the wild-type enzyme. The half-life of thermal inactivation of the mutant was found to be 2-4 times longer than that of the wild-type enzyme. The optimum temperature of the mutant was 55 degrees C, which was 5 degrees C higher than that of the wild-type enzyme. Under reducing conditions, however, the characteristics of the mutant enzyme reverted to those of the wild-type enzyme. Similar results were obtained for another Cys mutant as to position 194 (wild-type, Ser), which is the same surface residue as Ser161. Possible reasons for the enhanced thermostability of the single-Cys mutant subtilisins E under oxidizing conditions are discussed in terms of two different mechanisms.

Amino Acid Substitution↗
PubMed2000

Modifying effects of ethinylestradiol but not methoxychlor on N-ethyl-N-nitrosourea-induced uterine carcinogenesis in heterozygous p53-deficient CBA mice.

It is unknown whether endocrine-disrupting chemicals (EDCs) with estrogenic activities have any modifying effects on uterine carcinogenesis. In our previous study, we established a uterine-carcinogenesis model that is useful for detecting tumor-modifying effects of EDCs by the administration of N-ethyl-N-nitrosourea (ENU) to female heterozygous p53-deficient CBA mice [p53 (+/-) mice]. To investigate the effects of ethinylestradiol (EE) and methoxychlor (MXC) on development of ENU-induced uterine tumors, female p53 (+/-) mice and their wild-type littermates [p53 (+/+) mice] received an intraperitoneal injection of 120 mg/kg body weight (bw) of ENU, followed, in Group 1, by no further treatment; in Group 2, by a diet containing 1 ppm EE; in Group 3, by a diet containing 5 ppm EE for 4 weeks and 2.5 ppm EE thereafter; and in Group 4, by a diet containing 2000 ppm MXC for 26 weeks. Uterine proliferative lesions that were induced were composed of both endometrial-stromal and epithelial-cell types. Endometrial stromal sarcomas were induced in p53 (+/-) mice of Groups 1 to 4, and the incidence (87%) in Group 3 was significantly increased compared to Group 1 (47%). Atypical hyperplasias (clear-cell type) of the endometrial gland in p53 (+/-) mice were seen at incidences of 0, 14, 60, and 0% in Groups 1, 2, 3, and 4, respectively, while their incidence in p53 (+/+) mice was 0, 7, 53, and 0%, respectively, with a significant difference between Groups 1 and 3 in both cases. One p53 (+/-) mouse in Group 3 also had an adenocarcinoma consisting of clear cells, and the PCNA labeling indices of the clear-cell atypical hyperplasias, and this endometrial adenocarcinoma, were higher than those of glandular hyperplasias. The present study suggests that 2.5 ppm EE, but not MXC, exerts tumor-promoting effects on stromal and epithelial proliferative lesions of the uteri in p53 (+/-) mice initiated with ENU.

Adenocarcinoma, Clear Cell↗
PubMed2000

Saccharomyces cerevisiae sigma 1278b has novel genes of the N-acetyltransferase gene superfamily required for L-proline analogue resistance.

We discovered on the chromosome of Saccharomyces cerevisiae Sigma 1278b novel genes involved in L-proline analogue L-azetidine-2-carboxylic acid resistance which are not present in the standard laboratory strains. The 5.4 kb-DNA fragment was cloned from the genomic library of the L-azetidine-2-carboxylic acid-resistant mutant derived from a cross between S. cerevisiae strains S288C and Sigma 1278b. The nucleotide sequence of a 4.5-kb segment exhibited no identity with the sequence in the genome project involving strain S288C. Deletion analysis indicated that one open reading frame encoding a predicted protein of 229 amino acids is indispensable for L-azetidine-2-carboxylic acid resistance. The protein sequence was found to be a member of the N-acetyltransferase superfamily. Genomic Southern analysis and gene disruption showed that two copies of the novel gene with one amino acid change at position 85 required for L-azetidine-2-carboxylic acid resistance were present on chromosomes X and XIV of Sigma 1278b background strains. When this novel MPR1 or MPR2 gene (sigma 1278b gene for L-proline analogue resistance) was introduced into the other S. cerevisiae strains, all of the recombinants were resistant to L-azetidine-2-carboxylic acid, indicating that both MPR1 and MPR2 are expressed and have a global function in S. cerevisiae.

Amino Acid Sequence↗
PubMed2000

Activation of the interferon-inducible 2'-5'-oligoadenylate synthetase gene by hepatitis C virus core protein.

The effects of hepatitis C virus (HCV) proteins on several signal transduction pathways in human nonneoplastic hepatocyte PH5CH8 cells were investigated using expression vectors encoding HCV proteins derived from HCV-infected human nonneoplastic cultured T-lymphocyte and hepatocyte cells (MT-2C and PH5CH7), which could support HCV replication. The amino acid sequences of HCV proteins obtained from HCV-infected human cells were identical or very close to the consensus sequences of the proteins derived from the original inoculum used for HCV infection. During the course of the study, we found that HCV core protein specifically activated the 40/46-kDa 2'-5'-oligoadenylate synthetase (2'-5'-OAS) gene promoter in a dose-dependent manner in different human hepatocyte cell lines (PH5CH8, HepG2, and PLC/PRF/5). We also found that the activation by core protein was further enhanced in the cells treated with alpha interferon. The expression of E1 or E2 envelope protein or nonstructural NS5A protein did not activate the 2'-5'-OAS gene promoter. We demonstrated that the activation by core protein in the hepatocyte cells was suppressed by antisense RNA complementary to core-encoding RNA. Deletion mutant analysis of core protein and deletion analysis of the 2'-5'-OAS gene promoter have been performed. Finally, we demonstrated that the activation of the 2'-5'-OAS gene occurred at the transcriptional level and furthermore demonstrated that the endogenous 2'-5'-OAS gene was also activated by core protein. This is the first report to show that a viral protein activated the 2'-5'-OAS gene.

2',5'-Oligoadenylate Synthetase↗
PubMed2000

Evolution of genetic codes through isologous diversification of cellular states.

Evolution of genetic codes is studied as change in the choice of enzymes that are used to synthesize amino acids from the genetic information of nucleic acids. We propose the following theory: the differentiation of physiological states of a cell allows for a choice of enzymes, and this choice is later fixed genetically through evolution. To demonstrate this theory, a dynamical systems model consisting of the concentrations of metabolites, enzymes, amino acyl tRNA synthetase, and tRNA - amino acid complexes in a cell is introduced and studied numerically. It is shown that the biochemical states of cells are differentiated by cell-cell interactions, and each differentiated type starts to use a different synthetase. Through the mutation of genes, this difference in the genetic code is amplified and stabilized. The relevance of this theory to the evolution of non-universal genetic code in mitochondria is suggested. The present theory is based on our recent theory of isologous symbiotic speciation, which is briefly reviewed. According to the theory, phenotypes of organisms are first differentiated into distinct types through the interaction and developmental dynamics, even though they have identical genotypes; later, with mutation in the genotype, the genotype also differentiates into discrete types, while maintaining the "symbiotic" relationship between the types. Relevance of the theory to natural as well as artificial evolution is discussed.

Biological Clocks↗
PubMed2000

Effect of gene disruption of succinate dehydrogenase on succinate production in a sake yeast strain.

Succinate dehydrogenase (SDH) of Saccharomyces cerevisiae consists of four subunits encoded by the SDH1, SDH2, SDH3, and SDH4 genes. We determined the effect of SDH deficiency on the productivity of organic acids in a sake yeast strain Kyokai no. 9. The SDH activity of single disruptants was retained at 30-90% of that of the wild-type strain, but the activity disappeared in double disruptants of the SDH1 and SDH2 or SDH1b (the SDH1 homologue) genes. Two double disruptants showed no growth on a medium containing glycerol as the sole carbon source, while the single disruptants could utilize glycerol. These results indicate that double disruption of the SDH1 and SDH2 or SDH1b genes is required for complete loss of SDH activity and that the SDH1b gene compensates for the function of the SDH1 gene. The sdh1 sdh1b disruptant showed a marked increase in succinate productivity of up to 1.9-fold along with a decrease in malate productivity relative to the wild-type strains under shaking conditions. Under both static and sake brewing conditions, the productivity of these organic acids in the disruptants was virtually unchanged from that in the wild-type strain. Furthermore, SDH activity was undetectable in the wild-type and the disrupted strains under static conditions. These results suggest that SDH activity contributes to succinate production under shaking conditions, but not under static and sake brewing conditions.

Journal Article↗
PubMed2000

Use of Bacillus brevis for synthesis and secretion of Des-B30 single-chain human insulin precursor.

A synthetic gene encoding a single chain human insulin precursor [B-chain (1-29)-A-chain] linked to the C-terminal lysine of human epidermal growth factor (1-28) (EGF-SCI) was constructed. This gene was expressed using Bacillus brevis. EGF-SCI was isolated from the supernatant of the culture broth. Treatment of EGF-SCI with lysyl endopeptidase resulted in the formation of des-B30 human insulin. The identification of the formed des-B30 human insulin was made by the measurement of molecular weight and amino acid analysis. The binding coefficient to anti-human insulin antibody was comparable to that of human insulin.

Amino Acid Sequence↗
PubMed2000

Efficient selection for thermostable protease in Thermus thermophilus.

An efficient procedure was established to select for thermostable proteases in an extreme thermophile, Thermus thermophilus. A non-protease-secreting mutant derived from T. thermophilus TH125 was used as host and the expression plasmid for aqualysin I from T. aquaticus YT-1 was constructed as a source of thermostable protease. T. thermophilus cells harboring the recombinant plasmid produced active aqualysin I into the medium and were able to grow on a minimal medium containing milk casein as the sole source of carbon and nitrogen.

Culture Media↗
PubMed2000

Effects of TAK-044, a nonselective endothelin receptor antagonist, on the spontaneous and indomethacin- or methylene blue-induced constriction of the ductus arteriosus in rats.

We studied the effects of TAK-044, a nonselective endothelin (ET) receptor antagonist, on the indomethacin- or methylene blue-induced constriction of the ductus arteriosus (DA) in rats and compared them with the effects on spontaneous DA constriction. Injection of TAK-044 into 21-day-old fetuses in utero was performed through the uterine wall of laparotomized mother rats under light ether anesthesia. The fetuses were autopsied 3 hr after treatment with TAK-044 (10 mg/kg) in utero and simultaneous administration to the laparotomized mother rats of indomethacin (3 mg/kg, p.o.) or methylene blue (100 mg/kg, i.p.). In the second experiment, pregnant rats were decapitated on day 21 of gestation to obtain newborn rats by cesarean delivery. Newborn rats which were given TAK-044 (2, 10 mg/kg) immediately after or 1 hr before cesarean delivery were autopsied at various times after birth. In both experiments, pups were rapidly frozen in an acetone-dry ice mixture at autopsy to evaluate the DA constriction by the whole-body freezing and shaving method. TAK-044 injection into the fetus 3 hr before autopsy completely inhibited the DA constriction induced by maternal treatment with indomethacin or methylene blue. TAK-044 caused dose-dependent inhibition of the spontaneous closure of the DA after birth. The inhibitory effect was more pronounced in pups which were given TAK-044 in utero 1 hr before birth; however, the inhibitory effect was incomplete in newborn pups. These results, together with the previous finding that BQ-123, an ETA-specific receptor antagonist, inhibits the ductal constriction induced by oxygen in vitro [Coceani et al., 1992], indicate that the ETA receptor plays a significant role in the indomethacin- or methylene blue-induced DA constriction as well as in the spontaneous DA constriction after birth, and also indicate that the inhibition of ETA receptor by TAK-044 was more easily achieved in fetuses than in neonates.

Animals↗
PubMed2000

Adaptation of the muscles of mastication to the flat skull feature in the polar bear (Ursus maritimus).

The muscles of mastication of the polar bear (Ursus maritimus) and those of the brown bear (U. arctos) were examined by anatomical approach. In addition, the examination of the skull was carried out in the polar bear, brown bear and giant panda (Ailuropoda melanoleuca). In the polar bear, the rostro-ventral part of the superficial layer of the M. masseter possessed the abundant fleshy portion folded in the rostral and lateral directions like an accordion. Moreover, the rostro-medial area of the superficial layer became hollow in the nuchal direction when the mouth was closed. The M. temporalis of the polar bear covered up the anterior border of the coronoid process of the mandible and occupied the almost entire area of the cranial surface. The M. pterygoideus medialis of the polar bear was inserted on the ventral border of the mandible and on the ventral part of the temporal bone more widely than that of the brown bear. As results of our measurements of the mandible, an effect of the leverage in the polar bear was the smallest in three species. In the polar bear, the skull was flat, and the space between zygomatic arch and ventral border of the mandible, occupied by the M. masseter was the narrowest. It is suggested that the muscles of mastication of the polar bear is adapted to the flat skull feature for supplementing the functions.

Animals↗
PubMed2000

Histological change after interferon therapy in chronic hepatitis C in view of iron deposition in the liver.

We examined the efficacy of interferon (IFN) therapy for chronic hepatitis C (CHC) in view of the change of liver histology and iron staining before and after IFN therapy. Enrolled in this study were 109 patients with CHC who completed IFN treatment and were followed for at least 1 yr after the end of IFN therapy. Serum iron, unsaturated-iron-binding capacity (UIBC), and total-iron-binding capacity (TIBC) were assessed before IFN therapy. Knodell's histological activity index (HAI) score and iron staining were examined in 55 patients in whom liver biopsy was performed at two points: before and. 1 yr after IFN therapy. Serum iron levels before IFN therapy did not correlate with the response to IFN. The HAI score significantly decreased after IFN therapy in complete responders (p < 0.01) and biochemical responders (p < 0.01). Three factors in the HAI, periportal necrosis, intralobular necrosis, and portal inflammation, but not fibrosis, were significantly decreased in complete responders (p < 0.01) and biochemical responders (p < 0.01). Of 55 patients, 23 (41.8%) were positive for iron staining before IFN therapy and 14 of 55 (25.5%) after IFN therapy. The positive rate for iron staining tended to decrease after IFN therapy, not correlating to the response to IFN, but the change was not statistically significant. In conclusion, the histological improvement by IFN therapy was mostly seen in necroinflammatory changes but not in fibrosis at least 1 yr after IFN, and iron staining tended to decrease after IFN therapy.

Adult↗
PubMed2000

Preliminary study of combination therapy with interferon-alpha and zinc in chronic hepatitis C patients with genotype 1b.

We have evaluated the efficacy of interferon-alpha (IFN-alpha) plus zinc therapy in hepatitis C patients with genotype 1b, poor responders for IFN alone. Ten patients were injected with 10 MU of IFN-alpha every day for 4 wk, followed by three times a week for 20 wk (control group). Nine patients took 300 mg of zinc sulfate a day orally during IFN-alpha therapy (zinc sulfate group), and 15 patients took IFN-alpha and 150 mg of polaprezinc (polaprezinc group). On the d 8 of IFN therapy, circadian zinc levels in serum elevated significantly in the polaprezinc group compared to the zinc sulfate group or control group. Serum ALT levels normalized in 73.3% of the polaprezinc group, 55.6% of the zinc sulfate group, and 40.0% of the control group at 6 mo after the end of IFN therapy. Sustained eradication for the hepatitis C virus RNA judged at the end of the 6-mo follow-up period was higher in the polaprezinc group than in the zinc sulfate group (53.3% vs 11.1%, p < 0.05) or the control group (20.0%). No clinical side effects of zinc were observed at the dose used. The data suggest that polaprezinc is expected to increase the therapeutic response of IFN-alpha for chronic hepatitis C with genotype 1b.

Carnosine↗
PubMed2000

Toxicity and blood concentrations of xylazine and its metabolite, 2,6-dimethylaniline, in rats after single or continuous oral administrations.

To cast light on whether the carcinogenic risk of 2,6-dimethylaniline (DMA), a metabolite of xylazine, may increase by ingestion of edible tissues from domestic animals treated with xylazine, the following studies of xylazine and DMA were performed. In Experiment I, male F344 rats received a single oral administration of 150 mg/kg of xylazine hydrochloride. Rats showed symptoms suggesting loss of sensation and pain immediately after the treatment. These signs had disappeared after 3 hr, but the animals died of hydrothorax and pulmonary edema by 9 hr. The plasma concentration of xylazine was 2.88 +/- 0.95 micrograms/ml at 15 min, and then decreased to 0.10 +/- 0.01 microgram/ml at 6 hr. The plasma level of DMA remained at 0.03 to 0.04 microgram/ml during the measurement period. In Experiment II, male F344 rats were fed a diet containing 1000 ppm of xylazine hydrochloride, regarded as the maximum tolerated dose, for 4 weeks. No clear clinical signs were evident and the plasma levels of xylazine and DMA were at the detection limit (0.02 microgram/ml) or less, although follicular cell hypertrophy of the thyroid was observed in all the treated animals. In Experiment III, male F344 rats were fed a diet containing 3000 ppm or 300 ppm of DMA for 4 weeks. Histological changes, such as atrophy of Bowman's gland and irregular arrangement of olfactory epithelial cells, were only observed in the olfactory epithelium of the 3000 ppm group. The plasma levels of DMA were 0.20 to 0.36 microgram/ml in the 3000 ppm group, but under the detection limit in the 300 ppm group. These results suggest that the probability of nasal carcinogenic effects of DNA on consumers via ingestion of edible tissues from food-producing animals treated with xylazine is extremely low, since DMA levels in the blood of rats subjected to continuous administration of high doses of xylazine remained under the detection limit.

Administration, Oral↗
PubMed2000

Effects of an agent inducing dominant lethals on rat sperm--examination with ethyl methanesulfonate.

Ethyl methanesulfonate (EMS), an alkylating agent which induces dominant lethals, was administered in oral doses of 100 mg/kg to Crj:CD(SD)IGS male rats for 5 consecutive days. At the termination of treatment and after a 28-day withdrawal, mating with untreated females and sperm analysis (motion, number, and morphology) were performed. The copulated females were sacrificed at 20 days of gestation. At the termination of treatment, no clinical signs related to EMS were observed except for a decrease in body weight. Gross pathology and sperm analysis revealed no abnormalities in treated males. However, females mated at the termination of treatment had a clearly higher fetal mortality. Females mated after the 28-day withdrawal exhibited lower fetal mortality than females mated at the termination of treatment. On the other hand, females mated after the 28-day withdrawal exhibited a lower implantation rate that was not observed in females mated at the termination of treatment. For males after a 28-day withdrawal, sperm analysis revealed both a decrease in sperm motion and number and an increase in morphological change. These findings indicate that two types of male reproductive toxicity induced by EMS can be distinguished. One induces a low implantation rate that can be detected by sperm analysis, while the other induces fetal lethals that could not be detected by sperm analysis in this study.

Animals↗
PubMed2000

Acute fatty liver of pregnancy showing microbial infection in the liver.

A 24-year-old, nulliparous woman in her 30th week of pregnancy was admitted due to threatened premature delivery. Ritodrin chloride relieved the premature contraction of the uterus but jaundice and drowsiness appeared 7 weeks later. Laboratory data revealed disseminated intravascular coagulation (DIC) with intrahepatic cholestasis, and ultrasound examination showed fatty liver. The patient was diagnosed with acute fatty liver of pregnancy (AFLP). Emergency delivery by Caesarean section was performed at 37 weeks of pregnancy and the liver function and DIC improved immediately. Liver biopsy 13 days after delivery showed nuclear swelling and cytoplasmic ballooning with mild fatty deposition. These findings were relatively compatible with acute AFLP. Higher magnification and electron microscopy revealed intracytoplasmic bacteria and fungus in the residual stage. The bacterial infection could be considered related to AFLP.

Acute Disease↗
PubMed2000

[An elderly case of ruptured aortic arch aneurysm with hemorrhagic cardiac tamponade].

We reported a case of successful aortic arch replacement using selective cerebral perfusion for ruptured distal aortic arch aneurysm (DAAA) with cardiac tamponade. A 80-year-old man who had preoperative episode of severe chest pain. Computed tomography showed saccular DAAA and pericardial effusion. He was diagnosed as ruptured DAAA with hemorrhagic cardiac tamponade. We performed urgent graft replacement of the aortic arch using selective cerebral perfusion. Postoperatively he had no complication. Thirty days after the operation he was discharged from the hospital and he is now leading a normal life.

Aged↗
PubMed2000

[A case of water intoxication during transcervical resection of the uterine myoma].

A 28-year-old woman underwent trans-cervical resection (TCR) of the uterine myoma. She had no history of complications except for anemia. Anesthesia was maintained with inhalation anesthesia. After 120 min from introducing the resectoscope, her serum sodium dropped to 86.1 mEq.l-1. But her heart rate and the systolic arterial pressure were stable. She was treated with steroid, mannitol, isotonic saline, and 7% NaHCO3. Soon after the end of the operation, she recovered consciousness. At two hours postoperatively, her serum sodium was 119.6 mEq.l-1. On the 1st postoperative day, her serum sodium returned to the normal range (137 mEq.l-1). We should be aware of asymptomatic water intoxication during TCR.

Adult↗