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Biomedical subjects

Y Sagara

Publications and source records attributed to Y Sagara.

At least 145 records · Page 8Linked to original sources
PubMed1993

Essential role of the Arg112 residue of cytochrome P450cam for electron transfer from reduced putidaredoxin.

Cytochrome P450cam (CYP101) of Pseudomonas putida PpG1 in which Arg112 is substituted by Cys was isolated by in vitro random mutagenesis of the camC gene DNA coding for P450cam. The absorption spectra of the purified mutant enzyme were similar to those of the wild type enzyme, but its substrate-dependent NADH oxidation activity in the presence of putidaredoxin (Pd) and putidaredoxin reductase (PdR) was extremely low. The rate constant of electron transfer from reduced Pd to the heme of the mutant P450cam, measured on an anaerobic stopped flow apparatus, was 1/400 of that of the wild type enzyme and the dissociation constant of the mutant P450cam for oxidized Pd was several fold higher than that of the wild type enzyme. A considerable decrease in mid-point potential of the mutant enzyme was also noted. We conclude that Arg112, which is located on the surface of the P450cam molecule and hydrogen-bonded to one of the heme propionate chains, plays an essential role in the electron transfer from Pd.

Arginine↗
PubMed1993

Requirement of phosphatidylglycerol for flagellation of Escherichia coli.

We report that phosphatidylglycerol is required for flagellation of Escherichia coli. Cells carrying the pgsA3 mutation did not form swarm rings in semisolid agar. P1 transduction experiments revealed that the potential for phosphatidylglycerol synthesis and for the formation of swarm rings was co-transducible. The pgsA3 mutant transformed with the wild type pgsA+ gene cloned into the R-plasmid vector had the potential for both phosphatidylglycerol synthesis and cell motility. Electromicroscopic and SDS-PAGE analyses showed that the pgsA3 mutation causes the lack of flagellation.

Electrophoresis, Polyacrylamide Gel↗
PubMed1993

Comparative studies of cardiac and skeletal sarcoplasmic reticulum ATPases. Effect of a phospholamban antibody on enzyme activation by Ca2+.

Vesicular fragments of skeletal (LSR) and cardiac (CSR) sarcoplasmic reticulum were compared with the aim of defining the effect of a monoclonal phospholamban (Pl) antibody (Ab). The Pl Ab has no effect on LSR, while enhancing the Ca2+ transport rates of CSR at Ca2+ concentrations below saturation. We found no direct effect of the Pl Ab on Ca2+ binding by the ATPase in the absence of ATP. Equilibrium measurements of Ca2+ binding yield positively cooperative isotherms which are best fit with a two-interacting sites equation. LSR and CSR display nearly identical affinities for Ca2+, and no effect of the Pl Ab is observed. Taking advantage of a stable CrATP-enzyme complex, we demonstrated that the stoichiometric ratio of occluded Ca2+ to catalytic sites is 2 in either LSR or CSR and that the addition of Pl Ab does not affect the Ca2+ concentration dependence of Ca2+ occluded after equilibration of the system. Interestingly, the cooperative interaction between the two Ca2+ sites is lost in the occluded state, with only one of the two sites acquiring lumenal exposure. The concentration dependence of Ca2+ inhibition of CSR ATPase phosphorylation with Pi is also unaffected by the Pl Ab. Contrary to the lack of Pl Ab effect on reactions measured at equilibrium, enhancement of phosphorylated intermediate formation by the Pl Ab is obtained in kinetic experiments in which nonsaturating Ca2+ and ATP are added to CSR preincubated with EGTA. Therefore, Ab binding to Pl reduces specifically the activation energy for a slow transition triggered by Ca2+ binding, with consequent enhancement of overall kinetics under conditions enhancing the rate-limiting contribution of this transition.

Adenosine Triphosphatases↗
PubMed1993

Complete nucleotide sequence of the 5-exo-hydroxycamphor dehydrogenase gene on the CAM plasmid of Pseudomonas putida (ATCC 17453).

We determined the complete nucleotide sequence of the first gene of Pseudomonas putida cytochrome P-450cam hydroxylase operon, camD, which encodes 5-exo-hydroxycamphor dehydrogenase. This dehydrogenase apparently consists of 361 amino acids and has a molecular mass of 38.4 kDa. Structural relationships to other zinc-containing alcohol dehydrogenases also became evident.

Alcohol Dehydrogenase↗
PubMed1993

Expression in Escherichia coli of functional cytochrome P450c17 lacking its hydrophobic amino-terminal signal anchor.

A truncated form of bovine microsomal 17 alpha-hydroxylase cytochrome P450 (P450c17) lacking its amino-terminal hydrophobic signal anchor sequence (delta 2-17) was expressed in Escherichia coli to give more than 600 nmol P450 per liter using XL1-blue as the host. The expression level of the truncated P450c17 showed variation among different host strains. When intact E. coli cells were used for analysis, the truncated P450c17 showed the typical cytochrome P450 CO-difference spectrum and type I substrate binding spectra for pregnenolone, 17 alpha-hydroxypregnenolone, progesterone, and 17 alpha-hydroxyprogesterone. The apparent Ks values for these steroids are comparable to those obtained under the same conditions with wild type P450c17. The truncated P450c17 is primarily associated with the membrane fraction from E. coli and remained with this fraction after treatment with 0.1 M Na2CO3. The 17 alpha-hydroxylase activity of the truncated form was observed using intact E. coli. Using isolated membrane fractions, the truncated P450c17 also showed 17,20-lyase activity upon reconstitution with rat liver microsomal NADPH-cytochrome P450 reductase. These results indicate that P450c17 can be associated with E. coli membranes not only via the amino-terminal hydrophobic region as expected for the wild type, nontruncated form, but also through other sequences within the polypeptide chain and that integration of the amino-terminal region into the membrane is not essential for the folding pathway leading to functional P450c17 in E. coli. This is in contrast to mammalian cells where the same truncated P450c17 is found to be inactive and the folding pathway leading to functional P450c17 appears to require a signal anchor sequence.

Animals↗
PubMed1993

Dynamical monitoring network system for perinatal care.

A new type of monitoring network system for perinatal care is proposed and has been developed. The patient monitoring system and data analyzing system are connected by a local area network (LAN). The doctor can retrieve past sampled data and results of data analysis, and make a detailed analysis at the same time that the patient is being monitored. The mainframe of the hospital information system (HIS) is connected with a data server in the perinatal care area via LAN. The database in perinatal care is supplied to the HIS and the doctor through this monitoring network system. If data sampled at maternity clinics or hospitals are once transmitted to the data server on LAN via the public telephone circuit, these data are available for the specialists on LAN. This function is utilized for supporting the obstetricians.

Computer Systems↗
PubMed1993

Role of melatonin in nocturnal prolactin secretion in women with normoprolactinemia and mild hyperprolactinemia.

OBJECTIVE: Our objective was to clarify the relationship between the nocturnal melatonin surge and the nocturnal prolactin release in women with normoprolactinemia and mild hyperprolactinemia. STUDY DESIGN: Nocturnal serum melatonin and prolactin levels were determined for a various awake/sleep and light/dark conditions in a total of 23 healthy normoprolactinemia and 9 mild hyperprolactinemia. Patterns of prolactin and melatonin levels were subject to analysis of variance. RESULTS: During sleep deprivation under continuous illumination in normoprolactinemia, neither melatonin nor prolactin increased in concentration. In contrast, during sleep deprivation in a dark environment, both hormones were increased. The maximal prolactin levels in mild hyperprolactinemia under physiologic sleep/dark were significantly higher (p < 0.01) and were reached 2 hours earlier than in normoprolactinemia. However, each woman with mild hyperprolactinemia had similar melatonin surges. Oral administration (1 mg) of melatonin to normoprolactinemia and mild hyperprolactinemia in the daytime resulted in release of prolactin in a fashion similar to that observed during the night. CONCLUSION: Melatonin can regulate nocturnal prolactin secretion independent of sleep-related factors. Furthermore, the nature of the response to exogenous melatonin administration suggests that mild hyperprolactinemia may be unusually sensitive to melatonin.

Adult↗
PubMed1993

Purification of a GTP-binding protein localized in mitochondria.

A particulate fraction consisting of heavy organelles such as nuclei and mitochondria was prepared from Ehrlich ascites tumor cells. From this fraction we have purified a GTP-binding protein with a molecular mass of 33 kDa (MTG33) by guanidine hydrochloride extraction followed by four steps of column chromatography. The Kd value of MTG33 for GTP was 17 nM. [alpha-32P]GTP-binding to MTG33 was inhibited by GTP and GDP, but not appreciably by ATP, CTP, UTP, or GMP. MTG33 hydrolyzed GTP to GDP at a rate of 4.5 mmol/min/mol protein. Subcellular fractionation analysis of mouse liver revealed that the heavy mitochondrial fraction contained the highest level of MTG33. Furthermore, dual immunofluorescence examination indicated that the staining of NIH 3T3 cells with anti-MTG33 antibody is coincident with the distribution of mitochondrial succinate dehydrogenase. Of the mouse organs examined, the heart contained the highest level of MTG33. These results strongly suggest that MTG33 is a GTP-binding protein located in mitochondria.

3T3 Cells↗
PubMed1993

Analysis of immunosuppressive mechanism to cytotoxic T lymphocyte generation in patients with advanced cancer.

In vitro activation of cytotoxic T lymphocytes (CTLs) in peripheral blood mononuclear cells (PBMCs) of patients with advanced cancer is extremely difficult. The proportion of monocytes among PBMCs is high in this type of patient. We investigated monocytosis and the mechanism of suppression of CTL generation and obtained the following findings. 1. The cell fraction ratios of PBMCs were divided into two groups: those with a higher proportion of T cells (Group I), and those with a rapid increase in monocytes and a decrease in T cells (Group II). 2. CTL generation occurred in Group I, but was either extremely low or not observed in Group II. 3. Supernatant from the culture of monocytes obtained from Group II remarkably suppressed CTL generation. 4. Statistical analysis revealed that the cancer recurrence rate over a 2-year period was significantly higher in Group II than in Group I. These findings show that monocytes are associated with the recurrence and worsening of cancer. Furthermore, the monocytes exert this effect by releasing a humoral factor that suppresses CTL activation.

Female↗
PubMed1993

Evidence for autoregulation of camR, which encodes a repressor for the cytochrome P-450cam hydroxylase operon on the Pseudomonas putida CAM plasmid.

The regulatory gene camR on the CAM plasmid of Pseudomonas putida (ATCC 17453) negatively controls expression of the cytochrome P-450cam hydroxylase operon (camDCAB) for the camphor degradation pathway and is oriented in a direction opposite to that of the camDCAB operon. In this study, we examined expression of the camR gene by monitoring the beta-galactosidase activity of camR-lacZ translational fusions in P. putida camR and camR+ strains. We found that the camR gene was autogenously regulated by its own product, CamR. To search for an operator site of the camR gene, a cam repressor (CamR)-overproducing plasmid, pHAOV1, was constructed by placing the camR gene under the control of a pL promoter. The translational initiation codon of CamR was changed by site-directed mutagenesis from GTG to ATG to improve translation efficiency. Judging from sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis, the CamR protein was expressed up to about 10% of the soluble protein of CamR-overproducing Escherichia coli JM83/pHAOV1 cells. Results of DNase I footprinting assays using the cell lysate indicated that the CamR repressor covered a single region between the camR gene and the camDCAB operon. Our findings also suggest that the camR gene autogenously regulates its own expression by binding of the gene product, CamR, to the operator, which also serves as an operator of the camDCAB operon.

Base Sequence↗
PubMed1993

Role of the free radical-scavenger system in aromatase activity of the human ovary.

The present study was undertaken to clarify the physiological role of the free radical-scavenger system in the control of estrogen production in the human ovary with age. Both superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) activities in the homogenate of the ovary significantly decreased from the premenopausal period to the menopausal period. The lipid peroxide content, however, showed a small and gradual decrease from the premenopausal to the postmenopausal period. In the premenopausal women, aromatase (Ar) activity in the microsomal fraction of the ovary showed a sharp decline and a significant negative correlation with age. Significant positive correlations were found between SOD and Ar activity, and between GSH-Px and Ar activity. Treatments of homogenate of the ovary with 10, 50 and 100 microM of H2O2 resulted in a decrease in Ar activity in a dose-dependent manner. Moreover, treatment with 10 and 50 microM of H2O2 produced a more significant decrease in the Ar activity in the premenopausal women than that in the postmenopausal women. Treatments with 0.01, 0.1, 1.0 and 10.0 U of GSH-Px before administration of 50 microM of H2O2, on the other hand, induced a recovery of the Ar activity in a dose-dependent manner. The present study showed the inhibitory effect of peroxide on Ar activity, which was attenuated by GSH-Px. These results seem to suggest that these age-related, relatively free radical predominant environments in the human ovary may have an important role in the decrease of estrogen production through the effects on the Ar activity.

Adult↗
PubMed1993

Effect of gentle massage on regression of sensory analgesia during epidural block.

We investigated the effect of gentle epigastric massage on the regression of the sensory analgesia of epidural block. Sixteen patients, who underwent minor obstetric or gynecologic surgery under epidural block with lidocaine, were divided into two groups. Group A was the control group. Group B received gentle massage of the epigastric area for 30 min. The proximal extent of sensory analgesia before massage and 0 and 30 min after the massage was T9 +/- 1, T10 +/- 1, and T10 +/- 1 in group A, and T9 +/- 1, T11 +/- 1 and L1 +/- 1 in group B (mean +/- SD), respectively. The regression of sensory analgesia in group B was significantly (P < 0.001) faster than in group A 30 min after the massage. We conclude that peripheral sensory stimulation as weak as gentle massage may initiate a series of indirect mechanisms that lead to accelerated regression of sensory analgesia.

Abdomen↗
PubMed1993

Gene structure of bovine adrenodoxin reductase.

The gene structure of bovine adrenodoxin reductase, a component of the mitochondrial steroid hydroxylating system in the adrenal cortex, was determined. When we screened a bovine genomic DNA library, using bovine adrenodoxin reductase cDNA as a probe, we isolated 10 genomic clones covering a continuous 30 kilobase (kb) region of bovine chromosomal DNA in which the adrenodoxin reductase gene spanned approximately 12 kb. The adrenodoxin reductase gene consisted of 12 exons and all the donor and acceptor sites of the exon/intron junction followed the GT/AG rule. The transcription initiation site was found to be 79 bases upstream of the translation initiation site by primer extension analysis and putative CAAT and GC boxes, but no typical TATA box, were present in the 5'-flanking region. The 5'-flanking region of this gene showed several features characteristic of promoters of house-keeping genes. The CCAGGG sequence present in the 5'-flanking region of genes for adrenodoxin and the steroid hydroxylating cytochromes P-450 in the adrenal cortex was present 13 bases upstream from the transcription initiation site. We analyzed the promoter activity of the 5'-upstream region of this gene, using as a reporter the chloramphenicol acetyltransferase gene. The region between position -757 to -376 was proved necessary for expression of this gene in mouse Y-1 tumor cells. Adrenocorticotropin did not enhance the expression of this bovine gene in the cells.

Amino Acid Sequence↗
PubMed1993

A study of cis-diamminedichloroplatinum(II) suppositories for the treatment of rabbit uterine endometrial carcinoma.

cis-Diamminedichloroplatinum (II) (cisplatin: CDDP) suppositories containing NaCl at different concentrations were prepared as a local chemotherapeutic agent for the treatment of uterine endometrial carcinoma and were administered to rabbits implanted with uterine VX2 tumor. The intrauterine CDDP histological level, as well as the antitumor effects and side effects of the suppositories to the liver and kidney were studied. The results showed high intrauterine tissue CDDP level in all suppository administrations. In particular, the NaCl-added suppositories enhanced the intrauterine CDDP level. As for antitumor effects, while the tumor growth rate of the NaCl-added suppository group was likely to be suppressed, the suppositories could not suppress tumor growth completely. The plasma platinum (Pt) level was 1.5 micrograms/ml or less and that of the liver and kidney was as low as 0.31 to 0.48 micrograms/g. No difference in levels depending on NaCl concentration was observed, nor was any abnormality found in the biochemical analysis including glutamate oxaloacetate transaminase (GOT) and blood urea nitrogen (BUN). Histopathological study revealed the degeneration of tumor cells in the NaCl-added suppository group. Minimal congestion and hemorrhage were observed in the endometria, possibly resulting from CDDP. By adding NaCl to CDDP suppositories, the uterine CDDP level and antitumor effects increased while no serious renal dysfunction was noted. Therefore, we conclude that NaCl-added CDDP suppositories are a useful local chemotherapy for endometrial carcinoma.

Animals↗
PubMed1993

Direct expression of adrenodoxin reductase in Escherichia coli and the functional characterization.

A plasmid for direct expression in Escherichia coli of the mature form bovine adrenodoxin reductase was constructed from the full-size cDNA for the enzyme [Y. Sagara, Y. Takata, T. Miyata, T. Hara, and T. Horiuchi, J. Biochem. (Tokyo), 102, 1333 (1987)] and an expression vector pCWori+. The recombinant adrenodoxin reductase was purified from the transformed E. coli cell lysates using adrenodoxin-Sepharose affinity chromatography [T. Sugiyama and T. Yamano, FEBS Lett., 52, 145 (1975)] with a yield of 2.5 mg/l of culture. The purified recombinant enzyme showed a single band on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and migration was identical with that of the authentic enzyme purified from bovine adrenal cortex mitochondria. The recombinant enzyme had Ser at its amino-terminus and the sequence of the amino terminal 9 residues was identical with that of the authentic bovine enzyme. The absorption spectrum of the recombinant enzyme showed peaks at 270, 376, and 450 nm and shoulders at 425 and 475 nm. Flavin content of the recombinant enzyme was 0.8 mol FAD/mol. The apparent Km value for bovine adrenodoxin in NADPH-cytochrome c reductase activity using a reconstitution system was 16 nM, a value comparable with that of the authentic bovine enzyme (17 nM). The cholesterol side chain cleavage activity with a reconstitution system was about 75% of that obtained when the authentic enzyme was used.

Adrenal Cortex↗
PubMed1993

[Reexpansion pulmonary edema after removal of a huge localized mesothelioma].

A 70-year-old female with hypoglycemia was admitted to our hospital for progressive dyspnea. In chest X-ray film, a mass occupied two thirds of the left hemi-thorax followed by the right shift of the mediastinum. Thoracotomy revealed that a huge tumor grew from the left upper lobe and the diaphragm without invasion. The resected tumor was 2550 g in weight. Histological diagnosis was benign fibrous mesothelioma. A large amount of expectoration started soon after the resection of the tumor. Measurement of protein in tracheal fluid and serum revealed that the event resulted from the reexpansion of the left lung after the huge mass removal. The patient recovered from pulmonary edema followed by steroid administration and respiratory management. We consider that it is useful to measure the protein in tracheal fluid and serum for diagnosis of reexpansion pulmonary edema.

Aged↗