Biomedical subjects

Y Nishimoto

Publications and source records attributed to Y Nishimoto.

At least 19 recordsLinked to original sources

PubMed1996

Isolation and characterization of cDNA for DREF, a promoter-activating factor for Drosophila DNA replication-related genes.

DREF, a transcription regulatory factor which specifically binds to the promoter-activating element DRE (DNA replication-related element) of DNA replication-related genes, was purified to homogeneity from nuclear extracts of Drosophila Kc cells. cDNA for DREF was isolated with the reverse-transcriptase polymerase chain reaction method using primers synthesized on the basis of partial amino acid sequences and following screening of cDNA libraries. Deduced from the nucleotide sequences of cDNA, DREF is a polypeptide of 701 amino acid residues with a molecular weight of 80,096, which contains three characteristic regions, rich in basic amino acids, proline, and acidic amino acids, respectively. Deletion analysis of bacterially expressed DREF fused with glutathione S-transferase (GST-DREF) indicated that a part of the N-terminal basic amino acid region (16-115 amino acids) is responsible for the specific binding to DRE. A polyclonal and four monoclonal antibodies were raised against the GST-DREF fusion protein. The antibodies inhibited specifically the transcription of DNA polymerase alpha promoter in vitro. Cotransfection experiments using Kc cells demonstrated that overproduction of DREF protein overcomes the repression of the proliferating cell nuclear antigen gene promoter by the zerknüllt gene product. These results confirmed that DREF is a trans-activating factor for DNA replication-related genes. Immunocytochemical analysis demonstrated the presence of DREF polypeptide in nuclei after the eighth nuclear division cycle, suggesting that nuclear accumulation of DREF is important for the coordinate zygotic expression of DNA replication-related genes carrying DRE sequences.

Amino Acid Sequence↗

PubMed1996

Depth dependence of the perturbing effect of placing a bulky group (oxazolidine ring spin labels) in the membrane on the membrane phase transition.

Electron paramagnetic resonance (EPR) and differential scanning calorimetry (DSC) have been used to study the effect on the phase transition of dimyristoylphosphatidylcholine membranes of incorporating various stearic acid spin labels (SASL's) that contain the bulky oxazolidine ring at various positions along the stearyl chain. SASL's lowered the phase transition temperature and decreased the size of the cooperative unit, with the effects stronger in the order of 9- > 12- > 5- > 16-SASL > stearic acid (no label). Incorporation of stearic acid without the spin label slightly increases the phase transition temperature. Incorporation of 9-SASL (3 mol% of lipid) decreased the transition temperature by 1.8 degrees C and the cooperative unit to 1/5 of that without the spin label, while the effect of 16-SASL was slight. The effect on transition enthalpy was small. It is concluded that the perturbing effect of placing a bulky group on the alkyl chain on phase transition is through inducing packing defects in the gel-phase.

Calorimetry, Differential Scanning↗

PubMed1995

Dynamic interactions of rabbit liver cytochromes P450IA2 and P450IIB4 with cytochrome b5 and NADPH-cytochrome P450 reductase in proteoliposomes.

Purified liver microsomal cytochrome P450IA2 or P450IIB4 was co-reconstituted with cytochrome b5 or NADPH-cytochrome P450 reductase in phosphatidylcholine-phosphatidylethanolamine-phosphatidylserine vesicles at a lipid to P450 weight ratio of 2 by cholate dialysis procedures. The proteoliposomes catalyzed drug oxidation. Rotational diffusion of cytochrome P450 was measured by observing the decay of absorption anisotropy, r(t), after photolysis of the heme.CO complex. Analysis of r(t) was based on a "rotation-about-membrane normal" model. The absorption anisotropy decayed within 1 ms to a time-independent value, r3. Different rotational mobility for the two cytochrome P450s was observed. Though 20% of cytochrome P450IA2 was immobile, all cytochrome P450IIB4 molecules were rotating. The rotational relaxation time, phi, of the mobile population was 237 microseconds for cytochrome P450IA2 and 160 microseconds for cytochrome P450IIB4. The two cytochrome P450s have shown very different interactions with cytochrome b5 and NADPH-cytochrome P450 reductase. By the presence of the redox partner, the mobile population of cytochrome P450IA2 was increased significantly from 80% to 96% (plus cytochrome b5) and to 89% (plus NADPH-cytochrome P450 reductase) due to dissociation of P450 oligomers. On the other hand, the mobility of cytochrome P450IIB4 was not considerably affected by the presence of cytochrome b5 or NADPH-cytochrome P450 reductase as judged by little difference in phi and r3, keeping the mobile population of 100%. These results imply that cytochrome P450IA2 forms a transient association with cytochrome b5 and NADPH-cytochrome P450 reductase.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1995

A nucleotide sequence essential for the function of DRE, a common promoter element for Drosophila DNa replication-related genes.

Promoter regions of the Drosophila proliferating cell nuclear antigen (PCNA) gene and the DNA polymerase alpha 180-kDa catalytic subunit gene contain a common 8 base pair (bp) promoter element, 5'-TATCGATA (DRE, Drosophila DNA replication-related element). We have generated various base substitutions and internal deletions in and around DRE (nucleotide positions -93 to -100 with respect to the transcription initiation site) of the PCNA gene in vitro and subsequently examined their effects on the binding to DREF (DRE-binding factor) and PCNA gene promote activity in cultured Drosophila Kc cells as well as in living flies. Gel mobility shift assays using nuclear extracts of Kc cells with and without competitor DNA fragments carrying the mutations indicated that the 10-bp sequence from positions -91 to -100 is essential for complex formation with DREF. Transient expression assays of chloramphenicol acetyl-transferase (CAT) in Kc cells transfected with PCNA promoter-CAT fusion genes carrying the mutations revealed that the 8-bp sequence from -93 to -100 is essential for activation of the promoter in Kc cells. Examination of lacZ expression from PCNA promoter-lacZ fusion genes carrying the mutations, introduced into flies by germ-line transformation, revealed that the 8-bp sequence is also important for DRE function during development. However, we obtained two exceptional mutations in the 8-bp sequence that did not or only marginally affected the PCNA gene promoter activity in transgenic flies. Both of these mutations effectively reduced the promoter activity in CAT transient expression assay in Kc cells and the binding to DREF in vitro. Therefore, the 8-bp sequence requirement for DRE function appears to be less stringent in living flies than in the cultured cell or in vitro cases.

Animals↗

PubMed1995

Distribution of PCNA during postblastoderm cell division cycles in the Drosophila melanogaster embryo: effect of a string- mutation.

We used immunocytochemical methods and a specific antibody to identify proliferating cell nuclear antigen (PCNA) in Drosophila embryos during the postblastoderm cell division cycles. The strong nuclear staining observed during interphase disappeared at prophase, staining remained nil throughout the remaining mitotic (M) phases and reappeared in nuclei at the next interphase. As the embryos with the homozygous string- mutation sustained the strong staining signal in nuclei throughout embryonic development, disappearance of the staining signal probably depends on string function and is coupled with the onset of mitosis. When cells in embryos were arrested at M phase following treatment with TN-16 or Vinblastine, the staining signal with the anti-PCNA antibody was lost. However, Western blots showed that the level of PCNA protein in M phase-arrested embryos did not decrease. Therefore, the disappearance of staining signals is apparently due to reorganization of PCNA protein in the multiprotein complex in nuclei, rendering it inaccessible to the antibody, rather than to the degradation of PCNA protein. In contrast to findings in developing embryos, cultured Drosophila Kc cells stained strongly with the anti-PCNA antibody, during both interphase and the M phase. In genetic crossing experiments of transgenic flies carrying the lacZ gene under the control of the PCNA gene regulatory region (-607 to +137 with respect to the transcription initiation site) with string- mutant files, the PCNA gene promoter seems to function in a manner independent of cell cycle progression or of functions of the string gene.

Animals↗

PubMed1994

Crystallization of 31 kDa C-terminal fragment of rat DNA polymerase beta.

A 248 residue C-terminal fragment of rat DNA polymerase beta (335 amino acid residues), a eukaryotic DNA repair enzyme, has been crystallized from polyethylene glycol 6000 solution. The crystals are orthorhombic, space group P2(1)2(1)2 with cell dimensions a = 120.3 A, b = 64.2 A, c = 39.4 A, and contain a single 31 kDa fragment in an asymmetric unit. The crystals diffract to 2.8 A resolution with laboratory X-ray source, and to 2.3 A resolution with synchrotron X-ray source, and are suitable for detailed structural analysis.

Animals↗

PubMed1994

Neuroendocrine studies in dementia patients: responses of plasma GH and PRL following bromocriptine administration.

Bromocriptine stimulates growth hormone (GH) secretion at the hypothalamus and suppresses prolactin (PRL) secretion at the pituitary level. We administered bromocriptine to 30 patients with dementia of the Alzheimer type (DAT), 30 patients with multi-infarct dementia (MID) and 22 age matched healthy controls, and compared response patterns of GH and PRL. Incomplete PRL suppressive responses (suppression rate < 50%) were seen in 36.7% of DAT patients and in 30.0% of MID patients, indicating that both groups had the same degree of pituitary dysfunctions. Blunted GH responses (< 5 ng/ml) were seen in 93.3% of DAT patients, in 63.3% of MID patients and in 31.8% of the controls. The results indicate that neuroendocrine regulation of GH is more selectively and severely damaged in DAT patients than in MID patients at the hypothalamus.

Aged↗

PubMed1994

[Trial of self-management of outpatients with implanted reservoir for arterial infusion of anticancer drugs].

We attempted to instruct the outpatients with malignant liver tumor (metastatic liver cancer and advanced hepatocellular carcinoma) who received intermittent arterial infusion chemotherapy using implanted reservoir to detach the devices for arterial infusion by themselves at home. All patients who received our instruction could master self-detachment at home, which shortened the hospitalized day. And patients' desire for this treatment promoted with improvement of the recognition of their families to participate in treatment together. We experienced no trouble which disturb the treatment. This methods was considered to promote the improvement of QOL of these patients.

Ambulatory Care↗

PubMed1994

Relationship between sigma-like site and progesterone-binding site of adult male rat liver microsomes.

An increasing amount of evidence suggests that the sigma (sigma) sites, putative targets for a variety of psychotomimetic and antipsychotic drugs, exist not only in the brain but also in various peripheral organs. However, there are many ambiguities as to their biological roles, subcellular distributions, endogenous ligands and so on. We therefore performed our study for clarification of some of these ambiguities. As a result, we demonstrated that adult male rat liver microsomes, especially smooth endoplasmic reticulum, possessed a saturable haloperidol-binding site closely resembling the sigma site, with a high affinity (Kd 1.0 +/- 0.3 nmol/L) and high capacity (Bmax 9.3 +/- 1.5 pmol/mg protein) and with the rank order of affinity of the ligands: haloperidol > reduced haloperidol > clorgyline > ifenprodil > 1,3-di(2-tolyl)guanidine, (-)-butaclamol > GBR-12909 > SKF-525A > progesterone > 5 alpha-dihydrotestosterone > R(+)-3- (hydroxyphenyl)-N-propylpiperidine > testosterone >> corticosteroids, estradiol-17 beta, cholesterol and neuroactive compounds displaying high affinities for other neurotransmitter receptors such as dopamine D2, serotonin (5-HT1A and 5-HT2) and alpha 1-adrenergic and GABAA receptors. This rank order showed a high correlation (r = 0.908) with that of a large portion (approximately 85%) of specific progesterone-binding site (Kd 31.0 +/- 3.5 nmol/L, Bmax 5.7 +/- 0.2 pmol/mg protein) of the same source. Therefore, these two sites were suggested to be the same or closely related. Furthermore, we provide a strong suggestion that these sites neither are identical with some subforms of the microsomal cytochromes P-450 or other steroid/drug-metabolizing enzymes nor participate universally and directly in the progesterone-metabolizing processes.

Animals↗

PubMed1994

[The causes and timing of reoperation for coronary artery disease].

In eight hundred and sixteen consecutive patients undergoing coronary artery bypass grafting (CABG) within the past 19 years (1974 to 1993), repeated CABG were performed on 12 patients (1.47%). The main reasons for repeated CABG were graft failure (GF), progression of native disease (NP) and incomplete revascularization (IR). The incidence of GF only and GF combined NP were high either within a half year or around 5 years after CABG. When the recurrence of angina is noted after CABG, the catheter intervention should be chosen at first but the reoperation should be done not so late. Although all patients survived from reoperation, three patients continued to have mild angina pectories. If a reoperation is inevitable, maximum utilization of arterial graft and accomplishment of complete revascularization are emphasized.

Aged↗

PubMed1994

[Reoperation for mitral valve disease].

From 1978 to March 1994, 95 patients underwent repeated open heart surgery for mitral valve diseases. There were 48 men and 47 women. The mean age was 50 and the mean interval between initial and second operation was 8 years. The initial operations were OMC in 39, MAP in 13, OMC+MAP in 11, MVR in 30 and AVR in 2 patients. The hospital death occurred in 31 cases (32.6%). In recent cases, however the hospital death were reduced to 4 (14.8%). This was attributed to the change in myocardial protection from previous intermittent antegrade crystalloid to recent continuous combined antegrade and retrograde cardioplegia.

Adolescent↗

PubMed1993

Cytogenetic abnormalities of hematopoietic tissue in retired workers of the Ohkunojima poison gas factory.

A high incidence of cancer of the respiratory tract has been reported among former workers in a poison gas manufacturing plant which operated on Ohkunojima from 1927 to 1945. This report provides evidence of a high incidence of chromosome abnormality and sister chromatid exchange (SCE) rate among the former workers, as well as cytogenetic changes in two patients among the former workers with chronic myelocytic leukemia (CML). A chromosome study of seven former workers with chronic bronchitis revealed a stable type of aberration, the average abnormality being 10.9 +/- 4.4 percent, which is equivalent to those of atomic bomb survivors exposed at 1.2 km from the hypocenter. The SCE rate observed in 16 former workers ranged from 4.9 +/- 2.1 to 17.8 +/- 3.9, which was significantly higher than in the control group (p < 0.03). One of the CML patients showed an extremely high percentage of missing Y chromosomes along with t(9;22) translocation which is a specific chromosome aberration for the disease. Furthermore, the patient had almost a 3 times higher SCE rate compared to the control group and a high incidence of chromosome abnormality (12.1%) of the peripheral lymphocytes. These results suggest that the development of leukemia in this patient was strongly related to poison gas exposure.

Aged↗

PubMed1993

[Risk factors and therapeutic methods for sternal wound infection following cardiac surgery].

In a study of 1040 patients undergoing cardiac surgery at The Osaka Medical College Hospital from 1984 through 1991, we analyzed the correlation between a variety of preoperative and operative parameters and the risk of postoperative sternal wound infection, as well as the efficacy of various therapeutic method for this condition. Sternal infection or mediastinitis developed after cardiac surgery in 31 patients (3.0%). As the risk factors for wound infections, age, diabetes mellitus, reexploration, duration of cardiopulmonary bypass and use of bilateral internal thoracic artery grafting were listed. Application of pectoral muscle flaps significantly led to shorten a duration between diagnosis of infection and hospital discharge, and increased survival rate, than the other treating methods such as debridement or irrigation. We conclude that bilateral internal thoracic artery grafts should not be used in the cases with diabetes, and closure with pectoral muscle flaps is effective for sternal infection and mediastinitis.

Coronary Artery Bypass↗

PubMed1992

Effect of Nocardia rubra cell-wall skeleton on cancer prevention in humans.

The retired workers at the chemical weapons plant in Japan are regarded as a high-risk group for cancers. Under the Cancer Preventive Program, Nocardia rubra cell-wall skeleton (N-CWS) was administered to 80 workers directly involved in the production of sulfur mustard and 66 workers engaged in work related to sulfur mustard production. Untreated workers whose age, sex, duties and duration of work at this factory were individually matched to the N-CWS-treated workers were used as controls. During a 4.5-year observation period, development of cancers was found in 7 treated workers and 17 untreated controls. After elimination of the influence of the difference in smoking level, the incidence of subjects who developed cancers was compared statistically between the N-CWS-treated workers and the untreated controls and a significant suppression of development of cancers was noted in the N-CWS-treated workers. Thus, it was concluded that the administration of N-CWS could prevent cancer development in humans.

Aged↗

PubMed1992

[Coronary artery bypass utilizing vein grafts: why didn't we use an arterial graft?].

Internal thoracic artery (ITA) grafts for coronary artery bypass grafting (CABG) are superior to SVG in the long-term patency and survival. In spite of our effort to utilize ITA whenever possible, we still have some cases which have to receive only vein grafts. Among the consecutive 201 patients undergoing CABG in the past three years, 32 patients (16%) received only vein grafts. We compared these patients (SVG group) with those who received ITA grafts (ITA group). The SVG group consisted of all emergency cases and many cases with single and double vessel disease. In the SVG group, perioperative use of IABP was more frequent and operative mortality was higher, although the number of grafts was fewer (2.66 vs 3.61) and aortic cross clamping time was shorter in this group. Main reasons for selecting only vein grafts were emergency surgery, inadvertent injury of ITA, inadequate ITA free flow etc. Technical refinement in the preparation of ITA is important to make possible wider application of arterial grafts for CABG.

Aged↗

PubMed1991

Inactivation of DNA polymerase beta by in vitro phosphorylation with protein kinase C.

The Mr = 38,300 polypeptide of the purified recombinant rat DNA polymerase beta served as an excellent substrate for protein kinase C (PKC) in vitro but not for the catalytic subunit of cAMP-dependent protein kinase. The phosphorylation by PKC resulted in inactivation of DNA polymerase beta activity, and recovery was achieved by dephosphorylation with alkaline phosphatase. Since the phosphorylated DNA polymerase beta was retained with use of a single-stranded DNA-cellulose column, inactivation might occur at a site different from that for the DNA binding. Amino acid sequence analysis of the phosphopeptides revealed that the phosphorylated sites were 2 serine residues at positions 44 and 55 from the NH2 terminus, either or both of which might be involved in the catalytic activity of DNA polymerase beta. Thus, the inactivation of the DNA repair enzyme, DNA polymerase beta, by PKC may be an important process in the modification of DNA metabolism in the nucleus through signal transduction processes.

Alkaline Phosphatase↗

PubMed1991

Aspartic acid residues at positions 190 and 192 of rat DNA polymerase beta are involved in primer binding.

The sequence Gly-Asp-Met-Asp, spanning positions 189-192 of rat DNA polymerase beta, is similar to the sequence motif Gly-Asp-Thr-Asp that is highly conserved in a number of replicative DNA polymerases from eukaryotic cells, viruses, and phages. The role of this sequence in the catalytic function of rat DNA polymerase beta was investigated by individually changing each amino acid in this region by site-directed mutagenesis. The mutant enzymes DE190 and DE192, in which aspartic acid residues at positions 190 and 192, respectively, were replaced by glutamic acid, showed about 0.1% activity of the wild-type enzyme. On the other hand, the replacement of Gly-189 by alanine or Met-191 by isoleucine or threonine only slightly affected the enzyme activity. A gel mobility shift assay showed that DNA complexes with enzyme DE190 and especially with DE192 were less stable than the corresponding complex with the wild-type enzyme. Kinetic analysis with these mutant enzymes indicate that their Km's for primer DNA were about 10-fold higher than that of the wild type, while Km's for deoxyribonucleoside triphosphate were not changed. Since neither DE190 nor DE192 had any significant alteration in secondary structure, our results suggest that both Asp-190 and Asp-192 are located in the active site and are involved in the interaction of DNA polymerase beta with primer.

Amino Acid Sequence↗

PubMed1991

Gallium-67 citrate scintigraphy in the pre-operative evaluation of soft tissue tumors of the extremities.

We conducted a study of 90 patients with soft tissue tumor in their arms or legs, in order to determine the usefulness of scintigraphy with gallium-67 citrate as a diagnostic means. All patients had adequate scan images, and tumor tissues had been histologically confirmed by surgical resection. The subjects consisted of 19 patients with malignant tumors, 55 patients with benign tumors, and 16 patients with other disorders in which soft tissue tumor-like lesions occurred. When the activity of the tumor was more than the activity of the normal region of the contralateral extremity, it was estimated to be positive. The positive rate was found in 78% (15/19) of patients with malignant tumors, in 25% (14/55) of patients with benign tumors and in 31% (5/16) of patients with other disorders. Classified by diseases, high positive rates were observed in liposarcoma, leiomyosarcoma, malignant lymphoma, neurinoma, extra-abdominal desmoid and sarcoidosis. Out of 7 patients in which the activity of the tumor was equal to, or higher than that of the liver, 6 patients had malignant tumors and one patient was diagnosed as having an abscess. It seemed possible to distinguish between liposarcoma and lipoma by means of a gallium-67 scan. Furthermore, the gallium-67 scan was useful in detecting lesions of sarcoidosis as well as in evaluating the response to treatment.

Adolescent↗