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Biomedical subjects

S Ohi

Publications and source records attributed to S Ohi.

At least 37 records · Page 2Linked to original sources
PubMed2000

Mouse fetuses by nuclear transfer from embryonic stem cells.

At present, two methods for cloning mammals by nuclear transfer are employed. The first is based on cell fusion and has been applied to domestic animals, such as sheep, cows, and goats. While, nuclear microinjection has been used in mice only. Cloning by nuclear transfer has been reported mainly with cells from primary culture and freshly isolated cells. Here, using ES cell line TT2, we tried to produce clone mouse embryos by the two methods. With ES cell line TT2 (10-13 passaged), 16% of reconstructed oocytes microinjected with the nuclei developed in vitro to the morula/blastocycst stage, and 50% of these embryos developed to fetuses until 14 dpc when transferred to pseudopregnant females. At 20 dpc implanted sites were degenerated and absorbed. Also, in vitro development of embryos reconstructed by electrofusion shown similar results. But, when transferred to recipients, subsequent development of embryos showed lower rates, as compared with embryos microinjected and from recipients live-born pups could not be obtained.

Animals↗
PubMed2000

Cloned mice derived from somatic cell nuclei.

In 1997, a cloned sheep "Dolly" was produced by nuclear transfer of somatic cell. The first birth of cloned mice derived from some somatic cells were succeeded in 1998. At present, it is shown that somatic cells, cumulus cells, fibroblasts and Sertoli cells can be used to the study of cloned animal as nuclear donor. In this study investigation was designed to compare with efficiency on the production of cloned embryos by using the microinjection and the electrofusion methods for nuclear transfer. Oocyte enucleation was performed with a micromanipulator. The oocyte was held by holding pipette, and was enucleated using a beveled pipette. Microinjection method: Cell's nucleus injection was carried out by piezo-micromanipulator. Cytochalasin B treated cumulus cell was aspirated into a injection pipette, and was broken its plasma membrane using the injection pipette. Then, the cumulus cell was injected into the enucleated ooplasm directly. Electrofusion method: The cell was aspirated into a beveled pipette, and then an aspirated cell was inserted into perivitelline space. Then, the pair of enucleated oocyte and cell was fused using electrical cell fusion apparatus. The reconstituted embryos were activated after nuclear transfer using St2+. Reconstituted embryos had been produced by the microinjection showed the embryonic development to over 8-cell stages. But, the rate of fragmentation of reconstituted embryos by the microinjection showed a little high rate in comparison with the electrofusion. When some reconstituted embryos by the microinjection were transplanted to pseudopregnant females' oviduct, 9 fetuses were observed at 14 days post coitum.

Animals↗
PubMed2000

Developing protocols for recombinant adeno-associated virus-mediated gene therapy in space.

With the advent of the era of International Space Station (ISS) and Mars exploration, it is important more than ever to develop means to cure genetic and acquired diseases, which include cancer and AIDS, for these diseases hamper human activities. Thus, our ultimate goal is to develop protocols for gene therapy, which are suitable to humans on the earth as well as in space. Specifically, we are trying to cure the hemoglobinopathies, beta-thalassemia (Cooley's anemia) and sickle cell anemia, by gene therapy. These well-characterized molecular diseases serve as models for developing ex vivo gene therapy, which would apply to other disorders as well. For example, the procedure may become directly relevant to treating astronauts for space-anemia, immune suppression and bone marrow derived tumors, e.g. leukemia. The adeno-associated virus serotype 2 (AAV2) is a non-pathogenic human parvovirus with broad host-range and tissue specificity. Exploiting these characteristics we have been developing protocols for recombinant AAV2 (rAAV)-based gene therapy. With the rAAV constructs and hematopoietic stem cell (HSC) culture systems in hand, we are currently attempting to cure the mouse model of beta-thalassemia [C57BL/6- Hbbth/Hbbth, Hb(d-minor)] by HSC transplantation (HST) as well as by gene therapy. This paper describes the current status of our rAAV-gene therapy research.

Animals↗
PubMed1997

Long-term survival and differentiation of human peripheral blood CD34+ cells in SCID mice.

Human peripheral blood progenitor cells (PBPC) are currently used as a source of hematopoietic reconstitution by autologous transplantation after myeloabrative chemotherapy for malignancies. PBPC would also be useful for allogeneic transplantation since the collection of PBPC is much safer than that of bone marrow stem cells (BM). For allogeneic transplantation, it is imperative to confirm that PBPC contains self-renewable stem cells that can sustain a long lasting hematopoiesis. In the present study, we examined the reconstitution of human hematopoiesis in severe combined immunodeficiency (SCID) mice by transplanting peripheral blood CD34+ cells in which the neo gene was transduced as a marker. In 2 of 4 mice receiving PB-CD34+ cell transplantation, the neo gene appeared as early as 4 weeks and lasted as long as 24 weeks in all DNA preparations of bone marrow, peripheral blood and spleen cells from the SCID mice, while in 2 of 4 mice receiving BM-CD34+ cell transplantation, although the neo gene also lasted as late as 24 weeks, it did not appear as early as in the mice receiving PB-CD34+ cell transplantation. A similar observation was noted in clinical trials, i.e. the white blood cell and platelet recovered earlier by transplantation of PBPC than of BM. In mice who had the neo gene, we were also able to demonstrate by FACS the presence of human lineage specific antigen in the cells as late as 24 weeks after transplantation with PB-CD34+ cells, and the presence of human IgG in the sera 10 weeks after transplantation. These findings indicate that PB-CD34+ cells contain long-term repopulating stem cells which undergo differentiation in SCID mice.

Animals↗
PubMed1997

[A case of intrathoracic chondrosarcoma of the rib head--comparison between MRI shadow and histopathological specimen].

A 71-year-old man was admitted our hospital because of mediastinal tumor. On chest X-ray, the mass was about 10 cm in diameter, contact with the right side of No. 3-5 vertebra and extended to the right thorax. After about 8 months, he felt anterior chest and back pain. Tumor was growing on chest X-ray and, on chest CT, linear and spotty calcification existed in it. On MRI, T1 weighted image, most part of tumor was low signal area but high area was also existed and it was parenchymatous hemorrhage. On T2 weighted image, tumor was high signal and irregular low area was calcification. Operation was performed and a hard tumor, measured 12 x 9 x 6 cm, originated from the 3rd-4th rib was resected. Histopathological diagnosis was chondrosarcoma. MRI was effective for the diagnosis of this case.

Aged↗
PubMed1997

[Clinical significance of 123I-MIBG myocardial scintigraphy in patients with hypertrophic cardiomyopathy].

We studied the abnormality of myocardial sympathetic nervous system in patients with hypertrophic cardiomyopathy using 123I-metaiodobenzylguanidine (MIBG) myocardial scintigraphy in comparison with the parameters of other clinical examinations. In 50 patients with HCM, the heart to mediastinum 123I-MIBG uptake ratio (H/M) was significantly low and washout rate (WR) of 123I-MIBG was significantly high respectively compared with normal subjects (n = 8). H/M was negatively correlated with serum norepinephrine level, wall thickness of left ventricle, left ventricular mass index, left ventricular end diastolic pressure respectively, and WR was positively correlated with those parameters respectively. On the other hand, LF/HF calculated by spectral analysis in holter electrocardiogram was positively correlated with H/M, and negatively correlated with WR. In HCM, H/M in patients with subjective symptoms was significantly lower than that without subjective symptoms, and WR in patients with paroxysmal atrial fibrillation was significantly higher than that without paroxysmal atrial fibrillation. This study revealed that H/M and WR reflected the severity and the difference of disease type in HCM. In conclusion, 123I-MIBG contributes to evaluating more details in diagnosis and pathophysiology of HCM.

3-Iodobenzylguanidine↗
PubMed1996

Marked elevation of plasma carcinoembryonic antigen and stomach carcinoma.

BACKGROUND: This study was undertaken to clarify the relationship between marked elevation of plasma carcinoembryonic antigen (CEA) and signet ring cell carcinoma of the stomach. METHODS: To elucidate the contributing factor of extreme elevation of plasma CEA value, the histologic and biochemical records for 310 cases of stomach carcinoma, including 202 advanced and 108 early, collected between 1980 to 1994 from the San-in Rosai Hospital and Tottori University Hospital were studied. Immunohistochemical localization of CEA in the stomach was performed using a peroxidase antiperoxidase (PAP) staining technique. RESULTS: Among 310 cases of gastric carcinoma, 44 (14%) had abnormal plasma CEA values. The positivity rates of early and advanced gastric carcinoma were 3.7% (4/18) and 19% (40/202), respectively. Concerning advanced gastric carcinoma, 20 cases had more than 51 ng/mL, and 20 cases had between 5 ng/mL and 50 ng/mL. Four cases with plasma CEA values of more than 1,000 ng/mL had histological signet ring cell carcinoma (one case), and poorly differentiated adenocarcinoma with signet ring cell carcinoma (2 cases). Three cases of signet ring cell carcinoma or poorly differentiated adenocarcinoma of the stomach were massive local infiltration rather than hepatic metastasis. Among 40 cases with elevated plasma CEA, a multivariate regression analysis showed that only one variable (lymph node metastasis) was an independent factor (P < 0.05). Significantly higher rates of peritoneal metastasis (P < 0.0001) and lymph node metastasis (P < 0.05) were observed in patients with marked elevations of plasma CEA than in patients with moderate elevations of plasma CEA. No correlation was obtained between plasma CEA value and several biochemical tests. CONCLUSIONS: Marked elevation of plasma CEA may be found in the absence of liver metastasis from signet ring or poorly differentiated gastric carcinoma. Patients with marked elevations of CEA also had lymphatic and peritoneal dissemination.

Adenocarcinoma↗
PubMed1996

Synthesis of human globin polypeptides mediated by recombinant adeno-associated virus vectors.

Adeno-associated virus, serotype 2 (AAV2)-based chimeric plasmids that harbored a near-full-length human alpha- or beta-globin cDNA were constructed. The cDNAs were spliced into an AAV plasmid, pAAV delta K, downstream from the viral P40 promoter, substituting the capsid gene region. The correctness of the insertion with regard to the transcription polarity was ascertained by both restriction enzyme analysis and DNA sequencing. One of the constructs, pAAVcHBBLCR, contained the erythroid-specific enhancer elements, the locus control region, HS1 and HS2, to ensure an efficient and tissue-specific gene expression. Use of a defective complementing helper, pAVXB (Dixit, M.; et al. Gene 1991, 104, 253-257.) and adenovirus 2 made it possible to prepare recombinant AAVs (rAAVs). Infection of human 293 cells (embryonal kidney cell line) with the resultant rAAV (AAVcHBB) and cotransfection of mouse erythroleukemia (MEL) cells with the beta-globin construct (pAAVcHBBLCR) and an alpha-globin construct (pAAVcHAB) triggered efficient synthesis of human globin polypeptides in the cells, as analyzed by biochemical and immunohistochemical means. The LCR made the construct respond to an inducer, N,N-hexamethylenebisacetamide, the amount of expressed human beta-globin reaching a similar level as the endogenous mouse beta-globin in MEL cells. Electrotransfection of mouse bone marrow hematopoietic stem/progenitor cells with the constructs dramatically increased the number of benzidine-positive cells in liquid suspension culture, indicating expression and synthesis of a human hemoglobin in these cells. Thus, the rAAV constructs may be useful for gene therapy of hemoglobinopathies.

Animals↗
PubMed1995

Co-administration of IL3 with G-CSF increases the CFU-S mobilization into peripheral blood.

In an attempt to establish an efficient method of collecting peripheral blood stem cells and to utilize them for allogeneic peripheral blood stem cell transplantation, the effect of a combined administration of recombinant murine interleukin-3 (IL3) and recombinant human granulocyte colony-stimulating factor (G-CSF) to mobilize bone marrow stem cells into the circulation was examined in C57BL/6 mice. Some appreciable numbers (796 +/- 112/ml) of CFU-GM were recovered 6 days after G-CSF administration (500 micrograms/kg per day), while by IL3 administration (100,000 units/kg per day), the CFU-GM count was much lower (61 +/- 9/ml) with a small peak at day 4. By a combined administration of IL3 (100,000 units/kg per day) and G-CSF (500 micrograms/kg per day), the CFU-GM count at the peak of day 5 was significantly augmented (1178 +/- 277/ml) as compared to that of G-CSF or IL3 alone (P < 0.05). The CFU-S counts at day 5 (168 +/- 12/ml) and at day 6 (172 +/- 27) were also significantly higher than those of IL3 alone (day 5, 30 +/- 15/ml; day 6, 20 +/- 10/ml) or G-CSF alone (day 5, 114 +/- 14/ml; day 6, 112 +/- 19/ml). Thus the combined administration of IL3 and G-CSF appears to be promising for high yield collection of peripheral blood stem cells.

Animals↗
PubMed1995

Expression and phosphorylation of BiP/GRP78, a molecular chaperone in the endoplasmic reticulum, during the differentiation of a mouse myeloblastic cell line.

To determine the functional significance of endoplasmic reticulum chaperones in hematopoietic cells, we analyzed the expression and post-translational modification of BiP/GRP78 and GRP94 as well as the cytoplasmic chaperones HSP70 and HSC70 during the differentiation of a mouse myeloid leukemia cell line, M1. The amounts of BiP/GRP78 and GRP94 increased several-fold when M1 cells were induced to differentiate into macrophage-like cells by treatment with interleukin-6 (IL-6). Synthesis began to increase at 4 hr after IL-6 treatment. The phosphorylated form of BiP/GRP78 increased during the later stages of differentiation. These data suggested that the chaperone activity of BiP/GRP78 and GRP94 may be needed for differentiated macrophage-like cells or for the differentiation event itself, and that functionally different BiP/GRP78 accumulate during the differentiation of M1 cells.

Animals↗
PubMed1995

The role of renal dopamine in the reduction of high blood pressure by beta 1-selective beta-blocker with intrinsic sympathomimetic activity in spontaneously hypertensive rats.

The present experiments were undertaken to clarify the difference of renal dopamine production from beta 1-selective beta-blocker with and without intrinsic sympathomimetic activity (ISA). Either beta-blocker with ISA, celiprolol (100 or 300 mg/kg/day; CEL-100 or CEL-300) or beta-blocker without ISA, atenolol (50 mg/kg/day; ATE-50) was administered to the SHR from 19 to 26 weeks. Degrees of lowering blood pressure in CEL-300 SHR and in ATE-50 SHR were similar, but decrease in heart rate was significantly less in CEL-300 SHR than in ATE-50 SHR. Urine output, which was significantly less in control SHR than in control WKY, was significantly greater in CEL-100 SHR and CEL-300 SHR, but not in ATE-50 SHR. Urinary excretions of noradrenaline (u-NA) and dopamine (u-DA) were significantly higher in control SHR than in control WKY and a comparable u-DA/u-NA ratio was found in these two groups. U-DA and the ratio of u-DA/u-NA were significantly elevated in CEL-100 SHR and CEL-300 SHR, but not in ATE-50 SHR. There was a significant positive correlation between u-DA/u-NA ratio and urine output and a significant negative correlation between the ratio of u-DA/u-NA and change of blood pressure in control SHR, CEL-100 SHR and CEL-300 SHR. These results suggest that an enhancement of renal dopamine production by ISA (beta 2 stimulation) of beta 1-selective beta-blocker may contribute, at least in part, to the antihypertensive effect of this drug.

Adrenergic beta-1 Receptor Antagonists↗
PubMed1995

[Systemic and regional myocardial distribution of 123I-BMIPP in normal subjects].

We estimated the systemic and left ventricular (LV) regional myocardial distribution of 123I-BMIPP (beta-methyl-p-iodophenyl-pentadecanoic-acid) in normal subjects (n = 13, mean age 43.9). In planar studies, the count ratios of heart, lung and liver to mediastinum were 2.63, 1.28 and 3.80 in the early images, and 2.23, 1.20 and 2.26 in the delayed images, respectively. The uptake in liver was almost identical with that in heart in the delayed images. In SPECT studies, the regional relative counts in anterior, septal, posterior and lateral LV walls were 100, 98, 96 and 108 (%) in the initial images, and 100, 98, 99 and 107 (%) in the delayed images, respectively. The regional relative uptake was significantly higher in the lateral wall than those in the other parts of LV walls in both images. The relative counts in the basal, mid- and apical portions were 100, 111 and 87 (%) in the initial images, and 100, 113, 92 (%) in the delayed images, respectively. These results suggest that the myocardial regional distribution of BMIPP is not always uniform even in normal subjects. Thus, it is necessary to interpret with caution in the light of these findings, especially for detect in a myocardial lesion in an early phase of cardiomyopathy or a mild myocardial ischemia.

Adult↗
PubMed1994

Improved recovery of myelosuppression following chemotherapy in mice by combined administration of PSK and various cytokines.

Granulocyte-colony-stimulating factor (G-CSF), granulocyte/macrophage-colony-stimulating factor (GM-CSF) and interleukin-3 (IL-3) were used in combination with PSK, a protein-bound polysaccharide extracted from mycelium of Coriolus versicolor (strain CM101), in myelosuppressed mice. The myelosuppression model consisted of BDF1 mice who received 150 mg/kg 5-fluorouracil (5-FU) intravenously. The peripheral blood leukocyte count during the recovery stage was significantly increased when these cytokines were administered with PSK compared to when the cytokines were used individually. In vitro colony assay revealed that the combination of PSK and any of GM-CSF, IL-3 or stem cell factor (SCF) showed a greater increase in colony numbers than when these materials were administered individually, although G-CSF did not show a synergistic effect with PSK. When bone marrow cells were obtained from mice which had been given PSK or IL-3, the colony assays were made in the presence of PSK or IL-3 in vitro. The greatest increase in the numbers was observed in colonies of the cultured group in the presence of IL-3 after the PSK priming. However, the colony formation potential of PSK was not inhibited by addition of anti-SCF antibody. The above results indicate that the combined administration of PSK with G-CSF, GM-CSF or IL-3 increased the hematological recovery of myelosuppressed mice. Moreover, the phase at which PSK has effects on hematopoietic cells seems to be at a more immature level than with IL-3. The combined administration of PSK and the above cytokines may improve myelosuppression after chemotherapy in patients with malignancy.

Animals↗
PubMed1993

Cytokine mRNA expression of bone marrow stromal cells from patients with aplastic anaemia and myelodysplastic syndrome.

We studied mRNA expression of the cytokine granulocyte-colony stimulating factor (G-CSF), interleukin-1 beta (IL-1 beta), IL-6, IL-8 and stem cell factor of stromal cells derived from bone marrows of nine normal volunteers, eight patients with aplastic anaemia (AA) and seven patients with myelodysplastic syndrome (MDS). The proportion of endothelial cells, macrophages, fibroblast-like cells and adipocytes in stromal cells showed no differences between normal volunteers and the patients. Levels of cytokine mRNA expression were determined by reverse transcription-polymerase chain reaction. Spontaneous expression occurred and this was augmented by LPS stimulation in cells of all the normal volunteers and in most patients. When stimulated by LPS, the mean G-CSF and IL-1 beta mRNA expressions in patients with AA were significantly higher than normal volunteers, but there was one patient showing lower IL-1 beta, IL-6 and IL-8 expression with no response to LPS. LPS-induced IL-6 and IL-8 expression of two patients with MDS were significantly higher than normal. The spontaneous and LPS-induced protein concentration of G-CSF, IL-6 and IL-8 in culture supernatants from 15, 10 and four patients, correlated well with the mRNA expression. The correlation coefficients were 0 x 92, 0 x 78 and 0 x 91, respectively. In conclusion, there were a few patients whose aetiology appeared to be reduction of stromal cytokine expression in AA, but most patients with AA and MDS expressed normal or high levels of cytokine mRNA.

Adolescent↗
PubMed1993

Recombinant human granulocyte colony-stimulating factor can mobilize sufficient amounts of peripheral blood stem cells in healthy volunteers for allogeneic transplantation.

Peripheral blood stem cells (PBSC) are a good source for bone marrow reconstitution after intensive chemotherapy. The ability to transplant PBSC between allogeneic subjects would be a key step forward in the application of this procedure. For this purpose, we examined the mobilization and recovery of PBSC in healthy volunteers who were given recombinant human granulocyte colony-stimulating factor (G-CSF). Three informed healthy volunteers were injected with G-CSF subcutaneously at a dose of 2.5 micrograms/kg for 6 successive days and at 5.0 micrograms/kg for the following 4 days. The concentration of PBSC was assessed daily by CFU-GM and BFU-E assays, both of which peaked on the sixth to seventh day of G-CSF administration. Comparison between colony assays and hematological parameters revealed that flow cytometry analysis of CD34+ cells by mononuclear cell gating is a rapid and convenient method to quantify mobilized stem cells. The maximum numbers of CFU-GM were 432, 665, and 1386 colonies/ml blood. It is calculated that sufficient amounts of stem cells for transplantation (at least 1.0 x 10(5) CFU-GM/kg) could be obtained by leukapheresis of 5 to 201 blood when the peak was attained. This trial confirmed the feasibility of allogeneic transplantation using PBSC from healthy volunteers who have received G-CSF.

Adult↗
PubMed1992

A procedure for large-scale plasmid isolation without using ultracentrifugation.

An expedient procedure for large-scale plasmid isolation from Escherichia coli strains without using ultracentrifugation or special setups or reagents is described. The protocol, which utilizes a modified alkaline extraction procedure as well as differential precipitations by isopropanol and lithium chloride, is simple and rapid and yet produces plasmid DNA with a yield of about 2 mg/liter culture. The isolated plasmids consisted of mostly monomeric and dimeric covalently closed circular DNA. The plasmids could be digested by various restriction endonucleases and were compatible with gene cloning, transfection-gene expression, and viral production.

1-Propanol↗