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Biomedical subjects

N Kubota

Publications and source records attributed to N Kubota.

At least 127 records · Page 7Linked to original sources
PubMed1992

Detection of topoisomerase I gene point mutation in CPT-11 resistant lung cancer cell line.

CPT-11, a recently developed topoisomerase I (Topo I) inhibitor, attracts the attention not only of basic researchers but also of clinicians because of its high antitumor activity. The CPT-11 resistant human lung cancer cell line, PC-7/CPT, showed 10-fold resistance compared to parental cell line, PC-7. The total activity of Topo I in the resistant cell line was one fourth that of the parental sensitive cell line. The Topo I from the resistant cells was also 5-fold more resistant to the inhibitory effect of CPT-11 than that of the parental cells. We speculated that the alteration of the Topo I gene may be responsible for the change in topoisomerase activity of the CPT-11 resistant cell line. Therefore, we analyzed the mutation of Topo I gene using the method of single strand conformation polymorphism of polymerase chain reaction and the reverse transcriptase. We divided Topo I cDNA into ten fragments which overlapped each other and covered whole coding sequences of the Topo I cDNA. We observed mobility shift of two fragments in the PC-7/CPT, suggesting the presence of some mutations in these fragments. We performed the direct-sequencing of these portions by the dideoxy chain termination method and observed an altered sequence having a G to A base change in PC-7/CPT. This base substitution results in replacement of the conserved threonine at 729 position with alanine. These results suggest that the point mutation of Topo I gene is related to the decreases of Topo I activity and the sensitivity to Topo I inhibitor in PC-7/CPT cells.

Antineoplastic Agents, Phytogenic↗
PubMed1992

Application of immunocryoultramicrotomy to free cells: ultrastructural localization of myeloperoxidase in neutrophils of human peripheral blood.

Immunocryoultramicrotomy is generally considered the most suitable method for observing the immunostainings by monoclonal antibodies to intracellular antigens by electron microscopy. However, this procedure with the free cells such as peripheral blood cells or cultured cells is not widely used because of the difficulty in preparing ultrathin frozen sections. We report here a new technique for making ultrathin frozen sections of peripheral blood cells by positioning the cell in a semigelatinized medium for ultrasectioning. Monoclonal antibody against myeloperoxidase (MPO) was reacted with the ultrathin sections made with this new technique to observe the ultrastructural localization of intracellular MPO. MPO was observed in the primary granules of the neutrophils.

Antibodies, Monoclonal↗
PubMed1992

A case of splenic vein occlusion caused by the intravenous tumor thrombus of nonfunctioning islet cell carcinoma.

We report herein a case of successfully treated advanced, nonfunctioning islet cell carcinoma associated with left-sided portal hypertension. The splenic vein was obstructed by a huge intravenous tumor thrombus developing from the main pancreatic tumor. Direct invasion to adjacent organs such as the spleen, colon, left kidney and stomach was also observed, although liver metastasis was not present. Radical resection was carried out with removal of these five involved organs and the patient is alive without recurrence more than 5 years after surgery.

Adenoma, Islet Cell↗
PubMed1992

Alteration of type II regulatory subunit of cAMP-dependent protein kinase in human cisplatin-resistant cells as a basis of collateral sensitivity to 8-chloro-cAMP.

A cyclic adenosine 3',5'-monophosphate (cAMP) analogue, 8-chloro-cAMP (8-Cl-cAMP), had a collateral growth-inhibitory effect on a cis-diamminedichloroplatinum(II) (CDDP)-resistant human cancer cell lines (PC-14/CDDP). The non-selective analogues dibutyryl-cAMP, 8-bromo-cAMP and forskolin, which are cAMP agonists, showed far less cytotoxicity than 8-Cl-cAMP in both cell lines. There was no significant difference in cAMP content between PC-14 and PC-14/CDDP. Because 8-Cl-cAMP has been shown to bind selectively to the site I receptor of the type II regulatory subunit (RII) of cAMP-dependent protein kinase, we determined the level of expression of regulatory subunits in PC-14 and PC-14/CDDP cells by photoaffinity labeling. PC-14/CDDP cells had a higher RII level, low site I receptor of type I regulatory subunit (RI) level, and a lower RI/RII ratio than the parental PC-14 cells. Exposure to 8-Cl-cAMP increased the RI and RII level in PC-14/CDDP cells in dose- and time-dependent manners. On the other hand, in parental PC-14 cells, RII was not detected and the levels of RI and RII were not increased by exposure to 8-Cl-cAMP. These results suggested that the change in RI and/or RII levels caused by 8-Cl-cAMP was correlated with 8-Cl-cAMP-induced growth inhibition and that the collateral sensitivity to 8-Cl-cAMP in CDDP-resistant cells was due to the increased RII level. Our results suggest that 8-Cl-cAMP can be used in combination with CDDP and that measurement of RI and RII levels and/or the RI/RII ratio is a useful tool to predict CDDP sensitivity.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1992

[An epidemiogenetic study of typical retinitis pigmentosa in Japan--a preliminary report of nationwide, multicenter study].

We performed a nationwide, multicenter study of typical retinitis pigmentosa with reference to the inheritance patterns of the disease. A total of 253 probands were registered during two months of 1989, and an analysis of the parental consanguinity of 182 probands with the method of inbreeding coefficient enabled us to estimate the relative prevalence of genetic types; autosomal recessive trait: 47.6%; autosomal dominant trait: 17.3%; sporadic cases: 34.6%. A comparison of the results with previous studies has indicated a decrease in the prevalence of the autosomal recessive trait and an increase in the sporadic cases, as would be expected from the decrease in consanguineous marriages and offsprings in the past few decades in Japan. X-linked retinitis pigmentosa was rarely identified, but precise evaluation of its frequency needs further investigation.

Adolescent↗
PubMed1991

Effects of dietary konjac mannan on serum and liver cholesterol levels and biliary bile acid composition in hamsters.

We studied the effect of dietary konjac mannan on serum and liver cholesterol levels and biliary bile acid composition in hamsters. In hamsters fed a control diet supplemented with konjac mannan for 6 weeks, serum and liver cholesterol levels were decreased significantly compared to hamsters fed the control diet. Addition of konjac mannan to the cholesterol-enriched diet reduced the elevation of serum cholesterol caused by feeding cholesterol. However, the absorption of cholesterol from the intestine was not changed between the four groups. The increase in the proportion of chenodeoxycholic acid in biliary bile acids was significantly enhanced in the hamsters fed both cholesterol and konjac mannan compared to hamsters fed the control diet. The change of the bile acid composition may suggest that konjac mannan inhibits the intestinal absorption of bile acids.

Animals↗
PubMed1991

[Clinico-pathological studies on the effects of preoperative hyperthermo-chemoradiotherapy of advanced esophageal carcinoma].

We report clinico-pathological studies on the effect of preoperative hyperthermia and chemotherapy combined with radiotherapy (HCR) for progress of the local curability of advanced esophageal carcinoma. The subjects of these studies were 17 patients who underwent subtotal esophagectomy after preoperative irradiation 40 Gy from 1980 to 1989, of which 8 patients had HCR, 6 patients irradiation only (R), 3 patients both irradiation and chemotherapy (CR). The clinical response rate of the patients with R or CR was 33% (PR 3, MR 3, NC 3), and the histological effective (Ef3 or Ef2) rate was 56% (Ef3 1, Ef2 4, Ef1 4). The clinical response rate of the patients with HCR was 88% (PR 7, MR 1), and the histological effective rate was 100% (Ef3 1 Ef2 7). HCR was more effective than R or CR for the local lesion of esophageal carcinoma histopathologically (p less than 0.05). However, the survival rate of patients with HCR was similar to R and CR, respectively. These results suggest that further improvement of the heating methods and the methods of combining hyperthermia with irradiation and chemotherapy is needed.

Adult↗
PubMed1991

Changes in causes of visual handicaps in Tokyo.

Newly registered visually handicapped patients at the Tokyo Metropolitan Rehabilitation Center for the Physically and Mentally Handicapped were studied in 1968 (when the facility was opened), 1978, and 1988. Examinations were carried out by the same physicians and diagnoses were made by uniform criteria. Since approximately 10% of the total population of Japan lives in Tokyo, the statistics on the causes of visual handicaps in this area deserve to be reported. The changes observed over the past twenty years are as follows: There were few changes in the percentages of high myopia, retinal degeneration and senile cataract. The percentages of corneal opacity, retinopathy of prematurity, congenital cataract and optic atrophy decreased, while those of diabetic retinopathy, glaucoma and retinal macular degeneration increased. Advances in medical treatment and the shift into an aging society in Japan are responsible for these changes observed.

Adolescent↗
PubMed1991

Hyperthermic purging in vitro of murine leukemia cells (MK-8057): surviving fractions of normal and leukemic stem cells and the long-term survival of mice injected with the post-hyperthermic leukemia cells.

Hyperthermic purging of leukemic cells has been applied in clinical trials, although an accurate evaluation system to compare the effect on leukemia cells with that on normal hemopoietic cells has not been established. We evaluated the heat-sensitivity of murine leukemia cells, MK-8057, and compared differences in heat-sensitivity between surviving fractions of leukemic stem cells (leukemic spleen colony-forming units, L-CFU-S) and normal hemopoietic stem cells (spleen colony-forming units, CFU-S). Using the spleen colony assay, the survival fraction of L-CFU-S was compared with that of CFU-S after various hyperthermic treatments. One of the most efficient conditions, that is, hyperthermia at 42 degrees C for 3 h, allowed only 0.17% of L-CFU-S to survive, whereas 26.5% of normal CFU-S survived. Hyperthermia at 44 degrees C for 45 min further reduced the L-CFU-S (0.13%); however, the relative ratio of L-CFU-S to CFU-S was less than that at 42 degrees C for 3 h, because there was a larger reduction at 44 degrees C in normal CFU-S (5.1%). Recipient mice injected with MK-8057 cells treated with hyperthermia survived longer in proportion to the decreasing number of surviving L-CFU-S injected. This extension of the survival of recipient mice given MK-8057 cells after hyperthermia was also proportional to the estimated survival fraction of L-CFU-S. The survival fraction of MK-8057 cells after hyperthermia that was independently calculated through the extended survival of the recipients showed a good correlation with the surviving fraction of L-CFU-S, seen as the leukemic spleen colonies, at a correlation coefficient of r = 0.985. The number of surviving mice receiving the post-hyperthermic MK-8057 cells and the number of L-CFU-S calculated to have been injected had a relationship based on a Poisson distribution. Thus, the calculated results imply that the hyperthermia proportionally targets L-CFU-S, which are the only cells responsible for killing the recipient mice.

Animals↗
PubMed1990

[Characterization of ultra high energy neutron beam generated by 500 MeV proton beam].

An ultra high energy neutron facility was constructed at PARMS, University of Tsukuba, to produce a neutron beam superior to an X-ray beam generated by a modern linac in terms of dose distribution. This has been achieved using the reaction on a thick uranium target struck by 500 MeV proton beam from the booster-synchrotron of High Energy Physics Laboratory. The percentage depth dose of this neutron beam is nearly equivalent to that of X-rays at around 20 MV and the dose rate of 15 cGy per minute. Relative biological effectiveness of this neutron beam has been estimated on the cell killing effect by the use of HMV-I cell line. Resultant survival curve of cells after the neutron irradiation shows the shoulder with n and Dq of 8 and 2.3 Gy, respectively. RBE value at 10(-2) survival level for the present neutron, compared with 137Cs gamma-rays is 1.24. The result suggests that the biological effects of high energy neutrons are not practically large enough whenever the depth dose distribution of neutrons becomes superior to high energy linac X-rays.

Cell Survival↗
PubMed1990

Structural characterization of natural and recombinant human granulocyte colony-stimulating factors.

Granulocyte colony-stimulating factor (G-CSF) is a glycoprotein which stimulates predominantly neutrophilic granulocyte colony formation in mammals. Natural human G-CSF (hG-CSF) and recombinant hG-CSF produced in Chinese hamster ovary (CHO) cells transfected with the cDNA clone for hG-CSF have been purified to apparent homogeneity for structural and biological comparison. The amino acid sequence of recombinant hG-CSF, composed of 174 amino acid residues, was identical with that of natural hG-CSF and also with the sequence predicted from the cDNA. Both forms of hG-CSF have a free Cys-17 and two intramolecular disulfide linkages, between Cys-36 and Cys-42, and between Cys-64 and Cys-74. The O-glycosylation occurred at Thr-133 in both hG-CSFs. Similar CD spectra were obtained for both hG-CSFs. Additionally, both forms showed almost the same biological activities determined by in vitro colony-forming assay and in vivo assay. It is thus concluded that the recombinant hG-CSF is indistinguishable from its natural counterpart and that the former is valuable for more detailed characterization and clinical use.

Amino Acid Sequence↗
PubMed1990

Effect of multiple-dose irradiation on repair capacity in plateau phase C3H 10T1/2 cells.

Using plateau phase C3H 10T1/2 cells, we studied the effect of multiple-dose irradiation on the repair capacity of cells after further irradiation. Cells were irradiated with repeated doses of 2.5 Gy delivered two fractions per day at 6 to 7 hours interval. The cell survival after exposure to 1 to 9 fractions was lower above 5 fractions as compared to that predicted by calculating from single dose survival curve by assuming that cells retain their capacity to repair radiation damage after each fraction. Repair kinetics experiments showed that cells were less able to repair potentially lethal damage after test dose following multiple dose irradiations. There was, however, no difference in the ability to repair sublethal damage and potentially lethal damage sensitive to anisotonic treatment in preirradiated and untreated cells. Thus, it would appear that an enhancement of the lethal expression of potentially lethal damage of three types of damage may, at least in part, contribute to the difference between the cell survival curve after multiple fractions and that predicted by calculation.

Animals↗
PubMed1990

[The effect of repeated small doses of radiation on cell killing and repair capacity in plateau phase C3H 10T 1/2 cells].

Using plateau phase C3H 10 T 1/2 cells, we studied the effect of multiple-dose irradiation on the repair capacity of cells after further irradiation. Cells were irradiated with repeated doses of 2.5 Gy delivered two fractions per day at 6 to 7 hours interval. The cell survival after exposure to 1 to 9 fractions was lower above fractions as compared to that predicted by calculating from single dose survival curve by assuming that cells retain their capacity to repair radiation damage after each fraction. Repair kinetics experiments showed that cells were less able to repair only slow type potentially lethal damage after test dose following multiple dose irradiations. Thus, it would appear that an enhancement of the lethal expression of potentially lethal damage of three types of damage may, at least in part, contribute to the difference between the cell survival curve after multiple fractions and that predicted by calculation.

Animals↗
PubMed1989

Fixation and repair of radiation-induced potentially mutagenic damage sensitive to hypertonic treatment in human diploid fibroblasts.

The effect of hypertonic salt treatment on the repair of potentially lethal damage and potentially mutagenic damage in X-irradiated asynchronous and synchronous human diploid fibroblasts (IMR91) have been studied. Resistance to 6-thioguanine was used for the mutagenic end point. When cells in late-S-phase were treated with hypertonic salt solution immediately after X-irradiation, both cell killing and mutation induction were enhanced, as compared to X-irradiation alone. This suggests that X-irradiation of cells in late S phase induces both potentially lethal damage and potentially mutagenic damage and that both are sensitive to hypertonic salt solution. When cells were allowed 2 h for repair after exposure to X-rays, both types of damage were completely repaired. Almost the same results were obtained with asynchronous cells. These results are discussed in terms of the relationship between radiation damage leading to cell lethality and mutagenesis.

Aphidicolin↗
PubMed1989

Immunological unresponsiveness to hepatic allografts in rats. Immunological reactivities of the recipient to donor antigens.

Wistar (RT1bv1) rats transplanted orthotopically with ACI (RT1a) livers survive indefinitely without any immunosuppression, while heterotopic heart grafts or skin grafts are rejected acutely in this combination. Levels of alkaline phosphatase after liver allografting remain significantly higher than those found in controls receiving syngeneic grafts. We studied changes in immune responsiveness in rats receiving liver grafts. Local graft-versus-host reactivity was present at all times assayed. Delayed type hypersensitivity reactions were already positive 2 weeks after liver transplantation and increased in strength. Liver graft-bearing rats were subsequently grafted with donor or third-party skin. Third-party skin grafts survived significantly longer on liver-grafted rats than on untreated controls when grafted within the first week after grafting. Donor-type skin grafts survived longer than controls when grafted within the first 4 weeks after liver grafting, although the skin grafts were eventually rejected. Donor-type skin grafted more than 8 weeks after liver grafting was rejected acutely. In an adoptive transfer assay, ACI hearts survived significantly longer in Wistar rats given serum from Wistar donors 2-4 weeks after ACI liver grafts than in untreated controls. On the other hand, spleen cells obtained at any period after liver grafting were not capable of prolonging cardiac allograft survival after transfer to syngeneic recipients. Thus cellular responses to ACI antigen are not changed during the life-span of liver-grafted animals. Evidence suggests that a serum "enhancing" factor protects the donor liver from rejection in the initial period after liver transplantation. The long-term acceptance of liver grafts is discussed.

Animals↗