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Biomedical subjects

H Ohkawa

Publications and source records attributed to H Ohkawa.

At least 91 records · Page 5Linked to original sources
PubMed1994

Evidence for the uptake of a vitamin D analogue (OCT) by a human carcinoma and its effect of suppressing the transcription of parathyroid hormone-related peptide gene in vivo.

The present study was undertaken to clarify the pharmacokinetics of 22-oxa-1,25-dihydroxyvitamin D3 (22-oxa-1,25-(OH)2D3, OCT), a vitamin D3 analogue with little calcemic activity, and its effect on the transcription of parathyroid hormone-related peptide (PTHRP) gene in nude mice bearing a human carcinoma (FA-6) associated with humoral hypercalcemia. FA-6 tumor expressed vitamin D receptor (VDR) mRNA, and its nuclear extract contained a specific and saturable 1,25-(OH)2D3 binding activity. Although [3H]OCT administered intravenously into FA-6 tumor-bearing nude mice was cleared from the circulation more rapidly than [3H]1,25-(OH)2D3, the uptake of [3H]OCT into the tumor tissue, relative to the radioactivity in the circulation, was greater than that of [3H]1,25-(OH)2D3. Intravenous or oral administration of OCT reduced the steady-state levels of PTHRP mRNA in FA-6 tumor, and nuclear run-off assays demonstrated that the effect of OCT on PTHRP gene expression occurred at a transcriptional level. RNase mapping analysis revealed that both upstream and downstream promoters of the human PTHRP gene were down-regulated by OCT. Finally, OCT exerted a preventive as well as therapeutic effect on cancer-associated hypercalcemia with a marked prolongation of the survival time in tumor-bearing animals. These results suggest that OCT is effectively taken up by a VDR-positive human carcinoma in vivo and has a therapeutic potential for cancer-associated hypercalcemia through suppression of PTHRP gene transcription.

Animals↗
PubMed1994

Rotation and membrane topology of genetically expressed methylcholanthrene-inducible cytochrome P-450IA1 lacking the N-terminal hydrophobic segment in yeast microsomes.

A modified rat liver cytochrome P-450IA1, lacking amino acids 2-30, a proposed membrane anchor for cytochrome P-450, was expressed genetically in yeast microsomal membranes. This truncated cytochrome is practically active in the deethylation of 7-ethoxycoumarin. A full-length cytochrome P-450IA1 was also expressed in yeast microsomes. Rotational diffusion of P-450IA1 was examined by observing the flash-induced absorption anisotropy r(t) of the P-450.CO complex. The anisotropy decayed to a time-independent value within a 2-ms time range. Since the decay curve has the characteristics of a slow rotation of membrane-embedded cytochrome, the theoretical analysis of r(t) was performed based on a "rotation-about-membrane normal" model. 41% of the shortened P-450IA1 was rotating with the rotational relaxation time phi of 1020 microseconds, whereas 27% of the full-length P-450IA1 was mobile with phi = 1101 microseconds. The high salt treatment did not remove the shortened cytochrome from the membrane and also did not drastically weaken the interactions of the cytochrome with the membrane, as judged from the slow rotation characteristics (phi = 830 microseconds). These results demonstrate that the N-terminal shortened P-450IA1 is incorporated properly into the yeast microsomal membrane and that the N-terminal hydrophobic segment is not solely responsible for attachment to the membrane, providing evidence that additional segments of P-450IA1 are involved in the membrane binding.

Animals↗
PubMed1994

Kinetic studies on a genetically engineered fused enzyme between rat cytochrome P4501A1 and yeast NADPH-P450 reductase.

Three expression plasmids, pAMC1 for rat P4501A1, pAMR2 for P4501A1 and yeast NADPH-P450 reductase, and pAFCR1 for a fused enzyme between P4501A1 and the reductase, were constructed, and each was introduced into Saccharomyces cerevisiae AH22 cells. The microsomal fraction prepared from the recombinant yeast cells was subjected to kinetic studies of zoxazolamine 6-hydroxylation at 10 degrees C. The apparent Km and Vmax values for hydroxylation by the fused enzyme in AH22/pAFCR1 microsomes were 0.38 mM and 0.42 s-1, respectively. The rate constant for reduction of the fused enzyme with NADPH in the presence of 1 mM zoxazolamine was larger than 50 s-1 using a dual-wavelength stopped-flow spectrometer, indicating that electrons are rapidly transferred from NADPH through FAD and FMN to the heme iron of the fused enzyme. The rate constant kon for substrate binding to the fused enzyme was 25 mM-1.s-1, which is not much different from that of nonfused P4501A1. These results together with spectral data measured during the hydroxylation reaction in the steady state suggest that the rate-limiting step of the reaction by the fused enzyme might be the release of product. On the other hand, the apparent Km and Vmax values for the hydroxylation of P4501A1 in AH22/pAMC1 and AH22/pAMR2 microsomes were 0.32 and 0.33 mM, and 0.015 and 0.29 s-1, respectively. The rate constants for the reduction of P4501A1 were 0.025 and 0.40 s-1, respectively, for AH22/pAMC1 and AH22/pAMR2 microsomes.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

A murine model for bone marrow metastasis established by an i.v. injection of C-1300 neuroblastoma in A/J mice.

A reproducible tumor model for bone marrow metastasis has been developed by an injection of murine C-1300 neuroblastoma (C-1300 NB) cells into the tail vein of syngeneic A/J mice. The animals died with liver metastases at 18-21 days after an injection of 10(5) tumor cells and often had bone marrow metastasis in the femur. N-methylformamide (NMF), a maturational agent, was administered to inhibit liver metastases and to extend survival in mice with advancing bone metastasis. Histological examination of bone marrow metastasis, demonstrated lesions varying from a few small colonies of C-1300 NB cells either in metaphysis or diaphysis to large foci replacing normal hematopoietic bone marrow, simultaneously invading epiphysis or cortex of bone as bone metastasis. This assay demonstrated the ability to detect neuroblastoma cells in the bone marrow histologically and could determine bone marrow TD50 by extraction of bone marrow cells after treatment with various doses of drug. Fifty per cent of mice injected with cyclophosphamide (CY) developed bone marrow metastasis without liver metastasis. Treatment with tamoxifen, an anti-calmodulin drug, suppressed tumor takes in the recipient mice with tamoxifen-dose-dependent fashion. This experimental system allows for investigations into the therapeutic response and biology of neuroblastoma metastases in the bone marrow.

Animals↗
PubMed1994

Herbicide-resistant tobacco plants expressing the fused enzyme between rat cytochrome P4501A1 (CYP1A1) and yeast NADPH-cytochrome P450 oxidoreductase.

Transgenic tobacco (Nicotiana tabacum cv Xanthi) plants expressing a genetically engineered fused enzyme between rat cytochrome P4501A1 (CYP1A1) and yeast NADPH-cytochrome P450 oxidoreductase were produced. The expression plasmid pGFC2 for the fused enzyme was constructed by insertion of the corresponding cDNA into the expression vector pNG01 under the control of the cauliflower mosaic virus 35S promoter and nopaline synthase gene terminator. The fused enzyme cDNA was integrated into tobacco genomes by Agrobacterium infection techniques. In transgenic tobacco plants, the fused enzyme protein was localized primarily in the microsomal fraction. The microsomal monooxygenase activities were approximately 10 times higher toward both 7-ethoxycoumarin and benzo[a]pyrene than in the control plant. The transgenic plants also showed resistance to the herbicide chlortoluron.

Animals↗
PubMed1994

Donor stromal cells support hematopoiesis in CFU-S colonies.

To examine the effect of hematopoietic stromal cells on bone marrow transplantation (BMT), CF-1 cells, which were previously established from splenic stroma of a mouse that was administered recombinant human granulocyte colony-stimulating factor (rhG-CSF), were transplanted with bone marrow cells. The stromal cells were transfected with the plasmid DNA of beta-galactosidase (beta G-CF-1) cells for studying immunohistochemistry. Using immunohistochemistry, the spleen and bone marrow of the recipient mice were examined for beta-galactosidase on the 8th and 12th day after BMT. In the spleen, beta G-CF-1 cells were observed within and in the regions immediately surrounding the colony-forming unit-spleen (CFU-S) on the 8th and 12th day after BMT. In certain regions, beta G-CF-1 cells existed in or surrounded the CFU-S colonies that consisted of the undifferentiated hematopoietic cells, while in other regions the granulopoiesis increased, especially in correspondence with the existence of beta G-CF-1 cells. In the bone marrow cavity, beta G-CF-1 cells were also recognized, though hematopoiesis was lower than the normal bone marrow. These data demonstrate that CF-1 cells, that is, donor stromal cells, can be transplanted to the hematopoietic organs and can then support hematopoiesis.

Animals↗
PubMed1994

[Changes in rectal temperature during tympanoplasty under general anesthesia].

We measured rectal temperature of 35 patients who underwent tympanoplasty under various types of general anesthesia. They were allocated at random to three groups according to the types of general anesthesia; 10 patients of enflurane-N2O (enflurane group); 10 patients of neuroleptic anesthesia with droperidol, pentazocine-N2O (NLA group); 15 patients of total intravenous anesthesia with droperidol, fentanyl and ketamine (DFK group). After the induction of anesthesia, their rectal temperature was continuously monitored with an electric thermometer of NEC San-ei throughout the surgical procedure. Increase in rectal temperature was observed in all three groups, and increase in DFK group was significant compared with other two groups. It is possible that the significant increase in rectal temperature in DFK group is supposedly due to normally maintained hypothalamic thermoregulatory function as well as direct surgical stimulation to central nervous system.

Adult↗
PubMed1993

Passive immunization with anti-parathyroid hormone-related protein monoclonal antibody markedly prolongs survival time of hypercalcemic nude mice bearing transplanted human PTHrP-producing tumors.

Malignancy-associated hypercalcemia is mainly caused by excessive production of parathyroid hormone-related protein (PTHrP) by the tumor. Using anti-PTHrP-(1-34) monoclonal murine antibody (anti-PTHrP MoAb), we studied whether repeated injection of the homologous antibody would continuously decrease the serum calcium concentration in hypercalcemic nude mice bearing transplanted human PTHrP-producing tumors, leading to prolongation of their survival time. Daily SC injections of anti-PTHrP MoAb decreased the serum calcium concentration almost to within the normal range in nude mice bearing transplanted human PTHrP-producing tumors (T3M-1, EC-GI, PC-3, and FA-6) but not in a nude mouse bearing a transplanted parathyroid carcinoma. The antibody did not affect FA-6 tumor growth either in vitro or in vivo. Pancreatic carcinoma cells (FA-6), which caused the most severe hypercalcemia, were inoculated into 6-week-old nude mice. When severe hypercalcemia (approximately 19 mg/dl) had developed, daily SC injection of anti-PTHrP MoAb was started. Within 18 days of this time point, all untreated tumor-bearing mice (n = 10) died of hypercalcemia and cachexia, whereas all the treated mice (n = 10) showed an increase in body weight and survived for at least 25 days. Histologic examination of the treated mice revealed a marked decrease in osteoclastic bone resorption, without toxicologic findings in the kidney and liver. These results suggest that passive immunization against PTHrP can continuously ameliorate the hypercalcemia and markedly prolong the survival time of severely hypercalcemic, tumor-bearing mice. If a human monoclonal antibody against PTHrP-(1-34) could be developed, then passive immunization would be potentially one of the most effective therapies for patients with malignancy-associated hypercalcemia due to excessive production of PTHrP.

Animals↗
PubMed1993

Familial congenital diaphragmatic hernia in the pig--studies on pathology and heredity.

Thirteen cases of congenital diaphragmatic hernia were encountered in one of the three pig families, established by us as a disease model of ano-rectal malformations. Five were males and 8 were females. The age at the onset of symptoms ranged between one and 4 months after birth. On the laterality, 7 had right side, 5 had left side and one had bilateral hernias. No hernial sac was observed. Herniated organs were intestine in the right and stomach in the left side. Thus congenital origin with late onset was suggested as a cause of the disease. In the studies of the heredity, there was no linkage between ano-rectal malformations and diaphragmatic hernia. Double recessive genes were supposed as a mode of inheritance, according to the statistical calculation. An effort is being made to produce a model of congenital diaphragmatic hernia from the pig family.

Animals↗
PubMed1993

[Clinical study on intraoperative hyperketonemia in non-diabetic surgical patients under general anesthesia].

We measured plasma 3-hydroxybutyrate (3-OHBA) concentrations in 925 non-diabetic surgical patients who underwent various surgical procedures under various types of general anesthesia. Lactated Ringer's solution only was used as an intraoperative fluid. Among them, 46 patients (4.9%) developed high 3-OHBA levels of over 500 microM.l-1 during surgery. The causes of observed hyperketonemia would be surgical stress and preoperative fasting irrespective of 8 types of general anesthesia. Blood glucose, lactic acid, base excess and pH were not influenced by hyperketonemia. Prolonged recovery from anesthesia was not observed in any patients with hyperketonemia. Without any specific treatments, plasma 3-OHBA levels were unchanged or rather decreased in 70% of the patients whose 3-OHBA levels were over 500 microM. The results suggest that hyperketonemia below 500 microM.l-1 is not always disadvantageous in nondiabetic patients under general anesthesia.

3-Hydroxybutyric Acid↗
PubMed1993

[Clinical study on total intravenous anesthesia with droperidol, fentanyl, and ketamine--18. Effect on peripheral circulation as judged by core-peripheral temperature gradient].

Effect of total intravenous anesthesia with droperidol, fentanyl and ketamine (DFK) on peripheral circulation was studied by examining core-peripheral temperature gradient in twenty five patients who underwent abdominal surgery. A core temperature probe was attached on the forehead and peripheral probe on the palm of the hand of the side on which the blood pressure cuff was not applied. The temperature gradient was less than three degrees centigrade in 60% of the patients and the gradient was significantly less as compared with that of isoflurane anesthesia even at 300 minutes after the start of surgical operation. This advantage would have been caused by such factors as circulatory stimulating effect of ketamine, sympathetic blocking effect by droperidol and adequate postoperative analgesia by fentanyl and norketamine, a metabolite of ketamine. The results suggest that DFK would exert a beneficial effect on peripheral circulation, particularly during prolonged surgical procedures.

Abdomen↗
PubMed1992

Enhanced hematopoiesis in vivo and in vitro by splenic stromal cells derived from the mouse with recombinant granulocyte colony-stimulating factor.

Splenic stromal cells (CF-1 cells) were established from a mouse administered recombinant human granulocyte colony-stimulating factor (rG-CSF) to clarify the mechanism of splenic extramedullary hematopoiesis induced by the factor. The cells were negative for alkaline phosphatase, factor VIII-related antigen, mac I, and phagocytosis. They were positive for acid phosphatase, collagen type I, collagen type III, and fibronectin. CF-1 cells were not converted to adipocytes in a confluent culture with 10(-6) mol/L hydrocortisone. [35S]rG-CSF bound to CF-1 cells specifically in the growth phase but not in the resting phase. The CF-1 cells had greater colony-stimulating activities than the normal splenic stromal cells. When CF-1 cells were added to bone marrow cells in the spleen colony-forming cells (CFU-S) assay, the number of colonies in the spleen increased between 1.4 and 1.8 times the control without these stromal cells. On the other hand, the normal splenic stromal cells had no effect on increasing the number of CFU-S colonies. Therefore, these data suggest that a factor-dependent hematopoietic microenvironment is generated in the spleen by rG-CSF, and the stromal cells that have the hematopoietic potency become dominant in splenic extramedullary hematopoiesis induced by rG-CSF.

Acid Phosphatase↗
PubMed1992

Organella-targeted expression of rat liver cytochrome P450c27 in yeast. Genetically engineered alteration of mitochondrial P450 into a microsomal form creates a novel functional electron transport chain.

A modified rat cytochrome P450c27, whose mitochondrial targeting signal had been replaced by a possible microsomal targeting signal of bovine cytochrome P450c17, was expressed in yeast. The modified P450c27 hemoprotein was correctly localized on yeast microsomes and exhibited the P450c27-dependent monooxygenase activity by addition of bovine adrenodoxin (ADX) and NADPH-adrenodoxin reductase (ADR). Considering the previous observation that P450c27 with its own mitochondrial targeting signal was imported into yeast mitochondria (Akiyoshi-Shibata, M., Usui, E., Sakaki, T., Yabusaki, Y., Noshiro, M., Okuda, K., and Ohkawa, H. (1991) FEBS Lett. 280, 367-370), it is now suggested that the destination of P450c27 to either mitochondria or microsomes in yeast depends solely on the amino-terminal targeting signal. In addition, the modified P450c27 was simultaneously expressed in yeast with mature forms of bovine ADX and ADR. The recombinant yeast produced the P450 on the microsomes and mature forms of ADX and ADR in the cytoplasm, and showed the monooxygenase activity. Accordingly, a novel type of functional electron transport chain has been established between the cytoplasm and the microsomes in yeast.

Animals↗
PubMed1992

Synthetic studies of vitamin D analogues. X. Synthesis and biological activities of 1 alpha,25-dihydroxy-21-norvitamin D3.

1 alpha,25-Dihydroxy-21-norvitamin D3 (3) was synthesized from 1 alpha-hydroxydehydroepiandrosterone (4). Certain biological properties of 3 were examined in comparison with those of 1 alpha,25-dihydroxyvitamin D3 (1) and 1 alpha,25-dihydroxy-21-nor-20-oxavitamin D3 (2) to evaluate the effect of the 21-methyl substituent on biological activities. The differentiation-inducing activity of 3 towards human myeloid leukemia cells was approximately one-fifth of that of 1, while in the binding affinity with chick intestinal cytosolic receptor, 3 was about one-tenth of that of 1. The rather weak effect of 3 on serum calcium levels in normal mice at a dosage of 500 micrograms/kg (intravenous administration) indicates that the essential importance of the 21-methyl moiety may lie in its effect on the regulation of calcium metabolism.

Animals↗
PubMed1992

Insecticidal activity and processing in larval gut juices of genetically engineered 130-kDa proteins of Bacillus thuringiensis subsp. aizawai.

The 130-kDa insecticidal protein (IP) of Bacillus thuringiensis subsp. aizawai is proteolytically processed in the gut juice of susceptible insect larvae to yield an insecticidally active 60-kDa fragment. Twenty-seven mutant IP genes with the replacement of codons for Arg and Lys with codons for Gln in the active fragment and its adjacent regions of the 130-kDa IP were constructed by site-directed mutagenesis and expressed in Escherichia coli cells. The produced mutant IPs at Arg87, Arg131, Arg198, Arg311, Arg368, Arg402, Arg458, Arg502, Arg512, Arg524, Arg526, Arg528, and Arg601 had reduced insecticidal activity against Spodoptera litura larvae. The mutant at Arg601 was sensitive to proteolytic digestion in the gut juice of S. litura larvae. Although the mutants at Arg619, Lys622, and Lys637 had nearly the same activity as that of the wild type, the mutant with the triple replacement at Arg619, Lys622, and Lys637 was 2.5 times more active against S. litura larvae than the wild type. This triple mutant showed a slightly different processing profile in the gut juice than that of the wild type.

Amino Acid Sequence↗
PubMed1992

Parathyroid hormone-related protein as a cause of hypercalcemia in a B-cell type malignant lymphoma.

Hypercalcemia occurred in a patient with non-Hodgkin's (B-cell type) lymphoma when generalized lymphadenopathy developed. Despite low normal plasma parathyroid hormone (PTH), nephrogenous cAMP (NcAMP) was not suppressed, and serum and urine PTH-related protein (PTH-rP) levels were elevated. The plasma level of 1,25(OH)2D was within normal range. The combined chemotherapies successfully reduced the tumor size, serum Ca, PTH-rP, and lactic dehydrogenase. Serum osteocalcin was suppressed while the patient was hypercalcemic, and increased after chemotherapy. In the extract of the tumor tissue obtained post mortem, bioactivity stimulating the production of cAMP in osteoblasts was demonstrated along with the immunoreactive PTH-rP. This is the first report of a B-cell lymphoma producing PTH-rP and its association with humoral hypercalcemia of malignancy.

Aclarubicin↗
PubMed1992

[Clinical study on total intravenous anesthesia with droperidol, pentazocine and ketamine].

Fifty patients underwent various surgical procedures including abdominal, orthopedic, plastic and gynecological operations under total intravenous anesthesia with ketamine, pentazocine and droperidol. Neither nitrous oxide, inhaled anesthetics nor narcotics such as fentanyl were administered to the patients. Intraoperative muscle relaxation was achieved with vecuronium and the patients were ventilated manually throughout the surgical procedures. Thirty percent of the patients developed hypertension and tachycardia, but they were easily overcome with administration of calcium ion channel blocker. Their peripheral circulation as well as urine output was well maintained. No adverse effects on the liver and kidney were observed post-operatively. Their post-operative sedation and analgesia were evaluated excellent. A few patients had strange dream as if they might have missed their way into the "pink" tunnel. The data above described suggest that this anesthetic method would deserve further detailed clinical study.

Adult↗