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Biomedical subjects

M Okabe

Publications and source records attributed to M Okabe.

At least 127 records · Page 7Linked to original sources
PubMed1999

Image analysis of morphological change during arachidonic acid production by Mortierella alpina 1S-4.

The changes in mycelial morphology during arachidonic acid (AA) production by Mortierella alpina 1S-4 were investigated using an image analysis system. Cultivation was performed in a 10-kl fermentor, and the culture broth was separated into two fractions by sieving (0.5 mm aperture size): the filament fraction (F-fraction, <0.5 mm), and the pellet fraction (P-fraction, >0.5 mm). The effect of the mycelial morphology in each fraction on AA production was analyzed. As a result, a product distribution in the culture broth wherein the AA content in the mycelia of the P-fraction was observed to be higher than that in the mycelia of the F-fraction throughout the cultivation. Morphological analysis of the P-fraction revealed that the hairy pellets became smooth because the mycelia on the pellet surface were shaved off; some pellets were broken and reduced in size. The shaved-off mycelia from the hairy pellets surface moved into the F-fraction and aggregated there. From the above findings, it was likely that the low AA content in the F-fraction was due to mycelial damage during the cultivation. In addition, the morphology of the hairy pellets was found to contribute to an increase in the viscosity of culture broth.

Journal Article↗
PubMed1999

Visualization of a recombinant gene protein in the baculovirus expression vector system using confocal scanning laser microscopy.

Expression of the recombinant protein beta-galactosidase in the Spodoptera frugiperda Sf-9 insect cell line infected by the Autographa californica nuclear polyhedrosis virus expressing beta-galactosidase (AcNPV-betagal) was visualized using confocal scanning laser microscopy with fluorescent staining of both the recombinant protein and the cell nucleus. The average size of the insect cells and the intracellular DNA concentration both increased markedly, respectively reading 3.8- and 2.3-fold the values before infection. The average beta-galactosidase activity began to increase at 20-24 h post infection and finally reached 1.9 x 10(4) units/ml. As the post infection time increased, the stained nucleus images expanded and spread broadly. Beta-galactosidase was first identified by fluorescent staining at 12 h post-infection, filled the cell at 27 h, began to be released at 36 h, and finally spread out of the cell. The locations of the nucleus and expressed beta-galactosidase were identified from computerized tomograms and 3-dimensional images.

Journal Article↗
PubMed1999

Effect of soybean oil on oxygen transfer in the production of tetracycline with an airlift bioreactor.

Corn starch and soybean oil are suitable carbon sources for the production of tetracycline by Streptomyces aureofacience CG-1. However, it could not produce more than 6 g/l of tetracycline even if initial corn starch concentration was increased to more than 100 g/l. It was confirmed by shaking flask experiments that the k(L)a in a mixture of 2% soybean oil in water was four folds compared with that without soybean oil. With the addition of soybean oil to the starch medium in a shaking flask, tetracycline production was significantly improved. By scaling-up to a 5.5-l airlift bioreactor from 500-ml Erlenmeyer flask, more than 10 g/l of tetracycline was produced with the addition of 60 g/l of soybean oil to the medium containing 100 g/l of corn starch. The dissolved oxygen level in the airlift bioreactor containing soybean oil was higher than that without soybean oil. This suggests that soybean oil is not only a suitable carbon source but is also a surface-active agent which may accelerate the oxygen transfer. This may lead to the possibility of the enhanced production of tetracycline at a low cost in airlift bioreactor.

Journal Article↗
PubMed1999

Effect of nitrogen source on mycelial morphology and arachidonic acid production in cultures of Mortierella alpina.

The effects of nitrogen source on arachidonic acid (AA) production and morphological changes during the culture of Mortierella alpina were investigated using an image analysis system. When yeast extract, gluten meal, or corn steep liquor was used, a circular pellet morphology was obtained. However, when Pharmamedia, fish meal, or soybean meal was used, M. alpina formed radial filamentous mycelia. The radial filamentous area in the case of soybean meal was 75% of the total mycelial area. In a jar fermentor culture, M. alpina morphology varied with the cultivation period: (i) at 0-6 h culture, the inoculated pellet-like mycelia were adapted to the new environment, (ii) at 6 h-1 d culture, filamentous mycelia grew exponentially which yielded a feather-like morphology, (iii) at 1-2 d culture, the filamentous mycelia became disentangled as a result of the mechanical agitation; consequently, the proportion of filamentous mycelia was increased, (iv) at 2-4 d culture, mycelia showed stationary growth, but the AA concentration increased rapidly, and (v) at 4-6 d culture, hyphae grew thick radially with the AA concentration continuing to increase gradually. In the case of the cultures with feather-like morphology obtained using soybean meal, the AA yield was 0.14 g/g dry cell weight, which was two times higher than that in cultures grown using yeast extract. These results suggest that the feather-like morphology of culture of M. alpina is suitable for AA production.

Journal Article↗
PubMed1999

Differential expression of CD43 isoforms on murine T cells and their relationship to acute intestinal graft versus host disease: studies using enhanced-green fluorescent protein transgenic mice.

Three mAb (R2/60, S7 and 1B11) were used to study the expression of murine CD43 on peripheral T cells and intestinal intraepithelial lymphocytes (IEL) from normal mice, and from mice during acute graft versus host disease (GVHD). In the spleen, essentially all T cells expressed the R2/60 and S7 antigens, whereas the 1B11 antigen was expressed on about half of the CD8(+) cells and approximately 15% of CD4(+) T cells. Interestingly, a significant proportion of resting splenic B cells expressed the 1B11 and R2/60 antigens, but not the S7 antigen. The majority of IEL expressed R2/60 antigen; however, the S7 and 1B11 markers were differentially expressed on CD8alpha, CD8beta, TCRalphabeta and TCRgammadelta cells. Immunoprecipitation and Western blotting analyses identified characteristic 115 and 130 kDa reactive components from IEL lysates with mAb S7 and 1B11 respectively, and reactivity to both molecular entities by mAb R2/60. During acute intestinal GVHD induced by injecting CB6F(1) athymic nude mice with spleen cells from C57BL/6 enhanced-green fluorescent protein transgenic mice, 80-90% of donor T cells in the intestine epithelium expressed all CD43 isoforms; however, the level of expression of the 130 kDa CD43 antigen increased significantly and the level of the 115 kDa antigen decreased on GVHD donor T cells compared to cells at the time of transfer. Using EL4 cells, a similar shift in the expression of CD43 isoforms occurred experimentally following treatment with neuraminidase, suggesting that the type of CD43 isoform expressed on T cells is strongly influenced by conditions which affect membrane charge. The significance of these findings for intestinal immunopathology is discussed.

Animals↗
PubMed1999

Arrest of spermatogonial differentiation in jsd/jsd, Sl17H/Sl17H, and cryptorchid mice.

The nature of the spermatogenic arrest in cryptorchid C57Bl mice and in jsd/jsd and Sl17H/Sl17H mutant mice was identified by studying whole mounts of seminiferous tubules. In all three types of mice, virtually only A spermatogonia were found, topographically arranged in clones of 1 to 16 (rarely more) cells. These clonal sizes are typical for undifferentiated spermatogonia. The proportion of these cells lying in chains of more than 2 cells (50-70%) was comparable to that seen in epithelial stages VII-VIII in the normal epithelium. It is concluded that in all three types of mice, spermatogenesis is arrested at the point where the undifferentiated A spermatogonia, specifically A(al) spermatogonia, differentiate into the first generation of the differentiating-type spermatogonia, the A1 spermatogonia. The remaining A spermatogonia were proliferating, but no accumulation of spermatogonia was present, as spermatogonial apoptosis also took place. Spermatogonial clones of all sizes were seen to undergo apoptosis, but there were relatively many large apoptotic clones, indicating that the clones became more vulnerable when they became larger. In contrast to what is seen in the normal epithelium, odd-numbered clones, not composed of 2(n) cells, were present, as well as clumps of 2 or more spermatogonial nuclei in the same cytoplasm, in all three types of mice. This indicates a lack of integrity of spermatogonial clones, also observed in other situations with a relative paucity of cells on the basal membrane. It is concluded that the differentiation of the undifferentiated spermatogonia, affected in all three types of mice as well as in vitamin A-deficient animals, is a rather vulnerable point in the spermatogenic developmental pathway.

Animals↗
PubMed1999

Differential effects of UCN-01, staurosporine and CGP 41 251 on cell cycle progression and CDC2/cyclin B1 regulation in A431 cells synchronized at M phase by nocodazole.

UCN-01 (7-hydroxystaurosporine) and CGP 41 251 (4'-N-benzoyl staurosporine), both of which were discovered as protein kinase C selective inhibitors, have entered in phase 1 clinical trials as anti-cancer drugs. In this study, we have directly compared the effects of these drugs as well as staurosporine (STP) on cell cycle progression of A431 human epidermoid carcinoma cells synchronized at M phase by treatment with nocodazole. The nocodazole-synchronized cells progressed from M to G1 phase in the absence of the drug, which was accompanied by a decrease of cyclin B1 protein expression, disappearance of the complex formation of CDC2 with cyclin B1 and reduction of the kinase activity. Treatments of the M phase cells with UCN-01, STP and CGP 41 251 at 80% growth-inhibitory concentrations (IC80S) resulted in specific G1 block, G2M block and polyploidy, respectively. Decreases of cyclin B1 protein expression was partially prevented by treatments with STP and CGP 41 251 but not with UCN-01 at IC80S. Reductions of active complex and kinase activity of CDC2/cyclin B1 were also observed in the presence of the three drugs. In addition, augmentation of CDC2 protein tyrosine phosphorylation was induced only when the cells were treated with STP. These observations demonstrated that higher concentrations of UCN-01, STP and CGP 41 251 showed different effects on cell cycle progression as well as CDC2/cyclin B1 regulation in A431 cells synchronized at M phase. The data suggest that UCN-01 and CGP 41 251 may act at quite different points on the cell cycle.

Alkaloids↗
PubMed1999

Antitumor activity of KF22678, a novel thioester derivative of leinamycin.

KF22678, a novel thioester derivative of leinamycin with the 1-oxo-1,2-dithiolane-3-one moiety, was examined for anti-tumor activity, toxicity in mice and activation mechanism. KF22678 showed a broad antitumor spectrum against human carcinoma xenografts (lung, colon, ovary and prostate). The efficacy of KF22678 was significantly higher than that of cisplatin. KF22678 exhibited low cross-resistance against various drug-resistant cell lines of MDR1 or MRP overexpressing human tumors, and, in addition, exhibited more potent antitumor activity in vivo than ADM against A2780/ADM and KB/MRP xenograft. DL-Buthionine sulfoximine (BSO) pretreatment significantly reduced intracellular glutathione (GSH) level in human lung carcinoma A549 cells, leading to decrease in the cytotoxicity of KF22678, whereas the cytotoxicity of melphalan was augmented by BSO pretreatment. DNA single-strand breaks (SSB) were observed in A549 cells treated with KF22678 and bleomycin. DNA SSB induced by KF22678 was greatly reduced in the presence of BSO in the cells, whereas DNA SSB induced by bleomycin was not. In addition, the antitumor activity of KF22678 against BSO-pretreated human lung carcinoma PC-9 tumor was significantly decreased. These results suggest that the activation of KF22678 by intracellular GSH might be important for DNA SSB and antitumor activity in vitro and in vivo.

Animals↗
PubMed1999

Role of metallothionein on Ag accumulation in hepatic and renal cytosol after Ag injection to rats.

To examine the role of metallothionein on Ag accumulation in liver or kidney of rat after Ag injection, the relative Ag-binding capacity of Ag-induced metallothionein in hepatic or renal cytosol of rat after Ag injection was determined. The greater part of Ag increment in hepatic cytosol was attributable to a low molecular weight protein, while the main part of Ag increment in renal cytosol was ascribed to high molecular weight proteins. The low molecular weight, metal-binding protein was identified as metallothionein using ELISA. The maximal levels of hepatic and renal metallothionein mRNA induced by Ag occurred at 7 hr after Ag injection. There was a close relationship between Ag contents in the hepatic or renal cytosol and metallothionein after Ag injection. In dose-response and time-course studies, approximately 60-70% of the Ag increments in hepatic cytosol and approximately 30% of the Ag increments in renal cytosol were bound to metallothionein. These results suggest that the role of metallothionein on Ag accumulation in the liver after Ag injection is different from that of metallothionein on Ag accumulation in the kidney.

Animals↗
PubMed1999

The effect of heavy metal-induced metallothionein on Zn, Cu and Cd accumulation in rat kidney.

To examine the role of metallothionein on heavy metal accumulation in kidneys of rats after Zn, Cd or Cu injection, the relative Zn, Cd or Cu-binding capacity of heavy metal-induced metallothionein in renal cytosol of rats after Zn, Cd or Cu injection was determined. The Zn or Cu increment in renal cytosol after Zn or Cu injection was attributable to low and high molecular weight proteins, while most of the Cd increment was attributable to a low molecular weight protein. The low molecular weight, metal-binding protein was identified as metallothionein using a competitive ELISA. There was a close relationship between heavy metal contents in the renal cytosol and metallothionein of all heavy metal-injected rats. In dose-response and time-course studies, approximately 45, 40 and 85% of the Zn, Cu and Cd increments in renal cytosol were bound to metallothionein after Zn, Cu and Cd injection, respectively. Therefore the order of relative binding capacities of Zn, Cu and Cd-induced metallothionein in kidney was determined to be Cd>Zn approximately Cu. These results suggest that the role of metallothionein in Zn or Cu accumulation in the kidney after Zn or Cu injection is different from that of metallothionein in Cd accumulation in the kidney after Cd injection.

Animals↗
PubMed1999

Postnatal growth failure, short life span, and early onset of cellular senescence and subsequent immortalization in mice lacking the xeroderma pigmentosum group G gene.

The xeroderma pigmentosum group G (XP-G) gene (XPG) encodes a structure-specific DNA endonuclease that functions in nucleotide excision repair (NER). XP-G patients show various symptoms, ranging from mild cutaneous abnormalities to severe dermatological impairments. In some cases, patients exhibit growth failure and life-shortening and neurological dysfunctions, which are characteristics of Cockayne syndrome (CS). The known XPG protein function as the 3' nuclease in NER, however, cannot explain the development of CS in certain XP-G patients. To gain an insight into the functions of the XPG protein, we have generated and examined mice lacking xpg (the mouse counterpart of the human XPG gene) alleles. The xpg-deficient mice exhibited postnatal growth failure and underwent premature death. Since XPA-deficient mice, which are totally defective in NER, do not show such symptoms, our data indicate that XPG performs an additional function(s) besides its role in NER. Our in vitro studies showed that primary embryonic fibroblasts isolated from the xpg-deficient mice underwent premature senescence and exhibited the early onset of immortalization and accumulation of p53.

Animals↗
PubMed1999

Suppression of STAT5 functions in liver, mammary glands, and T cells in cytokine-inducible SH2-containing protein 1 transgenic mice.

Various cytokines utilize Janus kinase (JAK) and the STAT (signal transducers and activators of transcription) family of transcription factors to carry out their biological functions. Among STATs, two highly related proteins, STAT5a and STAT5b, are activated by various cytokines, including prolactin, growth hormone, erythropoietin, interleukin 2 (IL-2), and IL-3. We have cloned a STAT5-dependent immediate-early cytokine-responsive gene, CIS1 (encoding cytokine-inducible SH2-containing protein 1). In this study, we created CIS1 transgenic mice under the control of a beta-actin promoter. The transgenic mice developed normally; however, their body weight was lower than that of the wild-type mice, suggesting a defect in growth hormone signaling. Female transgenic mice failed to lactate after parturition because of a failure in terminal differentiation of the mammary glands, suggesting a defect in prolactin signaling. The IL-2-dependent upregulation of the IL-2 receptor alpha chain and proliferation were partially suppressed in the T cells of transgenic mice. These phenotypes remarkably resembled those found in STAT5a and/or STAT5b knockout mice. Indeed, STAT5 tyrosine phosphorylation was suppressed in mammary glands and the liver. Furthermore, the IL-2-induced activation of STAT5 was markedly inhibited in T cells in transgenic mice, while leukemia inhibitory factor-induced STAT3 phosphorylation was not affected. We also found that the numbers of gamma delta T cells, as well as those of natural killer (NK) cells and NKT cells, were dramatically decreased and that Th1/Th2 differentiation was altered in transgenic mice. These data suggest that CIS1 functions as a specific negative regulator of STAT5 in vivo and plays an important regulatory role in the liver, mammary glands, and T cells.

Animals↗
PubMed1999

Sprouty: a common antagonist of FGF and EGF signaling pathways in Drosophila.

Extracellular factors such as FGF and EGF control various aspects of morphogenesis, patterning and cellular proliferation in both invertebrates and vertebrates. In most systems, it is primarily the distribution of these factors that controls the differential behavior of the responding cells. Here we describe the role of Sprouty in eye development. Sprouty is an extracellular protein that has been shown to antagonize FGF signaling during tracheal branching in Drosophila. It is a novel type of protein with a highly conserved cysteine-rich region. In addition to the embryonic tracheal system, sprouty is also expressed in other tissues including the developing eye imaginal disc, embryonic chordotonal organ precursors and the midline glia. In each of these tissues, EGF receptor signaling is known to participate in the control of the correct number of neurons or glia. We show that, in all three tissues, the loss of sprouty results in supernumerary neurons or glia, respectively. Furthermore, overexpression of sprouty in wing veins and ovarian follicle cells, two other tissues where EGF signaling is required for patterning, results in phenotypes that resemble the loss-of-function phenotypes of Egf receptor. These results suggest that Sprouty acts as an antagonist of EGF as well as FGF signaling pathways. These receptor tyrosine kinase-mediated pathways may share not only intracellular signaling components but also extracellular factors that modulate the strength of the signal.

Animals↗
PubMed1999

Mutation in ankyrin repeats of the mouse Notch2 gene induces early embryonic lethality.

Notch family genes encode transmembrane proteins involved in cell-fate determination. Using gene targeting procedures, we disrupted the mouse Notch2 gene by replacing all but one of the ankyrin repeat sequences in the cytoplasmic domain with the E. coli (beta)-galactosidase gene. The mutant Notch2 gene encodes a 380 kDa Notch2-(beta)-gal fusion protein with (beta)-galactosidase activity. Notch2 homozygous mutant mice die prior to embryonic day 11.5, whereas heterozygotes show no apparent abnormalities and are fully viable. Analysis of Notch2 expression patterns, revealed by X-gal staining, demonstrated that the Notch2 gene is expressed in a wide variety of tissues including neuroepithelia, somites, optic vesicles, otic vesicles, and branchial arches, but not heart. Histological studies, including in situ nick end labeling procedures, showed earlier onset and higher incidence of apoptosis in homozygous mutant mice than in heterozygotes or wild type mice. Dying cells were particularly evident in neural tissues, where they were seen as early as embryonic day 9.5 in Notch2-deficient mice. Cells from Notch2 mutant mice attach and grow normally in culture, demonstrating that Notch2 deficiency does not interfere with cell proliferation and that expression of the Notch2-(beta)-gal fusion protein is not toxic per se. In contrast to Notch1-deficient mice, Notch2 mutant mice did not show disorganized somitogenesis, nor did they fail to properly regulate the expression of neurogenic genes such as Hes-5 or Mash1. In situ hybridization studies show no indication of altered Notch1 expression patterns in Notch2 mutant mice. The results indicate that Notch2 plays an essential role in postimplantation development in mice, probably in some aspect of cell specification and/or differentiation, and that the ankyrin repeats are indispensable for its function.

Animals↗
PubMed1999

Postmortem evaluation of morphologic changes in the infarcted myocardium that predict ventricular septal rupture in acute anteroseptal infarction.

Although thinning of the ventricular wall due to infarct expansion (septal aneurysm) may contribute to ventricular septal rupture (VSR), spatial factors predisposing to this mechanical complication have not been fully demonstrated. To identify the morphologic predictors of VSR, a retrospective postmortem study was performed on 17 hearts with acute anteroseptal myocardial infarction, comprising 7 with VSR and 10 without rupture. Infarct size and the extent of wall thinning were quantified. Wall thinning was defined as a decrease of less than 50% of thickness of the noninfarcted wall. The total infarct size did not differ among the groups. In the free wall (FW), the infarct was smaller in hearts with VSR than in those with a ruptured FW (p<0.05) or no rupture (p<0.01). The septal involvement was more extensive in patients with VSR than in those with FW rupture (p<0.05). Septal thinning was more extensive in hearts with VSR than in those with FW rupture (p<0.05) or non-rupture (p<0.05). A combination of a small infarct of the FW and a large septal infarct may contribute to the formation of septal aneurysm, which is believed to predispose to VSR. The presence of a small infarct of the anterior septum may be another setting for postinfarction septal rupture.

Aged↗
PubMed1998

Familial essential thrombocythemia associated with one-base deletion in the 5'-untranslated region of the thrombopoietin gene.

Familial essential thrombocythemia (ET) is inherited in an autosomal-dominant manner. This finding implies that familial ET may arise as a consequence of a mutation(s) that activates platelet production. In 1994, the thrombopoietin (TPO) gene was isolated and cloned. The TPO-TPO receptor, encoded for by the c-mpl gene, are essential regulators of thrombopoiesis. Alterations of TPO or c-Mpl thus may constitute a pathogenic event leading to familial ET. In a case of familial ET presented in our institute, serum TPO levels were significantly elevated in affected members of the family as compared with nonaffected members. Moreover, we identified a one-base deletion in the 5'-untranslated region of the TPO gene in affected but not in nonaffected family members. In vitro experiments showed that the identified mutation increased TPO production. Based on our findings, we propose that this region of the TPO gene may play a crucial role in regulating TPO expression. Our results strongly suggest that the identified mutation leads to familial ET.

DNA Mutational Analysis↗
PubMed1998

Cardioprotective effects of quinapril after myocardial infarction in hypertensive rats.

Although angiotensin-converting enzyme inhibitors are beneficial for patients with congestive heart failure, the appropriate timing and dosage in acute myocardial infarction are still controversial. We examined the hemodynamic effects of quinapril administered before acute myocardial infarction in spontaneously hypertensive rats (SHR). Quinapril (10 mg/kg per day in drinking water) was started 1 week before infarction and continued for 4 weeks after infarction (total duration 5 weeks). The hemodynamic parameters were evaluated by cardiac catheterization 4 weeks after coronary ligation. Sham-operated SHR served as controls. After infarction, left ventricular end-diastolic and right atrial pressures were increased (P < 0.01) and blood pressure and cardiac index were decreased (P < 0.01); the magnitude of blood pressure reduction was similar in the treated and untreated rats with infarction. Quinapril improved these hemodynamic parameters significantly and decreased left and right ventricular weight. These results suggest that a prior treatment with quinapril in SHR with acute myocardial infarction is hemodynamically beneficial.

Angiotensin-Converting Enzyme Inhibitors↗