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M Okabe

Publications and source records attributed to M Okabe.

At least 199 records · Page 11Linked to original sources
PubMed1996

Visualization of yellowish-orange luminescence from cuprous metallothioneins in liver of Long-Evans Cinnamon rat.

We describe the first use of an emission probe, based on the cuprous thiolate chromophore, for direct microscopical observation of cuprous metallothioneins located in liver of 15-week-old (just before spontaneous hepatitis) Long-Evans Cinnamon rats. The rats show remarkable accumulations of copper and cuprous metallothioneins. In the mildly fixed liver, we visualized the same yellowish-orange luminescence as the specific emission from cuprous metallothioneins, following excitation in 330-385 nm region. In liver from Long-Evans Agouti rat, a counter part of Long-Evans Cinnamon rat, no similar luminescence was found. So, it was thought that cuprous metallothioneins accumulated in the Long-Evans Cinnamon rat liver might emit the yellowish-orange light. To verify this presumption, we tentatively defined three histochemical criteria, quenching tests by oxidation, protonation and mercury treatment, based on the coordination chemical characteristics of metallothioneins. The emission completely satisfied these criteria. Furthermore, the reliability of these criteria was supported by immunocytochemical and biochemical results. Consequently, all results sufficiently indicate that the yellowish-orange luminescence in the Long-Evans Cinnamon rat liver is the emission from cuprous metallothioneins.

Animals↗
PubMed1996

argos Is required for projection of photoreceptor axons during optic lobe development in Drosophila.

The Drosophila argos gene encodes a secreted protein with an epidermal growth factor (EGF) motif, which acts as an inhibitor of cell recruitment in the developing eye and wing. Here, we have analyzed the role of argos during optic lobe development. argos expression was observed in the optic lobes throughout the developmental stages. In argos mutants, neuropiles failed to develop normally during embryonic and larval stages, and photoreceptor axons did not project properly into the lamina. Ubiquitous expression of argos, under control of the hsp70 promoter, rescued the defects in optic lobes. We have found that glial cells failed to differentiate in the larval optic lobes of argos mutants. Correspondingly, in loss-of-function repo mutants, whose glial cells also fail to differentiate, photoreceptor axons showed the impaired projection pattern similar to the argos phenotype. These results suggest that glial cells play a role for guidance of photoreceptor axons. The loss-of-function Star mutation (StarX155) dominantly suppressed the defects in the argos optic lobes, suggesting that these two genes act in an antagonistic fashion during optic lobe development.

Animals↗
PubMed1996

Reversal effect of itraconazole on adriamycin and etoposide resistance in human leukemia cells.

Itraconazole is a triazole antifungal agent that inhibits cell membrane serol biosynthesis. Currently, itraconazole is a potent candidate for in vivo use to revert multidrug resistance in acute leukemias, with the added benefit of its antifungal effect. As previously reported, itraconazole, as well as verapamil, reversed adriamycin-resistant K562 cells (K562/ADR) and HL60 cells (HL60/ADR) in dosages compatible to the plasma levels achieved by the therapeutic dosages used for the treatment of fungal infections. By RT-PCR analysis of mdr1, mdr3, and mrp mRNA, these adriamycin-resistant cells showed a higher expression of the transcript of these genes than those of the parent cells. By FACS analysis, both the adriamycin-resistant cells showed a higher expression of P-glycoprotein on their cell surfaces. These results suggested the involvement of itraconazole in the mdr gene and/or mrp gene product-associated resistance. Furthermore, itraconazole partially reversed etoposide resistance in both the K562 and K562/ADR cells. The present study suggests that itraconazole may reverse multidrug resistance, at least in part, via a classical MDR-associated mechanism.

Antibiotics, Antineoplastic↗
PubMed1996

Roles of the conserved serines of metallothionein in cadmium binding.

The sequence of six amino acid residues -Ser-Cys-Cys-Ser-Cys-Cys- is present in all mammalian metallothionein sequences and has been highly conserved during evolution, although the metallothioneins have divergent primary sequences. To determine whether two serines in the sequence play a crucial role in metal-binding of metallothioneins, a mutant metallothionein with these two serines replaced by leucines was obtained using an Escherichia coli expression system. The expressed protein was analyzed for its chemical and spectroscopic properties. It was confirmed that the mutant metallothionein (MT) bound cadmium through a metal-thiolate complex and that there was no strong difference between the mutant and the wild-type MTs in retaining the metal-binding cluster. However, the metal-binding cluster of the mutant metallothionein was more unstable than that of the wild-type metallothionein. The two conservative serines could play a role in the stability of metal-binding ligands.

Amino Acid Sequence↗
PubMed1996

Metallothionein in the ovaries of laying hens exposed to cadmium.

The presence of metallothionein (MT) and its mRNA in the ovaries of laying hens is reported. In laying hens injected with cadmium (Cd), Cd accumulated in the follicle walls of the ovaries not in the follicle yolks. In the follicle walls, (Cd, Zn)-MT and its mRNA were found. This indicates that MT bound to Cd is biosynthesized in the follicle walls. We suggest that MT might play a role in sequestering Cd. On the other hand, zinc (Zn) was found in the follicle walls and yolks in the Cd-treated laying hens. The relationship between Zn and (Cd, Zn)-MT is also discussed.

Animals↗
PubMed1996

Independent self-assembly of cadmium-binding alpha-fragment of metallothionein in Escherichia coli without participation of beta-fragment.

We examined the independent self-assembly of the alpha- and beta-fragments of human metallothionein (MT) into cadmium-binding conformation in an Escherichia coli expression system, in addition to wild-type MT expression. The expressed alpha-fragment formed independently the structure of a metal-binding cluster without the aid of the beta-fragment. The alpha-fragment and wild-type MT expressed in E.coli were purified and analyzed for their biochemical and spectroscopic properties. The apparent cadmium binding of the alpha-fragment was approximately 12-fold greater than that for the wild-type MT, whereas in other respects the studied biochemical properties were similar. In contrast, we were unable to obtain any independently expressed beta-fragment as the cadmium-binding form in this study. Possible explanations for this phenomenon are discussed.

Amino Acid Sequence↗
PubMed1996

Generation of new intercellular junctions between cardiocytes. A possible mechanism compensating for mechanical overload in the hypertrophied human adult myocardium.

Intercellular dehiscence is a common cardiocytic response to pathological conditions. However, little consideration has been given to the possibility of new intercellular junctions developing between cardiocytes within developed myocardium. To examine this possibility as it may relate to useful compensation for hemodynamic overloads, changes in cardiocytic connection were evaluated by scanning electron microscopy in hypertrophied myocardium of adult human hearts. Transmural myocardium of left ventricle was obtained at autopsy from five hearts with concentric hypertrophy, five hearts with eccentric hypertrophy, and five control hearts (noncardiac death). After formalin fixation, the number of cardiocytes connected to an individual cardiocyte was counted in tissues from the middle portion of the transmural samples by scanning electron microscopy. Cardiocytic diameter and connective tissue volume fraction were measured on the transmural sections by light microscopy. In concentrically hypertrophied hearts present both increased cardiocytic diameter and connective tissue volume fraction, the number of other cardiocytes connected to an individual cardiocyte (4.60 +/- 0.10 [mean +/- SE] was significantly increased (P < .05) compared with control hearts (4.19 +/- 0.12) or eccentrically hypertrophied hearts (4.11 +/- 0.10). The increase in junctions per cardiocyte in concentrically hypertrophied hearts suggests that new connections had been generated. More junctions developing during hypertrophy could add another structural advantage to those of cardiocytic hypertrophy and connective tissue proliferation as compensatory adjustments to hemodynamic overload in concentrically hypertrophied hearts.

Adult↗
PubMed1996

Direct visualization of copper-metallothionein in LEC rat kidneys: application of autofluorescence signal of copper-thiolate cluster.

We report on the histochemistry of copper-metallothionein (Cu-MT) in the kidneys of Long Evans Cinnamon (LEC) rats. We used the visualization principle of histochemistry based on the autofluorescence emission from the fluorophore of Cu(+)-thiolate clusters in proteins. Intense autofluorescence signals were observed with a ring at the outer stripe of the outer medulla. Orange fluorescence signals were observed in the nuclei and cytoplasm of proximal straight tubular (PST) cells of segment 3 (S3) at the outer stripe of the outer medulla, and yellow-orange signals were detected in lysosome-like organelles in the proximal convoluted tubule (PCT) cells of segments 1 and 2 (S1 and S2) adjacent to the glomeruli in the cortex. These fluorescent materials were identified as Cu-MT because both signals were quenched by withdrawing Cu+ or by blocking cysteine residues, the distributions of cysteine residues and immunoreactive MT showed identical patterns to the localization of the fluorescence signals, and the fluorescent proteins containing Cu were eluted at the same Kd value of purified Cu-MT by gel filtration chromatography. However, a high level of MT mRNA was detected only in the outer stripe of the outer medulla where the orange fluorescence signals were detected, but not in the cortex. This difference in localization between the protein and the mRNA suggested that synthesis of renal MT occurs do novo in the outer stripe of the outer medulla. The yellow-orange fluorescent Cu-MT located in the lysosomal organelles at S1 and S2 of the PCT cells in the cortex could be Cu-MT of nonrenal origin, i.e., Cu-MT transported from other organs.

Animals↗
PubMed1996

The diabetic state increases the activity but not the number of peritoneal macrophages in the GK rat promoting the tube formation of cultured endothelial cells in rat aorta.

The influence of the diabetic state on the number and activity of intraperitoneal macrophages was investigated in the GK rat. This rat is a model of non-insulin-dependent diabetes mellitus and was established from the normal Wistar rat. The cell number of a macrophages preparation in diabetic GK rats (10-15 weeks of age) did not differ from that in normal Wistar rats (9-10 weeks of age). The co-cultured macrophages in diabetic GK rats (9-12 weeks of age) significantly increased the tube formation of aortic endothelial cells (EC) compared with that in age-matched Wistar rats. The conditioned medium obtained from a culture of macrophages from GK rats also increased tube formation more than that from a culture of macrophages from Wistar rats. The effect of macrophages in the GK rat was not influenced by interferon-gamma (160 and 460 pM), but macrophages in normal rats were activated by these concentrations of interferon-gamma. In conclusion, the diabetic state increased the activity but not the number of macrophages in the GK rat, to stimulate tube formation of EC. The tube-forming effect of macrophages in the diabetic state may depend on released factors which differ from that of normal macrophages.

Animals↗
PubMed1996

Competence effect of basic fibroblast growth factor on cell cycle in subcultured endothelial cells of rat aorta by flow cytometry.

With flow cytometry, we investigated the effect of basic fibroblast growth factor (bFGF)-induced competence in subcultured endothelial cells (EC) (4-9-passage) of rat thoracic aorta. The cell population in each phase of the cell cycle was determined by a double staining technique with fluorescein isothiocyanate-conjugated mouse monoclonal antibody against the proliferation-associated nucleus antigen Ki-67 and propidium iodide for total DNA content. EC were cultured in medium containing 5% fetal bovine serum (FBS) for 6 days. After serum-starvation for 2 days, the treatment with bFGF (3-10 ng/ml) for 12 hr promoted the entry of cells into the G1 phase from the G0 phase concentration-dependently. bFGF (10 ng/ml) increased the cell population in the G1 phase by 5% of the total EC, compared with the control culture without bFGF. A further 12-15-hr culture with 1% FBS after bFGF treatment promoted the entry of the cell into the S phase. Thus flow cytometric analysis demonstrates that bFGF stimulates the entry of EC into the G1 phase from the G0 phase.

Animals↗
PubMed1996

Effect of ferrous ion on mildiomycin production by Streptoverticillium rimofaciens.

A specific regulatory effect of ferrous ion on a biosynthesis of mildiomycin by Streptoverticillium rimofaciens was investigated. The minimal concentration of ferrous ion necessary for the maximal production of mildiomycin was about 8 micrograms/ml. The physiological effects of ferrous ion on the mildiomycin production were examined in the media with and without the ferrous ion. Addition of ferrous ion to the culture medium increased both the mildiomycin production (10 times) and an assimilation of ammonium-nitrogen, activities of peptidase and protease.

Antifungal Agents↗
PubMed1996

Effect of phosphate ion on mildiomycin production by Streptoverticillium rimofaciens.

Effect of inorganic phosphate and ammonium nitrogen on mildiomycin production by Streptoverticillium rimofaciens was investigated in culture with and without addition of ferrous ion. In the presence of ferrous ion, the suitable supply of inorganic phosphate increased the intracellular ATP, but that was not observed without ferrous ion addition. The intracellular ATP remarkably affected ammonium nitrogen assimilation and mildiomycin production, and its concentration in the ferrous-sufficient culture was about 2 approximately 3 times higher than that in the ferrous-deficient culture. The low concentration of intracellular ATP in the ferrous-deficient culture resulted in the reduction of ammonium nitrogen assimilation and mildiomycin biosynthesis. This phosphate ion effect on the intracellular ATP concentration was demonstrated only when ferrous ion was added into the medium. These suggest that mildiomycin biosynthesis is regulated through the concentration of intracellular ATP related to the ammonium nitrogen assimilation.

Adenosine Triphosphate↗
PubMed1996

[Transverse lesion of the spinal cord due to mucormycosis in an AML patient].

A 54 year old man complained exertional dyspnea and palpitation since November 1989. As he was diagnosed with marked anemia, leukocytosis and thrombocytopenia by his work place doctor, he was admitted to our hospital. Acute myelogenous leukemia was diagnosed based on laboratory findings. BHAC-DMP, BHAC-MEP and A triple V therapies were only partially effective. Fine nodular shadows in all lung fields and a semicircular mass in the right lower lobe next to the thoracic vertebra were evident on the chest X-P since the end of March 1990. He was treated with antibiotics and amphotericin B but the abnormal lung shadows did not disappear. He had sudden onset of paraplegia and loss of all sensation below Th6 on May 1. Aparavertebral mass in the right lower lobe was detected by CT and MRI, for which radiotherapy was performed but without improvement. He died of respiratory failure on May 12. Autopsy showed that the semicircular paravertebral mass continued to the main pulmonary vein and epidural area of the thoracic cord (Th6-8). Microscopically, mucormycosis was found. Necrosis due to mucor embolism was found in the thoracic cord. It is usually difficult to diagnose mucormycosis in immunocompromised patients while they are alive. It is important to suspect mucormycosis if any infarction symptoms or infections resistant to antibiotics develop in immunocompromised patients.

Fatal Outcome↗
PubMed1995

A rapid and non-invasive selection of transgenic embryos before implantation using green fluorescent protein (GFP).

Non-invasive selection of transgenic mice was performed at the stage of preimplantation embryos. The morulae collected from wild female mated with hemizygous transgenic male expressing Aequorea victoria green fluorescent protein (GFP) under chicken beta-actin promoter could be classified as green or non-green under a fluorescent microscope. All the green embryos were shown to carry the transgene by PCR analysis. Taking advantage of the detection of GFP expression can be done non-invasively, the selected embryos were demonstrated to be able to developed to term with 100% of accuracy of the selection.

Animals↗
PubMed1995

Characterization of the testis-specific gene 'calmegin' promoter sequence and its activity defined by transgenic mouse experiments.

We have cloned the genomic DNA of calmegin [(1992) J. Biol. Chem. 269, 7744-7749] and analyzed its promoter region. It contained GC-rich sequences and potential binding sites for AP 2 and Sp 1, but lacked the TATA sequence. The 330 bp 5' flanking sequence of calmegin genomic DNA fused with the CAT gene was used for the study of promoter activity in transgenic mice. The CAT gene activity was detected exclusively in testes, indicating that the 330 bp calmegin 5' sequence was sufficient for the testis-specific expression. The existence of testicular nuclear factors specifically bound to the putative promoter sequence was also demonstrated.

Animals↗
PubMed1995

Diversity of sites for measles virus binding and for inactivation of complement C3b and C4b on membrane cofactor protein CD46.

The complement system membrane cofactor protein (MCP) CD46 serves as a C3b/C4b inactivating factor for the protection of host cells from autologous complement attack and as a receptor for measles virus (MV). MCP consists of four short consensus repeats (SCR) which are the predominant extracellular structural motif. In the present study, we determined which of the four SCR of MCP contribute to its function using Chinese hamster ovary cell clones expressing each SCR deletion mutants. The results were as follows: 1) SCR1 and SCR2 are mainly involved in MV binding and infection; 2) SCR2, SCR3, and SCR4 contribute to protect Chinese hamster ovary cells from human alternative complement pathway-mediated cytolysis; and 3) SCR2 and SCR3 are essential for protection of host cells from the classical complement pathway. These results on cell protective activity of the mutants against the human classical and the alternative complement pathways were compatible with factor I-mediated inactivation profiles of C4b and C3b, respectively, in the fluid-phase assay using solubilized mutants and factor I; the results were mostly consistent with those reported by Adams et al. (Adams, E. M., Brown, M. C., Nunge, M., Krych, M., and Atkinson, J. P. (1991) J. Immunol. 147, 3005-3011). SCR2 and SCR3 were required for C3b and C4b inactivation, and SCR4-deleted MCP showed weak cofactor activity for C4b cleavage but virtually no cofactor activity for C3b cleavage. The functional domains of MCP for the three natural ligands C3b, C4b, and MV, therefore, map to different, although partly overlapping, SCR domains.

Animals↗