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Biomedical subjects

K Ohki

Publications and source records attributed to K Ohki.

At least 55 records · Page 3Linked to original sources
PubMed1996

Cone-associated c-fos gene expression in the light-damaged rat retina.

PURPOSE: To examine whether light-controlled c-fos gene expression is mediated by a cone specific pathway in the rat retina. METHODS: To produce a cone-rich retina, rats were placed under continuous lighting for 21 days. The illuminance in the cages ranged from 1200 to 2000 lux. The presence of cones was determined by peanut agglutinin and rods by the monoclonal antibody 1-E7. The presence of c-fos transcripts in the retinas was analyzed by in situ hybridization using single-stranded RNA probes. RESULTS: After long-term exposure to continuous light, the 1-E7 immunoreactivity was not detected in the outer nuclear layer. The cone photoreceptors and cells in the ganglion cell (GCL) and inner nuclear layers (INL) survived. In normal retinas, there was strong hybridization for c-fos expression in the GCL and the INL 30 minutes after the onset of the light cycle. Light-damaged retinas also showed hybridization in the GCL but not in the INL under the same light regime. CONCLUSIONS: These results suggest that c-fos gene expression can be controlled through a cone specific pathway in the retina.

Animals↗
PubMed1995

Nitric oxide induces c-fos gene expression via cyclic AMP response element binding protein (CREB) phosphorylation in rat retinal pigment epithelium.

We have examined whether nitric oxide (NO) induces the expression of c-fos proto-oncogene and the phosphorylation of cyclic AMP response element binding protein (CREB) in the rat retina. NO donor sodium nitroprusside (SNP) was injected into the vitreal cavity of the eye, and the retina and retinal pigment epithelium (RPE) was analysed by in situ hybridization using single-stranded RNA probes for c-fos transcripts and by immunocytochemistry using an anti-phospho Ser-133 antibody 20 min after the injection of SNP, phosphorylated-CREB immunoreactivity was found in RPE cells and weakly in some cells of the INL. Forty-five min after the SNP injection, the expression of c-fos mRNA was detected in the RPE. These results suggested that NO induced the c-fos expression via the phosphorylation of CREB in RPE cells, as it has been demonstrated in PC12 cells that the transcription of c-fos gene was activated by the CREB phosphorylation.

Activating Transcription Factor 2↗
PubMed1995

Structural and thermotropic properties of calcium-dimyristoylphosphatidic acid complexes at acidic and neutral pH conditions.

Two kinds of calcium-dimyristoylphosphatidic acid (DMPA) complexes at acidic and neutral pH conditions were prepared in the following ways. The complex at pH 4 was obtained by adding Ca2+ to DMPA dispersion in pure water. On the other hand, the complex at pH 7.4 was obtained by adding Ca2+ to DMPA dispersion in the presence of NaOH. The stoichiometries of Ca2+ ion to DMPA molecule are 0.5-0.67 and approximately 1 for the complexes at pH 4 and 7.4, respectively. Static x-ray diffraction shows that the hydrocarbon chains of the Ca(2+)-DMPA complex at pH 4 at 20 degrees C are more tightly packed than those of the complex at pH 7.4 at 20 degrees C. Furthermore, the complex at pH 4 at 20 degrees C gives rise to several reflections that might be related to the ordered arrangement of the Ca2+ ions. These results indicate that the structure of the complex at pH 4 is crystalline-like. In the differential scanning calorimetry (DSC) thermogram, the complex at pH 7.4 undergoes no phase transition in a temperature range between 30 and 80 degrees C. On the other hand, in the DSC thermogram for the complex at pH 4, a peak appears at 65.8 degrees C in the first heating scan. In the successive second heating scan, a transition peak appears at 63.5 degrees C. In connection with the DSC results, the structural changes associated with these phase transitions were studied with temperature-scan x-ray diffraction. In the first heating scan, although a peak appears at 65.80C in the DSC thermogram, the hydrocarbon chain packing gradually converts from an orthorhombic lattice to a hexagonal lattice near 52 degree C, and successively the chain melting phase transition occurs near 670C. In the second heating scan, the hydrocarbon chains are packed in a hexagonal lattice over the whole temperature range and the chain melting phase transition occurs near 63.5 degree C. Therefore,the Ca2+-DMPA complex at pH 4 has a metastable state. The metastable state transforms to a stable state by maintaining the complex at pH 4 for about 90 h at 200C.

Calcium↗
PubMed1995

Interdigitated structure of phospholipid-alcohol systems studied by x-ray diffraction.

In the interdigitated structure of phosphatidylcholine/alcohol systems, the one-dimensional electron density profile in the direction normal to the membrane surface is generated from the x-ray diffraction pattern. The membrane thickness for these systems is expressed by the sum of the hydrocarbon chain lengths of phosphatidylcholine and alcohol molecules. For this study, various sets of phosphatidylcholines and 1-alcohols were used; a phosphatidylcholine has a carbon number from 14 to 18 in a hydrocarbon chain, and an alcohol has a carbon number from 1 (methanol) to 4 (1-butanol). Based upon the results, we propose a model for the interdigitated structure in which 1) two alcohol molecules occupy a volume whose surface is surrounded interstitially by the headgroups of phosphatidylcholine molecules, and 2) the methyl ends of both hydrocarbon chains in alcohol and phosphatidylcholine molecules face each other at the bottom of the volume.

1,2-Dipalmitoylphosphatidylcholine↗
PubMed1995

Surface components of shigellae involved in adhesion and haemagglutination.

Strains of Shigella species were studied for their ability to adhere and agglutinate mammalian erythrocytes. Shigella dysenteriae and Sh. flexneri exhibited haemagglutinating (HA) properties when cultured in Casamino Acids-Yeast Extract (CYE) broth in the presence of 1 mmol 1-1 calcium chloride, but other shigellae did not show this property under the same culture conditions. Repeated subcultivation of Sh. boydii, Sh. sonnei and HA negative strains of Sh. dysenteriae and Sh. flexneri in CYE broth medium induced adhesive and haemagglutinating properties that were inhibited by sodium periodate. HA activities of Shigella spp. were also inhibited by N-acetylneuraminic acid, alpha 1-glycoprotein and fetuin, but not by protease. Electron microscopy of Sh. dysenteriae 1, Sh. flexneri 2a, Sh. boydii 12 and Sh. sonnei 1 grown in CYE broth showed the presence of an extracellular slime layer that promoted agglutination of erythrocytes. The slime layer extracted from the cell surface of Shigella spp. showed HA properties, whereas lipopolysaccharide (LPS) obtained from the same strains, except Sh. dysenteriae 1, did not agglutinate erythrocytes. This evidence suggests that the cell surface haemagglutinin is a loosely bound slime layer which is expressed in CYE broth medium.

Animals↗
PubMed1995

Morphology of defensin-treated Staphylococcus aureus.

Defensins are a family of broad-spectrum antimicrobial peptides found abundantly in the cytoplasmic granules of mammalian neutrophils and Paneth cells of the small intestine. Defensins are known to form ion channels on the membranes of target cells. These channel formations and the cytotoxicity of defensins are intimately linked. We showed the morphological effects of defensins on the cytoplasmic membranes of Staphylococcus aureus by transmission electron microscopy. S. aureus exposed to defensins developed characteristic mesosome-like structures but did not show remarkable changes in cell walls. Defensins induced such structural changes not only at high concentration but also at low concentrations that were not bactericidal. We also showed that increasing the concentration of NaCl in the reaction mixture completely inhibited the occurrence of membranous changes of target cells exposed to defensins. These findings are, to our knowledge, the first report of morphological changes in gram-positive bacteria treated with defensins. Our results indicate that the first effect of defensins in S. aureus is to damage cytoplasmic membranes directly; they also support previous reports that the cell membrane is the principal target of defensins.

Animals↗
PubMed1995

In vitro differentiation of the murine macrophage cell line BDM-1 into osteoclast-like cells.

Osteoclasts are derived from hematopoietic stem cells, but details about their precursor are still obscure. We present here a mouse macrophage cell line, BDM-1 cells, that showed a high potential to differentiate into osteoclast-like multinucleate cells (MNCs) when cocultured with primary osteoblasts for 14 days in the presence of 10(-8) M 1 alpha,25-dihydroxyvitamin D3. These MNCs had tartrate-resistant acid phosphatase (TRAP) activity and strong ability to resorb dentine. In this culture system, 10(-10)-10(-8) M 12-O-tetradecanoylphorbol-13-acetate stimulated the formation of TRAP-positive MNCs, whereas salmon calcitonin inhibited it. Time-course effect studies showed that 12-O-tetradecanoylphorbol-13-acetate had an effect on the late phase of osteoclast differentiation but not on precursor proliferation. By immunocytochemical staining, all BDM-1 cells expressed Mac-1, Mac-2, and MOMA-2 antigens, and a large number of them expressed F4/80 antigen, but the rest of them were negative for this antigen. To select subclones able to differentiate into TRAP-positive MNCs, we sought to isolate several subclones from BDM-1 cells by mean of different specificity for F4/80 antigen expression. TRAP-positive MNCs were not generated from F4/80-positive subclones, but were obtained from subclones containing F4/80-negative cells. These results suggest that an F4/80-negative macrophage subpopulation is responsible for the differentiation of this cell line into osteoclasts.

Acid Phosphatase↗
PubMed1995

Hydrolytic cleavage of pyroglutamyl-peptide bond. I. The susceptibility of pyroglutamyl-peptide bond to dilute hydrochloric acid.

The susceptibility of the pyroglutamyl-peptide bond in some biologically active peptides, dog neuromedin U-8 fragment (pGlu-Phe-Leu-Phe-Arg-Pro-Arg-OH), human big gastrin fragment (pGlu-Leu-Gly-Pro-OH) and thyrotropin releasing hormone (TRH) fragments (pGlu-His-Pro-OH, pGlu-His-OH), to 1 N HCl under mild conditions and/or at 60 degrees C was studied. It was found that the N-terminal portion of pGlu-peptides is extremely labile to acid hydrolysis, giving not only the ring-opened product of the pyrrolidone moiety of the pGlu residue, but also the cleavage product of the pGlu-peptide linkage. The ring-opening reaction predominated over the cleavage reaction in hydrolysis of the four peptides in 1 N HCl at 60 degrees C. The ring-opening reaction and the cleavage reaction of pGlu-peptide linkage proceeded faster than the cleavage of internal peptide bonds. The rate of hydrolysis was affected by the reaction temperature, and the ring-operating reaction was greatly diminished at 4 degrees C in comparison with the cleavage reaction. Thus, the phenomenon that the pGlu-peptide bond is susceptible to dilute HCl as compared to the other peptide bond appears to be a general one.

Amino Acid Sequence↗
PubMed1995

[Study on surfactin, a cyclic depsipeptide. I. Isolation and structure of eight surfuctin analogs produced by Bacillus natto KMD 2311].

Crude surfactin was simply prepared from the culture filtrate of Bacillus natto KMD 2311 twice by acidification of the filtrate and extraction of the precipitate with ethanol. Eight surfactin analogs were isolated from the crude surfactin by RP-HPLC and gel filtration. The structure of each analog was deduced by means of amino acid composition of the acid hydrolysate and FAB-MS measurement to be a cyclic depsipeptide containing a hydroxyfatty acid. The structure of the hydroxyfatty acid moieties was elucidated as n- or iso- or anteiso-3-hydroxyfatty acid composed of carbon number 13-16 by GC analysis and EI-MS after the methanolysis of the analogs. The amino acid sequence of the peptide portion was assigned as acyl-Glu-Leu-Leu-Val-Asp-Leu-Leu by EI-MS for eight analogs. The isolated four compounds were found to be identical with the known surfactin analogs, A1, B1, B2 and C1. Although surfactin A2 and C2 had not been isolated, their structures were deduced to be a surfactin analog. Surfactin A3 and D were novel analogs. The acyl groups of surfactin A2, A3, C2 and D were anteiso-3-hydroxytridecanoic acid, n-3-hydroxytridecanoic acid, anteiso-3-hydroxypentadecanoic acid and iso-3-hydroxyhexadecanoic acid, respectively.

Amino Acid Sequence↗
PubMed1995

Relaxation effect of an audiovisual system on dental patients. Part 2. Palus-amplitude.

Dental care services include relaxation of patients to reduce their displeasure at and tension during treatment. For this purpose, it is common to use audio or audiovisual programs, that is, to let them listen to music or watch TV. However, the former utilizes only the auditory sense, and the latter is disadvantageous in that a TV set is a rather large-scale unit. In the present study, we determined the relaxation effect of an audiovisual instrument on dental patients. The instrument, which gives virtual vision, was composed of a lightweight eyeset of the snow goggle type that has a built-in TV screen, with earphone and accessories. The relaxation effect was determined using a polygraph.

Adult↗
PubMed1995

jun-B gene expression in rat retinal cells following focal retinal injury.

We examined jun-B gene expression in the rat retina after a focal retinal injury by in situ hybridization. 30 min after producing a penetrating retinal wound, the retina showed the expression of jun-B mRNA intensively in the inner nuclear layer (INL) and relatively sparsely in the ganglion cell layer. The double-labeled retina by immunocytochemistry and in situ hybridization demonstrated that jun-B mRNA within the INL surrounded S-100 protein immunoreactive cytoplasm in the middle sublayer of the INL. These results suggest that the jun-B expression is induced mainly in Müller cells following retinal damage.

Animals↗
PubMed1995

C-fos gene expression in rat retinal cells after focal retinal injury.

PURPOSE: To examine the expression of c-fos proto-oncogene in the rat retina after focal retinal injury. METHODS: A penetrating wound was made by pushing a 22-gauge hypodermic needle through the retina into the vitreous. The retinas were analyzed by in situ hybridization using single-stranded RNA probes for c-fos transcripts and by immunocytochemistry using anti-S100 protein antibody. RESULTS: Thirty minutes after the penetrating wound, c-fos mRNA was expressed in the inner nuclear layer (INL) and the ganglion cell layer (GCL) of the retina. Immunocytochemistry before in situ hybridization demonstrated c-fos mRNA surrounding the S-100 protein immunoreactive cytoplasm in the middle layer of the INL. CONCLUSIONS: Focal retinal injury of the rat retina induced the expression of c-fos mRNA in retinal cells. In the INL, it is suggested that the major cells that express c-fos mRNA after focal retinal injury were the Müller cells. These results suggest that c-fos may be involved in the transcription in the Müller cells after injury to the retina.

Animals↗
PubMed1994

The carboxyl-terminal region of HIV-1 Nef protein is a cell surface domain that can interact with CD4+ T cells.

Our previous studies have shown that the HIV-1 Nef Ag is expressed, at least in part, on the surface of infected cells. We demonstrated this by using membrane immunofluorescence and flow cytometry with Nef murine mAbs. To identify the domain of Nef exposed on the cell surface, epitope mapping of these and a new mAb was performed by ELISAs by using several recombinant truncated Nef fusion proteins and synthetic peptides. The results showed that mAbs F1, E7, E9, and 4H4 recognized Nef epitopes located at amino acid residues 148-157, 192-206, 158-206, and 1-33, respectively. The intensity of cell surface Nef staining was stronger with mAbs E7 and E9 than with F1, and there was no staining by 4H4, which indicates that the carboxyl-terminal region of Nef is predominantly exposed on the surface of HIV-1-infected T cell lines and PBMC. This surface Nef domain displayed high affinity for the surface of uninfected CD4+ T cells, because the binding of a soluble form of recombinant Nef protein to the cell surface was specifically blocked by the E7 and E9 mAbs or by synthetic peptides that contained the carboxyl-terminal region of Nef. In addition, syncytium formation between infected and uninfected cells also was specifically reduced by the same mAbs or peptides. Thus, the cell surface domain of Nef seems to play an important role in the interaction between HIV-1-infected and CD4+ uninfected T cells.

Antibodies, Monoclonal↗
PubMed1994

Multiple effects of CD4 CDR3-related peptide derivatives showing anti-HIV-1 activity on HIV-1 gp120 functions.

The interaction of the human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein gp120 with CD4 CDR3-related peptide derivatives showing anti-HIV-1 activity has been studied. Conformational changes in gp120, which could affect its interaction with CD4 and its shedding from virions, were detected by fluorescence spectrum analysis of tryptophan residues after addition of peptide representative of the CD4 CDR3-related region, but not the CD4 CDR2-related region. Interestingly, the addition of scrambled peptide, S1 (with altered amino acid sequence compared with the native CDR3-related peptide but unaltered overall composition), which we recently showed to have stronger anti-HIV-1 activity than the original CDR3-related peptide, had no effects on the conformational change in gp120 or on its interaction with CD4 and its shedding from HIV-1 virions. However, all of the CDR3-related peptides, including S1, showed blocking effects on the binding of antibodies against gp120 V3 loop and C-terminus regions. Thus, we concluded that there were at least two separable activities of the CDR3-related peptides in anti-HIV-1 activity, i.e. induction of conformational changes in gp120, which could affect its binding to CD4 and to gp41 (as observed in native CDR3-related peptides), and inactivation of V3 loop and C-terminus regions in gp120 (as observed in all of the CDR3-related peptides, including S1).

Amino Acid Sequence↗
PubMed1994

Contact hemolysin production by strains of enteroaggregative Escherichia coli isolated from children with diarrhea.

Forty-five strains of enteroaggregative Escherichia coli were tested for hemolytic activity with different culture media and erythrocytes from different species. Thirty-seven strains showed proteinase K-sensitive contact hemolysin activity with sheep erythrocytes when cultured in Casamino Acids-yeast extract broth supplemented with 1 mM calcium chloride and when cultured in nutrient broth media. The production of contact hemolysin was dependent on temperature, pH, and culture conditions.

Animals↗
PubMed1994

Laminin promotes proliferation of bone marrow-derived macrophages and macrophage cell lines.

We examined the effect of laminin on DNA synthesis or cell growth of bone marrow-derived macrophages (BMM) and five macrophage cell lines including two factor-dependent macrophage cell lines (BDM-1 and BDM-1W3) and three factor-independent ones (BAM3, J774.1, and P388D1). Laminin stimulated the M-CSF-dependent proliferation of BMM and BDM-1 cells in a dose-dependent manner, but it had no effect on the proliferation of BDM-1W3 cells. This stimulatory effect was modified by increasing the concentration of fetal calf serum in the culture medium. Laminin also stimulated the proliferation of BAM3 and J774.1 cells, whereas it could not stimulate that of P388D1 cells. Taken together, these results suggest that the differences in the response of these macrophage cell lines to the presence of laminin may be derived from the difference in the state of maturation of these cells. Although laminin acted as a potent mitogenic factor when it was added to the culture medium, the stimulation was two- to threefold lower when dishes were coated with equivalent amounts of laminin. When BDM-1 cells were cultured with laminin, they underwent drastic morphological changes. A monoclonal antibody that recognizes the integrin alpha 6 subunit only partially inhibited the mitogenic effect of laminin. The results in this report suggest that laminin may participate in the regulation of macrophage populations in vivo.

Amino Acid Sequence↗