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Biomedical subjects

M Okamoto

Publications and source records attributed to M Okamoto.

At least 325 records · Page 18Linked to original sources
PubMed1998

Affinity isoelectric focusing of human serum apolipoprotein B-containing lipoproteins on agarose gels with cyclodextrins.

In an attempt to subfractionate apolipoprotein B-containing lipoproteins (apoB-Lp), agarose isoelectric focusing (IEF) of apoB-Lp in human sera was carried out in the presence of two kinds of cyclodextrins (CDs), which have different affinities for cholesterol. ApoB-Lp in the agarose gels was detected by immunofixation, followed by protein staining. In the presence of both 11.4 mg/mL alpha-CD and 3.51 mg/mL beta-CD, but not either one alone, IEF separated a maximum of six apoB-Lp bands. Electrophoresis failed to separate the apoB-Lp subfractions under similar conditions with CDs. The patterns of apoB-Lp in IEF with the CDs appeared to differ in some sera. Since the subfractions of apoB-Lp can be separated without ultracentrifugation of serum, the present method serves for the screening of apoB-Lp heterogeneity on native serum lipoproteins.

Apolipoproteins B↗
PubMed1998

Enhanced expression of thrombospondin-1 and hypovascularity in human cholangiocarcinoma.

Cholangiocarcinoma (CCC) is relatively hypovascular, in contrast to hepatocellular carcinoma (HCC), which is often highly vascular. We investigated if the diminished vascularity of CCC is related to altered expression of thrombospondin-1 (TSP-1), an antiangiogenic factor, and/or vascular endothelial growth factor (VEGF), a potent angiogenic factor, comparing the relationships with those of high- and low-vascular HCC. We also investigated the relationship between the mutation of the p53 gene and TSP-1 expression or VEGF expression. Northern blot analysis and immunohistochemical staining were performed on surgically resected human CCC and HCC. The ratios of TSP-1 mRNA level in cancer cells versus adjacent noncancerous cells (T/N ratios) were significantly higher in CCC (n = 11) than in HCC with high vascularity (n = 15). In contrast, T/N ratios of VEGF mRNA level in CCC (n = 11) were comparable with those in HCC with low vascularity (n = 5). In CCC, the cancer cells and fibroblasts were positively stained with anti-TSP-1 antibody. We observed that T/N ratios of VEGF mRNA level, but not those of the TSP-1 mRNA level, were significantly correlated with vascularity in HCC. The relative increase in TSP-1 and the relative decrease in VEGF in tumors compared with normal tissue may underlie the limited angiogenesis of CCC. The p53 gene did not affect the expression of TSP-1 in CCC or VEGF in HCC.

Adult↗
PubMed1998

Maintained function of primary human hepatocytes by cellular interactions in coculture: implications for liver support systems.

The application of primary hepatocytes in hybrid artificial liver systems has been hampered by the gradual loss of differentiated morphology and function in vitro. Therefore, we have established a coculture model of autologous human hepatocytes and biliary epithelial cells (BEC) in collagen gel in the presence of hepatotrophic growth factors. Furthermore, we examined the effect of hepatocyte cell perfusion in a women multicompartment capillary membrane system. Normal hepatocytes isolated from human liver produced albumin for more than 2 weeks in serum-free media, and were further stimulated by conditioned medium. When cocultured with BEC, albumin secretion was greatly enhanced, suggesting that cellular interactions promote tissue-specific differentiation. When perfused in bioreactors, albumin levels were maintained at steady state for longer than 2 weeks. These data indicate that differentiation of primary hum hepatocytes can be maintained by coculture interactions and three-dimensional hybrid organ devices, providing appropriate growth factors and matrix for tissue regeneration.

Cell Communication↗
PubMed1998

cis-Diamminedichloroplatinum and 5-fluorouracil are potent inducers of the cytokines and natural killer cell activity in vivo and in vitro.

It has been reported that certain chemotherapeutic agents exhibit effects that enhance the antitumor host responses in the patients with malignant diseases. In the present study, we investigated whether cis-diamminedichloroplatinum (cisplatin) and 5-fluorouracil (5-FU) may induce cytokines and effector cells with antitumor efficacy in vivo and in vitro. The cultivation of human peripheral blood mononuclear cells (PBMC) in the presence of cisplatin (0-1.0 microg/ml) or 5-FU (0-5.0 microg/ml) resulted in the significant augmentation of natural killer (NK) and lymphokine-activated killer (LAK) cell activities as well as generation of interferon (IFN) gamma, tumor necrosis factor (TNF) alpha, beta interleukin(IL)-1beta, IL-6 and IL-12 in vitro. In addition, all of these activities were almost completely neutralized by addition of anti-asialoGM1 antibody and complement (P < 0.05). In an in vivo model, the administration of anti-asialoGM1 antibody significantly shortened the survival time extended by the treatment with cisplatin or 5-FU (P < 0.05), both on nude mice bearing salivary gland tumors and on syngeneic MethA-tumor-bearing BALB/c mice. Furthermore, high levels of NK and LAK activities and significant increases of the numbers of cells positive for asialoGM1, IFNgamma, TNFalpha, or IL-1beta were detected in the spleen cells derived from animals given cisplatin or 5-FU as compared with those given saline (P < 0.001-0.05). These findings clearly indicate that cisplatin and 5-FU are potent inducers of several types of cytokines and effector cells carrying antitumor activity mediated by asialoGM1-positive cells (mainly NK cells) for the most part, and that these abilities are closely associated with the in vivo antitumor effect of these agents.

Adenocarcinoma↗
PubMed1998

Upregulation of HIV-1 replication in chronically infected cells by ingenol derivatives.

We have previously reported that ingenol derivatives are highly potent inhibitors of human immunodeficiency virus type 1 (HIV-1) replication in acutely infected cells. In this study, however, we have found that some ingenol derivatives strongly enhance the replication of HIV-1 in chronically infected cells at nanomolar concentrations. One of the derivatives could activate nuclear factor kappa B (NF-kappa B), a potent inducer of HIV-1 replication, through the activation of protein kinase C (PKC). Whereas another derivative, which affected neither PKC nor NF-kappa B, significantly enhanced HIV-1 replication, suggesting that a PKC-independent mechanism may also exist in ingenol derivative-induced HIV-1 upregulation.

Butyrates↗
PubMed1998

RD6-2198, a novel betain-type fluoroalkylated oligomer, inhibits the replications of human immunodeficiency virus type 1 and other enveloped viruses.

We have examined a novel betain-type fluoroalkylated oligomer, RD6-2198, for its inhibitory effects on the replication of human immunodeficiency virus type 1 (HIV-1) and other enveloped viruses, including herpes simplex virus types 1 and 2 (HSV-1 and HSV-2, respectively) and respiratory syncytial virus (RSV) in cell cultures. We have found that the compound is a potent and selective inhibitor of these viruses. RD6-2198 inhibited the replication of HIV-1IIIB at a concentration of 0.85 microg/ml with a selectivity index greater than 59 in MT-4 cells. Furthermore, its 50% effective concentration (EC50) values for HSV-1, HSV-2 and RSV, were 0.51, 0.94 and 3.0 microg/ml, respectively. We found that the RD6-2198 suppressed the gp120-CD4 interaction (as monitored by an enzyme-linked immunosorbent assay (ELISA) method). RD6-2198 also inhibited the binding of anti-gp120 monoclonal antibody to gp120 expressed on MOLT-4/IIIB cells (MOLT-4 cells chronically infected with HIV-1IIIB). However, the compound did not inhibit the interaction of anti-CD4 antibody with CD4. These results suggest that RD6-2198 interacts with the viral envelope glycoprotein and thereby inhibits the viral adsorption process. In addition, RD6-2198 was also found to suppress the proliferation of MOLT-4/IIIB cells. When applied topically, RD6-2198 at a concentration of 10 mg/ml completely protected mice from an intravaginal HSV-2 infection.

Animals↗
PubMed1998

Mint 3: a ubiquitous mint isoform that does not bind to munc18-1 or -2.

Mint 1 and 2 are proteins that bind to munc18-1, an essential component of the synaptic vesicle fusion machinery, and are detectably expressed only in neurons [Okamoto and Südhof, J. Biol. Chem. 272, 31459-31464 (1997)]. Mint 1 and 2 are composed of a variable N-terminal region that includes a conserved munc18-1-binding site, and a constant C-terminal region that contains one PTB and two PDZ domains. We have now identified a third mint isoform, mint 3. Similar to mint 1 and 2, the C-terminal half of mint 3 is composed of one PTB domain and two PDZ domains. However, in contrast to mint 1 and 2, mint 3 lacks an N-terminal munc18-binding domain and does not interact with munc18-1 in yeast two-hybrid assays. Mint 3 is ubiquitously expressed in all tissues, with lowest levels in brain and testis whereas mint 1 and 2 appear to be brain-specific. Our data suggest that mints form a diverse family of proteins with specialized neuronal and ubiquitous isoforms.

Adaptor Proteins, Signal Transducing↗
PubMed1998

Reduced macrophage phagocytic activity in Wolffian lens regeneration of the newt after nickel subsulfide administration.

The inhibition of newt lens regeneration by intraocular injection of nickel subsulfide (alpha Ni3S2), a potent carcinogen, was shown in our previous studies. In the present study, we examined the effect of nickel subsulfide on macrophages involved in depigmentation in the early stages of regeneration using light and electron microscopic observations on semithin (1.5 microns) and ultrathin sections. Phagocytic activity of macrophages in ingestion of pigment granules from pigmented dorsal iris epithelial cells was much reduced in the carcinogen-treated eyes, whereas the change in their number and pattern accumulating in the iris during lens regeneration was generally the same as in untreated eyes.

Animals↗
PubMed1998

Vibrio vulnificus septicemia in a patient with severe aplastic anemia.

A 53-year-old man with severe aplastic anemia developed sporadic Vibrio vulnificus septicemia 1 day after eating raw fish and shellfish. Although V. vulnificus infection is potentially fatal, he was saved by immediate and sensitive antibiotic administration. Patients with chronic hematologic disease are susceptible to infection by this organism and are prone to developing septicemia when they eat raw seafood. It is necessary for a patient with this infection to be given effective antibiotics as quickly as possible.

Anemia, Aplastic↗
PubMed1998

Difference between dorsal and ventral iris in lens producing potency in normal lens regeneration is maintained after dissociation and reaggregation of cells from the adult newt, Cynops pyrrhogaster.

In Wolffian lens regeneration, lentectomized newt eye can produce a new lens from the dorsal marginal iris, but the ventral iris has never shown such capabilities. To investigate the difference of lens regenerating potency between dorsal and ventral iris epithelium at the cellular level, a transplantation system using cell reaggregates was developed. Two methods were devised for preparing the reaggregates from pigmented iris epithelial cells. One was rotating cells in an agar-coated multiplate on a gyratory shaker and the other was incubating cells in a microcentrifuge tube after slight centrifugation. Reaggregates made of dorsal iris cells that had been completely dissociated into single cells were phenotypically transformed into a lens when placed in the pupillary region of the lentectomized host eye. None of the ventral reaggregates produced a lens. Even dorsal reaggregates could not transdifferentiate into lens when they were placed away from the pupil. The produced lenses from the reaggregates were morphologically and immunohistochemically identified. To obtain evidence whether produced lenses really originated from singly dissociated cells, we labeled dissociated cells with a fluorescent dye (PKH26) before reaggregate formation and then traced it in the produced lens.

Animals↗
PubMed1998

Antisense RNA-mediated reduction of p53 induces malignant phenotype in nontumorigenic rat urothelial cells.

p53 mutation is commonly associated with high-grade, high-stage human urothelial carcinomas. Recent studies suggest that p53 mutation in low-grade, low-stage bladder carcinomas may be correlated with the progression of the disease. In the present study, we used antisense RNA methodology in vitro to evaluate the significance of the loss of p53 function at an early stage of urinary bladder carcinogenesis. An immortalized nontumorigenic rat urothelial cell line (MYP3) that strongly expresses wild-type (WT) p53 was transfected with a plasmid (pcDL-SR alpha-296) containing a rat WT p53 cDNA in antisense orientation. The transfection resulted in a significant reduction in p53 mRNA expression and protein synthesis, in stimulation of anchorage-dependent growth, and in acquisition of anchorage-independent growth potential. Three such clones, when tested in athymic nude mice, all formed muscle-invasive, high-grade transitional cell carcinomas at s.c. injection sites. When cells were inoculated into an orthotopic site (urinary bladder), one of two antisense transfectants tested formed bulky tumors in the bladder in all seven nude mice and metastases to lungs in three of the seven mice. Analysis of these cells revealed a decrease in the expression of p21 (WAF1, sdi1, or CIP1) and retinoblastoma (Rb) gene product. Phosphorylation of Rb protein was not inhibited when the cells were starved. No significant difference was observed in the expression of p16 protein. In cell cycle analysis, all antisense transfectants tested escaped from G1 arrest by starvation. Furthermore, secretion of interleukin (IL)-6 into culture medium was increased significantly. Treatment with anti-IL-6 antibody suppressed anchorage-dependent growth. This study directly demonstrates that the loss of p53 function at an early stage of urothelial carcinogenesis may result in acquisition of a malignant phenotype by regulating IL-6 production as well as cell cycle related genes.

Animals↗
PubMed1998

Prodigiosins uncouple mitochondrial and bacterial F-ATPases: evidence for their H+/Cl- symport activity.

Prodigiosin, metacycloprodigiosin, and prodigiosin 25-C all inhibited the acidification activity of submitochondrial and bacterial (Escherichia coli) F-ATPases (FoF1-ATPases) strongly (IC50 = 20-30 and 24-30 pmol/mg protein, respectively), without affecting significantly the ATP hydrolysis activity. Their effect on the acidification activity was rapid and reversible, showing non-competitive apparent Ki values of the order of nM to sub-nM. However, unlike FCCP (an ordinary uncoupler of oxidative phosphorylation), they showed no protonophoric activity, as demonstrated by the absence of acceleration of ATP hydrolysis. Prodigiosins also inhibited the acidification of proteoliposomes reconstituted from phospholipids and purified F-ATPase of E. coli, suggesting that their acidification-inhibitory effect is not due to the inhibition of anion channels. They did not, however, inhibit the ATP-dependent formation of membrane potential of F-ATPase vesicles. Furthermore, they inhibited and quickly reversed acidification by F-ATPase only in the presence of chloride, and not in the presence of gluconate anion. Finally, they induced swelling of liposomes and submitochondrial particles in isotonic solution of ammonium chloride but not ammonium gluconate, suggesting that intravesicular entry of Cl- is promoted by prodigiosins. These results suggest that prodigiosins uncouple F-ATPases through promotion of H+/Cl- symport (or OH-/Cl- exchange) across vesicular membranes.

Animals↗
PubMed1998

Origin and diversification of hindwingless Damaster ground beetles within the Japanese islands as deduced from mitochondrial ND5 gene sequences (Coleoptera, Carabidae).

Genealogical trees have been constructed using mitochondrial ND5 gene sequences of 78 Damaster (s. str.) specimens from all over the Japanese Islands. Eight lineages (called races in this paper) have been recognized. The races are tightly linked to geography with sharp distribution boundaries between them. The races and their distribution ranges do not coincide with those of classical morphology. Based on the observed distribution of the mitochondrial DNA haplotypes and the geohistorical data, we propose a diversification scenario of Damaster.

Animals↗
PubMed1998

Enhanced expression of an antimicrobial peptide sarcotoxin IA by GUS fusion in transgenic tobacco plants.

To enhance the disease resistance of plants expressing a foreign peptide, the gene for sarcotoxin IA, which is an antimicrobial peptide from an insect consisting of 39 amino acid residues, was introduced into tobacco (Nicotiana tabacum) under the control of a high expression promotor via Agrobacterium-mediated transformation. In transgenic plants, sarcotoxin IA mRNA accumulated to detectable levels, however, the amount of the peptide produced was so small that we could scarcely detect it by protein gel blot analysis, probably because of the instability of short peptides in plant cells. To improve the expression efficiency, genes for four types of amino-terminal and carboxyl-terminal translational fusions of sarcotoxin IA together with the GUS gene were introduced into tobacco. In all four types of transgenic tobacco plants, high level transcripts similar to that in the direct expression sarcotoxin IA construct were found. Protein gel blot analysis with both anti-sarcotoxin IA and GUS antibodies showed production of high levels of fusion protein in all transgenic plants. Among them, three types had abnormal membranes and phenotypes, although no such abnormalities were found in transgenic plants in which only sarcotoxin IA was expressed in a secretable form. All together, these results indicated that, for stable and effective expression of a foreign short peptide in transgenic plants, expression as a fusion protein is useful and that secretion of sarcotoxin IA outside of cells is necessary for generation of useful antimicrobial transgenic plants.

Anti-Bacterial Agents↗