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Biomedical subjects

K Kohno

Publications and source records attributed to K Kohno.

At least 145 records · Page 8Linked to original sources
PubMed1998

Nuclear expression of YB-1 protein correlates with P-glycoprotein expression in human osteosarcoma.

The Y-box-binding protein, YB-1, is a member of the DNA-binding protein family. It binds to the Y-box, an inverted CCAAT box, in the promoter region of the human multidrug resistance 1 gene, which encodes P-glycoprotein (P-gp). Nuclear localization of YB-1 protein has been reported to be associated with the intrinsic expression of P-gp in human breast cancer. We studied the immunohistochemical expression of YB-1 protein in 69 untreated biopsy specimens of conventional osteosarcomas and compared it with the expression of P-gp. Furthermore, cell proliferation, as determined by the MIB-1-labeling index (MIB-1-LI), was measured by immunohistochemistry. In all 69 untreated osteosarcomas, YB-1 protein was expressed in the cytoplasm. In 32 of 69 (46%) cases, YB-1 was also localized in the nucleus. The expression of P-gp was evident in 23 of these 32 cases, and there was a significant correlation between the nuclear expression of YB-1 and P-gp expression (P < 0.0001). Chondroblastic osteosarcoma expressed YB-1 in the nucleus more frequently (eight of nine cases) than did other types of osteosarcoma, whereas P-gp was also frequently expressed in chondroblastic subtype. There was no correlation between the nuclear expression of YB-1 and histological grade. The MIB-1-LI was significantly higher in cases showing the nuclear expression of YB-1 (MIB-1-LI averaged 22.56 in cases with only cytoplasmic expression of YB-1 but averaged 28.20 in cases with cytoplasmic and nuclear expression of YB-1; P = 0.0477). In human osteosarcoma, nuclear localization of YB-1 protein was associated with the expression of P-gp, suggesting that YB-1 could be a prognostic marker for multidrug resistance in osteosarcoma.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1997

Identification of the human leptin 5'-flanking sequences involved in the trophoblast-specific transcription.

Leptin is an adipocyte-derived blood-borne satiety factor that is involved in the regulation of energy homeostasis. We have recently demonstrated nonadipose tissue production of leptin; leptin is synthesized in and secreted from placental trophoblasts (Nature Med. 3: 1029-1033, 1997). To understand the transcriptional regulation of the human leptin gene in placental trophoblasts, we examined the promoter activity of various lengths of the human leptin 5'-flanking sequences in BeWo cells, a human trophoblastic cell line. The 2080-bp human leptin gene promoter region (-2080 to +108) showed a high-level transcription activity in BeWo cells. When DNA sequences between -1885 and -1830 were deleted, the promoter activity was reduced dramatically in BeWo cells. No significant changes in the promoter activity were noted when tested in primary cultures of rat mature adipocytes. Electrophoretic mobility shift assays revealed the presence of nuclear protein(s) binding to the sequences in BeWo cells but not in isolated rat mature adipocytes. The present study provides new insight into the trophoblast-specific transcription of the human leptin gene.

Adipocytes↗
PubMed1997

Nuclear translocation of the Y-box binding protein by ultraviolet irradiation.

The Y-box binding protein, YB-1, is a member of a DNA binding protein family with a structurally and functionally conserved cold shock domain. Using Western blotting and immunohistochemical methods, larger amounts of YB-1 were detected in the cytosol, particularly at the perinuclear region, than in the nucleus of human cancer cells. UV irradiation increased accumulation of YB-1 in the nucleus at 20 min and thereafter. This translocation of YB-1 into the nucleus by UV irradiation was blocked by the protein kinase inhibitor H-7, but not HA-1004. Both green fluorescent protein (GFP)-YB-1 and GFP-YB-1C with the C-terminus (248-317) of YB-1 were located mainly in the cytosol, but GFP-YB-1deltaC with a deletion at the C-terminus of YB-1 was located in the nucleus. YB-1 is translocated into the nucleus by UV irradiation, possibly through a protein kinase C-mediated signal transduction pathway, and the C-terminal region of YB-1 might be important for cytoplasmic retention of YB-1.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1997

Involvement of caspase-1 and caspase-3 in the production and processing of mature human interleukin 18 in monocytic THP.1 cells.

Recently, human interleukin 18 (hIL-18) cDNA was cloned, and the recombinant protein with a tentatively assigned NH2-terminal amino acid sequence was generated. However, natural hIL-18 has not yet been isolated, and its cellular processing is therefore still unclear. To clarify this, we purified natural hIL-18 from the cytosolic extract of monocytic THP.1 cells. Natural hIL-18 exhibited a molecular mass of 18.2 kDa, and the NH2-terminal amino acid was Tyr37. Biological activities of the purified protein were identical to those of recombinant hIL-18 with respect to the enhancement of natural killer cell cytotoxicity and interferon-gamma production by human peripheral blood mononuclear cells. We also found two precursor hIL-18 (prohIL-18)-processing activities in the cytosol of THP.1 cells. These activities were blocked separately by the caspase inhibitors Ac-YVAD-CHO and Ac-DEVD-CHO. Further analyses of the partially purified enzymes revealed that one is caspase-1, which cleaves prohIL-18 at the Asp36-Tyr37 site to generate the mature hIL-18, and the other is caspase-3, which cleaves both precursor and mature hIL-18 at Asp71-Ser72 and Asp76-Asn77 to generate biologically inactive products. These results suggest that the production and processing of natural hIL-18 are regulated by two processing enzymes, caspase-1 and caspase-3, in THP.1 cells.

Amino Acid Sequence↗
PubMed1997

Interleukin 18 induces the sequential activation of natural killer cells and cytotoxic T lymphocytes to protect syngeneic mice from transplantation with Meth A sarcoma.

We have recently reported that interleukin 18 (IL-18) pretreatment induces immunologically mediated antitumor effects in BALB/c mice injected i.p. with syngeneic Meth A sarcoma. In this study, mice were pretreated with IL-18 before Meth A transplantation, and immunocompetency in pretreated or untreated tumor-bearing mice (TBM) 3, 9, and 15 days after transplantation was compared with that of normal mice. On day 3, pretreated TBM mitogen-stimulated spleen cells produced significantly decreased levels of IL-2 and IFN-gamma during 24-h culture. In contrast, IL-10 and granulocyte macrophage colony-stimulating factor productions were significantly enhanced in pretreated TBM cultures, and natural killer (NK) cell activity was also significantly augmented. Splenomegaly was also observed in the pretreated TBM on day 3, and the proliferating cells were identified as asialo GM1+ cells by flow cytometry. Cytotoxic activity of pretreated TBM spleen cells after a 5-day mixed lymphocyte-tumor cell culture did not differ from that of untreated TBM and normal mice on day 3 but was significantly enhanced on days 9 and 15 compared with that observed in normal mice and untreated TBM. Concurrently, the production of IL-2 and of IL-10 recovered and decreased, respectively, and NK activity dropped to normal levels. The effects of IL-18 on cytokine production and NK activity observed on day 3 treated TBM were also reproduced in normal mice. In conclusion, IL-18 seems to enhance the generation of NK activity early after tumor transplantation and simultaneously induces an increase and a decrease in the production of IL-10 and IL-2, respectively. As NK activity subsides to normal levels and IL-10 synthesis decreases, IL-2 synthesis is restored, and cytolytic cell activity is significantly enhanced. These results provide new insight into the immunologically mediated antitumor effects of IL-18.

Animals↗
PubMed1997

Construction of a pilot human YAC library in a recombination-defective yeast strain.

Using high-molecular-weight DNA fragments from a human lymphoblastoid cell line, a pilot collection of 2500 YACs was constructed in YKK115, a recombination-deficient strain of Saccharomyces cerevisiae carrying mutations in both the rad51 and rad52 genes. Analysis of 520 clones from the current library by pulsed-field gel electrophoresis revealed more than 97% single YACs with an insert size averaging 340 kb. Fluorescent in situ hybridization (FISH) performed with 37 clones on metaphase chromosomes suggested a high proportion mapping at centromeric (7) or telomeric (4) locations. The results are consistent with the stabilization of YACs in strains disarmed in recombination functions [Kohno, K., Oshiro, T., Kishine, H., Wada, M., Takeda, H., Ihara, N., Imamoto, F., Kano, Y. and Schlessinger, D. (1997) Human YACs unstable in a rad52 single mutant strain become stable in rad51rad52 double mutant. Gene, 000, 000-000 (GENE 10429)], and further suggest that the YACs may include regions that have been difficult to clone in other strains.

Cell Line↗
PubMed1997

Construction and characterization of a rad51rad52 double mutant as a host for YAC libraries.

RAD52 or RAD51 recombination-deficient yeast strains stabilize otherwise unstable YACs containing ribosomal DNA or the human color vision locus (Kohno et al., 1994). Thus the RAD52RAD51 pathways(s) are apparently involved in the instability of YACs containing tandem repeat loci, presumably by promoting recombination-based deletion formation. Some other genomic loci are still unstable or unrecoverable in those strains, but we now find that greater stability is observed in a rad51rad52 double mutant strain that we have newly constructed. YACs containing a highly unstable region around DXS49 or centromeric regions throw off a variety of products in single mutants, but are much more stable in the rad51rad52 strain, which could therefore provide a better host for library construction and maintenance.

Chromosomes, Artificial, Yeast↗
PubMed1997

Cloning and expression of the rubredoxin gene from Desulfovibrio vulgaris (Miyazaki F)--comparison of the primary structure of desulfoferrodoxin.

A gene encoding rubredoxin from Desulfovibrio vulgaris (Miyazaki F) was cloned and overexpressed in Escherichia coli. A 1.1-kilobase pair DNA fragment, isolated from D. vulgaris (Miyazaki F) by double digestion with SmaI and SalI, contained two genes, the rubredoxin gene (rub) and the desulfoferrodoxin gene (rbo) which was situated upstream of rub. The deduced amino acid sequence of desulfoferrodoxin was homologous to those from other strains and Cys residues that are responsible to bind irons were also conserved. The expression system for rub was constructed under the control of the T7 promoter in E. coli. The purified protein was soluble and had a characteristic visible absorption spectrum. Inductively coupled plasma-atomic emission analysis and electron paramagnetic resonance analysis of the recombinant rubredoxin revealed the presence of an iron ion in a distorted tetrahedral geometry that was the same as native D. vulgaris rubredoxin. In vitro NADH reduction analysis indicated that recombinant rubredoxin was active, and its redox potential was determined as -5 mV.

Amino Acid Sequence↗
PubMed1997

Neuroprotective nitric oxide synthase inhibitor reduces intracellular calcium accumulation following transient global ischemia in the gerbil.

By observing the ultrastructural intracellular Ca2+ distribution with Ca(2+)-oxalate-pyroantimonate method, we examined whether the protective mechanism of the nitric oxide (NO) synthase inhibitor, N omega-nitro-L-arginine (LNNA), involves change of the intracellular Ca2+ movement in delayed neuronal death (DND) in gerbil hippocampal CA1 neurons following 5-min forebrain ischemia. In the group intraventricularly administered 5.0 mg/ml LNNA, 15 min after reperfusion the intracellular Ca2+ deposits and the mitochondrial Ca2+ uptake index increased to levels similar to those in the control group administered only artificial cerebro-spinal fluid, but by 180 min after reperfusion they had returned to the preischemic level. By 15 min after reperfusion Ca2+ deposits in the endoplasmic reticulum (ER) had almost disappeared in both groups, but at 180 min of reperfusion, the ER in only the LNNA group showed Ca2+ deposits. It is suggested that the neuronal toxicity of NO involves the dysfunction of the intracellular Ca2+ transport system including the mitochondria and ER.

Animals↗
PubMed1997

A novel mutation which enhances the fluorescence of green fluorescent protein at high temperatures.

Green fluorescent protein (GFP) from Aequorea victoria is widely used as a marker of gene expression and protein localization in living cells from prokaryotes to eukaryotes. However, the total fluorescent signal from wild-type GFP is very weak when expressed in cells cultured at 37 degrees C compared to 30 degrees C or below. This characteristic makes GFP poorly suited to use as a marker in mammalian cells. Here we describe a new variant of GFP which carries a substitution of Ser147 to Pro (S147P GFP) and which emits a stronger fluorescent signal than the wild-type GFP at high temperature. When S147P is combined with the Ser65 to Thr mutation (S65T GFP), the resulting double mutant emits fluorescence which is several-fold stronger than GFP with a single S65T modification in both bacterial or mammalian cells. This S147P mutation should be useful for constructing new GFP variants which stably emit strong fluorescence at a wide range of culturing temperatures.

Fluorescence↗
PubMed1997

IFN-gamma-inducing factor (IGIF) is a costimulatory factor on the activation of Th1 but not Th2 cells and exerts its effect independently of IL-12.

We have previously reported the cloning of a novel cytokine, IFN-gamma-inducing factor (IGIF), which shared some biologic activities with IL-12. In this study, we analyzed the effects of murine IGIF on the activation of T cells, and compared the effects with those of IL-12. IGIF alone had no effect on the activation of T cell lines or Th1 clones, while IGIF increased the IFN-gamma production by antigen-stimulated T cell lines, but had no effect on IL-4 or IL-10 production. As reported with IL-12, IGIF served as a costimulatory factor for Th1 clones stimulated with Ag on B cell APC, immobilized anti-CD3, Con A, or IL-2 to augment IFN-gamma production and to induce IL-2R alpha-chain expression and proliferation of the Th1 clones, whereas IGIF had little or no effect on the IL-4 production and proliferation of Th2 clones stimulated with anti-CD3 or Ag. However, IGIF synergized with IL-12 to further augment the IFN-gamma production of the Th1 clones. Even in the presence of saturated amounts of IL-12, IGIF still augmented the IFN-gamma production and proliferation and enhanced the IL-2R alpha-chain expression of the Th1 clones. In contrast with IL-12, IGIF induced IL-2 production by Ag- or anti-CD3-stimulated Th1 clones. These two findings indicate that IGIF and IL-12 are utilizing different signal transduction pathways. We also found that IGIF as well as IL-12 was endogenously released through interaction between Th1 cells and spleen cell APC in the presence of specific Ag, and that it regulated IFN-gamma production. These results further suggest that IGIF may act as an immunoregulatory factor in the immune response.

Animals↗
PubMed1997

Single LDL apheresis improves endothelium-dependent vasodilatation in hypercholesterolemic humans.

BACKGROUND: Although long-term lipid-lowering therapy improves endothelium-dependent vasodilatation in humans, it remains unknown whether the short-term removal of LDL per se ameliorates endothelial dysfunction. METHODS AND RESULTS: To examine the effects of a single session of LDL apheresis on endothelial function in patients with hypercholesterolemia, we measured forearm blood flow (FBF) by strain-gauge plethysmography before and after single LDL apheresis while infusing acetylcholine (ACh; 4 to 24 micrograms/min) and sodium nitroprusside (SNP; 0.2 to 1.2 micrograms/min). The single session of LDL apheresis reduced total LDL (from 142.2 +/- 15.0 to 32.6 +/- 5.0 mg/mL, P < .0005) and oxidized LDL (from 111.6 +/- 22.8 to 30.0 +/- 5.4 ng/mL, P < .005). Although ACh and SNP increased FBF dose-dependently before and after LDL apheresis, the endothelium-dependent vasodilatation responses to ACh were significantly augmented (P < .01) after the single session of LDL apheresis without changes in the endothelium-independent vasodilatation responses to SNP. The plasma levels of total and oxidized LDL correlated with the degree of ACh-induced vasodilatation. Furthermore, the local production of nitrate/nitrite, metabolites of NO, during ACh infusion was significantly (P < .05) augmented by LDL apheresis, and there was a significant correlation between the degree of ACh-induced vasodilatation and the production in nitrate/nitrite (r = .99, P < .0005). CONCLUSIONS: We demonstrated that even a single session of LDL apheresis with the reduction of total LDL and oxidized LDL improved endothelial function. Our results suggest that total LDL and/or oxidized LDL may directly impair endothelial function in the human forearm vessel.

Acetylcholine↗
PubMed1997

Maintenance of hypomethylation status and preferential expression of exogenous human MDR1/PGY1 gene in mouse L cells by YAC mediated transfer.

Selection of cells for resistance to vincristine or doxorubicin often induces overexpression of the multidrug resistance (MDR) genes, which encode the cell surface P-glycoproteins, as a result of gene amplification, transcriptional activation, or mRNA stabilization. The LMD1 and LMD4 cell lines were established after the transfer into mouse L cells of two independent yeast artificial chromosome clones containing 300 and 850 kb, respectively, of the human MDR locus. The human MDR1/PGY1 gene, but not the endogenous mouse mdr1a and mdr1b genes, was overexpressed as a result of gene amplification and transcriptional activation in various sublines of LMD1 and LMD4 cells selected for resistance to vincristine. Then we asked why human MDR1/PGY1 gene, but not mouse relevant gene, was expressed. Determination of the methylation status of cytosine residues at Msp I/Hap II cleavage sites (CCGG) in the promoter regions of human MDR1/PGY1 and mouse mdr1a revealed hypomethylation and hypermethylation of the human and mouse genes, respectively in LMD1, LMD4, and their vincristine-resistant derivatives. Various vincristine-resistant sublines were also established after exposure of LMD1 cells for 48 h to 5-aza-2'-deoxycytidine, an inhibitor of DNA methyltransferase. These sublines exhibited overexpression of mouse mdr1a and mdr1b, but not of human MDR1/PGY1, as well as hypomethylation of the mouse mdr1a promoter region. Thus, the selective expression of human or mouse MDR genes in this cell system appears to be related to the methylation status of the respective gene promoter regions.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1997

Endovascular treatment for acute thrombotic occlusion of the middle cerebral artery: local intra-arterial thrombolysis combined with percutaneous transluminal angioplasty.

We report our experience in treating 15 patients with acute thrombotic occlusion of the M1 or M2 segment of the middle cerebral artery who underwent intra-arterial thrombolytic therapy alone or in combination with percutaneous transluminal angioplasty (PTA). The results were compared with those of 30 patients with acute embolic occlusion of the same artery. Intra-arterial thrombolysis was performed in 10 patients and thrombolysis combined with PTA in 5 in whom symptoms reappeared due to restenosis or reocclusion, or in whom recanalisation was not successfully accomplished by thrombolysis alone. In the patients with embolism recanalisation was observed in 28 (93 %) and there was no patient with reocclusion. In the patients with thrombosis recanalisation immediately after thrombolysis alone was observed in 9 of 15 (60%). Restenosis, with reappearance of symptoms, occurred in 2 of these (22 %). In the patients who also underwent PTA, angiography after 1 month did not demonstrate any restenosis or reocclusion. Thrombolysis combined with PTA for acute thrombotic stroke may provide an effective procedure for restoring patency and preventing reocclusion of the occluded artery.

Aged↗
PubMed1997

In vivo antitumor effects of murine interferon-gamma-inducing factor/interleukin-18 in mice bearing syngeneic Meth A sarcoma malignant ascites.

Interferon-gamma-inducing factor/interleukin-18 is a novel cytokine that reportedly augments natural killer (NK) activity in human and mouse peripheral blood mononuclear cell cultures in vitro and has recently been designated IL-18. In this study, IL-18 exhibited significant antitumor effects in BALB/c mice challenged intraperitoneally (i.p.) with syngeneic Meth A sarcoma when administered i.p. on days 1, 2 and 3 after challenge. Intravenous (i.v.) administration also induced antitumor effects in the tumor-bearing mice; however, subcutaneous (s.c.) administration did not. When mice were twice pretreated with 1 microg IL-18 3 days and 6 h before tumor challenge, all mice survived whereas control mice died within 3 weeks of challenge. Inhibitory effects on Meth A cell growth in vitro were not observed with either IL-18 or interferon gamma. The effects of IL-18 pretreatment were abrogated by abolition of NK activity after mice had been injected with anti-asialo GM1 antibody 48 h before and, 24 h and 72 h after tumor challenge. Mice pretreated with IL-18 and surviving tumor challenge resisted rechallenge with Meth A cells but could not reject Ehrlich ascites carcinoma, and spleen cells from the resistant mice, but not control mice, exhibited cytotoxic activity against Meth A cells in vitro after restimulation with mitomycin C-treated Meth A cells for 5 days. The effector cells in the spleen cell preparations from resistant mice appear to be CD4+ cells because cytolytic activity was significantly inhibited after depletion of this subset by monoclonal antibodies and complement. In conclusion, IL-18 exhibits in vivo immunologically (primarily NK) mediated antitumor effects in mice challenged with syngeneic Meth A sarcoma and induces immunological memory and the generation of cytotoxic CD4+ cells.

Animals↗
PubMed1997

Migration of neutrophils from blood to tissue: alteration of modulatory effects of prostanoid on superoxide generation in rabbits and humans.

Alteration of neutrophil function is associated with their migration from blood into tissue. We evaluated this alteration in both human and rabbit neutrophils, by comparing the inhibitory effects of prostanoids on formylmethionyl-leucyl-phenylalanine (fMLP)-stimulated superoxide generation in human circulating blood neutrophils with those in saliva, and also comparing rabbit circulating blood neutrophils with those exudated into peritoneal cavity. We showed that EP-receptor agonists (PGE1) EP2/EP3 agonist (misoprostol), EP2-receptor agonist (butaprost) and DP-receptor agonist (PGD2) inhibited fMLP-stimulated superoxide production from human blood neutrophils in a concentration-dependent manner. In contrast, these prostanoids produced a significantly smaller maximum inhibition of fMLP-stimulated superoxide production in salivary neutrophils compared to those in blood neutrophils. Similar differences were observed for rabbit blood and peritoneal neutrophils. The inhibitory effect of EP2 agonist (butaprost) on the fMLP-stimulated superoxide generation in human blood neutrophils was significantly higher than that of EP3 agonist (ONO-AP-324). The EP1 antagonist (SC-51322) and EP4 antagonist (AH23848B) employed in this study could not antagonize the inhibitory effect of PGE2. TP agonist (U-46619) failed to show any inhibitory effect in either blood or salivary neutrophils. These results indicated that EP2 and DP receptors are the primary receptors mediating the prostanoids inhibition of fMLP-stimulated superoxide generation from neutrophils. Furthermore, it can be concluded that neutrophils become less responsive to prostanoids in terms of fMLP-stimulated superoxide production in association with their migration from blood to tissue.

Alprostadil↗