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Biomedical subjects

Y Ozeki

Publications and source records attributed to Y Ozeki.

At least 37 records · Page 2Linked to original sources
PubMed2001

Development and assessment of enzyme immunoassay for platelet-derived microparticles.

Platelet-derived microparticles (PMPs) are released from platelets through the platelet activation by high shear stress, collagen, or calcium ionophore (A23187). PMPs are observed in patients with acute myocardial infarction, thrombotic thrombocytopenic purpura, hemolytic uremic syndrome, heparin-induced thrombocytopenia and other thrombotic disorders, but the importance of circulating PMPs in the pathogenesis of these diseases is still debated. Numbers of PMPs are usually determined by flowcytometry (FCM), but easier and reproducible PMP assay systems are needed. To develop a better ELISA for PMPs, we used antibodies against the platelet antigens anti-GPIb (NNKY5-5), anti-GPIIb/IIIa (NNKY2-11, anti-CD41), anti-GPIX (KMP-9), and anti-CD9 (NNKY1-19). PMPs were detected with all combinations of these antibodies, but the ELISA having the highest and most specific absorbance was obtained with a combination of KMP-9 (capture antibody) and NNKY5-5 (detecting antibody). PMPs in blood samples were measured by ELISA and FCM. ELISA correlated with PMPs quantitated by FCM. By shaking ELISA plates during incubation, nonspecific binding of platelets was eliminated. The level of PMPs was not increased in diabetes mellitus, thrombotic thrombocytopenic purpura, antiphospholipid syndrome, or sepsis. The concentration of PMP was elevated in hemolytic uremic syndrome. Activated PMPs were absorbed to 0.8 microm filter, but circulating PMPs were not absorbed. These results suggest that activated PMPs are likely to adhere to leukocytes or endothelial cells at the activation site and that the circulating form of PMPs are likely to be a residue of activated PMPs. To detect only the activated form of PMPs, a new ELISA needs to be developed, and it will likely use a combination of antibodies that detect platelet activation markers such as P-selectin (CD62P) or activated GPIIb/IIIa.

Antibodies, Monoclonal↗
PubMed2000

Genetic polymorphisms of human melatonin 1b receptor gene in circadian rhythm sleep disorders and controls.

Recent studies suggest that melatonin 1b (Mel1b) receptor, as well as melatonin 1a (Mel1a) receptor, is involved in the modulation of circadian rhythms in mammals. Mutational analysis was performed in the entire coding region of the human Mel1b receptor gene using genomic DNA from sleep disorder subjects. We have identified two missense mutations, G24E and L66F. However, neither is likely to be associated with sleep disorders in our study population. One of the subjects with non-24-h sleep-wake syndrome carries missense mutations in both the Mel1a and Mel1b receptor genes.

Adult↗
PubMed2000

Ribozyme approach to downregulate vascular endothelial growth factor (VEGF) 189 expression in non-small cell lung cancer (NSCLC).

The aim of this study was to further clarify the role of the cell-associated isoform of vascular endothelial growth factor (VEGF189) on tumour growth and vascularity. Five isoforms of VEGF have been identified with different biological activities. VEGF121, VEGF145, VEGF165, VEGF189, VEGF206 are generated by alternative splicing. We used a hammerhead-type ribozyme (V189Rz) to suppress VEGF189 mRNA. The V189Rz specifically cleaved exon 6 of VEGF189 mRNA, but showed no activity against the VEGF121 or VEGF165 isoforms. The V189Rz was introduced into the human non-small cell lung cancer (NSCLC) cell line (OZ-6/VR). The expression level of VEGF189 mRNA was decreased in the OZ-6/VR cells, while VEGF121 and 165 expression was unaltered. The OZ-6/VR cells xenotransplanted into nude mice showed markedly reduced vascularisation and growth, whereas the cell line did not show any decreased growth under tissue culture conditions. The OZ-6/VR cells (1 x 10(5) cells/mouse) formed no tumours, whereas the parental OZ-6 cells formed large tumours within 8 weeks. The specific suppression of VEGF189 by the ribozyme decreased vascularity and xenotransplantability of the lung cancer cell line. Thus, the cell-associated isoform of VEGF, VEGF189, might have a key role in stromal vascularisation and the growth of NSCLC xenografts in vivo.

Animals↗
PubMed2000

Nerve stimulating system for peripheral nerve tumor resection under the thoracoscope.

The authors report a newly devised nerve stimulator and its clinical application in thoracoscopic surgery. The stimulator is a monopolar system, that has a long extension between the electrodes and the handle. The stimulator is inserted into the chest cavity through a portal, and is used to distinguish motor nerves from the surrounding tissue and to determine whether there is nerve involvement with tumor. The tumor is then safely and rapidly resected by thoracoscopic technique and the major nerves are spared. This method was used in two clinical cases, with excellent results obtained, and with no major complications.

Adult↗
PubMed2000

Enhanced ability of heparin-carrying polystyrene (HCPS) to bind to heparin-binding growth factors and to inhibit growth factor-induced endothelial cell growth.

Heparin-carrying polystyrene (HCPS) consists of low-molecular-weight heparin chains enriched in trisulfated disaccharide structures linked to a polystyrene core. In this study, the interactions between HCPSs of various molecular weights and heparin-binding growth factors, VEGF(165), FGF-2, and HGF, were compared to the interactions of the same factors with native heparin, periodate-oxidized heparin (IO(4)-heparin) and periodate-oxidized alkaline-degraded heparin (IO(4)-LMW-heparin). The binding of each growth factor to heparin-agarose beads (heparin-beads) was more strongly inhibited by HCPSs in a molecular weight-dependent manner than by native heparin or the modified heparins, indicating a stronger interaction between HCPS and these growth factors. HCPSs also inhibit heparin-binding growth factor-induced endothelial cell growth in a molecular weight-dependent manner much more strongly than the native or modified heparins. However, HCPSs did not inhibit the mitogenic activity of VEGF(121), which has a non-heparin-binding nature. Thus, HCPSs exhibit enhanced abilities to interact with each of the heparin-binding growth factors studied and to inhibit heparin-binding growth factor-induced endothelial cell proliferation in a molecular weight-dependent manner. These effects might be ascribed to the heparin-clustering effect of HCPSs.

Coronary Vessels↗
PubMed2000

Inhibition of 12(S)-hydroxyeicosatetraenoic acid (12-HETE) production suppressed the intimal hyperplasia caused by poor-runoff conditions in the rabbit autologous vein grafts.

The efficacy of OPC-29030, a newly developed inhibitor of 12(S)-hydroxyeicosatetraenoic acid (12-HETE) production, was evaluated on intimal hyperplasia of experimental autologous vein grafts in a distal poor-runoff model and a hyperlipidemic model in rabbits. First, rabbits were divided into two groups, the distal poor-runoff group (PR group) and the hyperlipidemic group (HL group). After 4 weeks preparing the PR model and the HL model, the femoral vein was implanted into the ipsilateral femoral artery. Then they were subdivided into two groups, depending on the diet provided; diet group with 0.1% OPC-29030 (OPC-29030 group) and normal diet group (control group). At 4 weeks, the grafts were harvested, and intimal hyperplasia of the graft was measured with an ocular cytometer. Intimal cell proliferation was determined by bromodeoxyuridine (BrdU) incorporation at 2 weeks after surgery. In addition, the effect of OPC-29030 on the proliferation or migration of rat aortic smooth muscle cells in culture was investigated. In the in vivo study in the PR group, the intimal hyperplasia and the plasma 12-HETE levels in the OPC-29030 group were significantly inhibited, compared with those of the control group. However, in the HL group, the intimal hyperplasia in both the OPC-29030 and control groups showed a remarkable degree of intimal hyperplasia. There was no significant difference between those two groups. Furthermore, there was no significant difference in the plasma 12-HETE levels in the HL group irrespective of the presence of OPC-29030. The BrdU labeling index at 2 weeks after grafting was significantly lower in the OPC-29030 group compared with that in the control group in the PR group. In the in vitro study, OPC-29030 did not inhibit smooth muscle cell proliferation; however, OPC-29030 inhibited the migration. These results demonstrate the efficacy of OPC-29030 in reducing the degree of intimal hyperplasia under PR conditions, but not under hyperlipidemic conditions. The mechanism of reducing the intimal hyperplasia may be that OPC-29030 inhibited 12-HETE production, which did not inhibit proliferation while inhibiting migration of the smooth muscle cell. These results suggested the possible involvement of 12-HETE with the intimal hyperplasia under PR conditions.

12-Hydroxy-5,8,10,14-eicosatetraenoic Acid↗
PubMed2000

Increased platelet sensitivity to collagen in individuals resistant to low-dose aspirin.

BACKGROUND AND PURPOSE: The purpose of this study was to assess individual differences in the pharmacological effects of acetylsalicylic acid (ASA) on bleeding time as measured by in vitro platelet aggregation and to examine the consistency of responses over time. METHODS: We measured template IIR bleeding time and platelet aggregation in 8 healthy male volunteers before and 2 hours after ingestion of 324 mg of ASA. An individual was considered a nonresponder if his post-ASA bleeding time was not 2 SDs above his baseline bleeding time, where SD was estimated from the baseline bleeding times of the 8 volunteers. The same experiment was done after a 30-month interval. RESULTS: Five volunteers were identified as ASA responders, and 3 were identified as nonresponders. Bleeding time before and after ingestion of ASA was 408+/-121 seconds (mean+/-SD) and 720+/-225 seconds, respectively, in ASA responders and 330+/-30 seconds and 330+/-52 seconds, respectively, in ASA nonresponders. The mean ED(50) for collagen-induced platelet aggregation, that is, the mean concentration of collagen that caused a response at 50% of maximum, was 0.91 microg/mL (95% CI, 0.73 to 1. 14) in ASA responders and 0.48 microg/mL (95% CI, 0.38 to 0.60) in nonresponders. When optimum concentrations of collagen, ie, concentrations that yielded 90% maximum aggregation, were used as stimuli, the mean IC(50) for ASA, that is, the mean concentration that yielded 50% inhibition, was 322.5 micromol/L (95% CI, 264.8 to 392.6) in ASA responders and 336.1 micromol/L (95% CI, 261.0 to 432. 8) in nonresponders. The variability in individual responsiveness in the second experiment remained consistent with that in the first experiment. CONCLUSIONS: ASA resistance may be caused by an increased sensitivity of platelets to collagen. A platelet aggregation study specific for collagen dose response may be useful for strict selection of ASA responders for low-dose ASA therapy and for identifying ASA nonresponders for high-dose ASA therapy.

Adult↗
PubMed1999

Alleic variants of human melatonin 1a receptor: function and prevalence in subjects with circadian rhythm sleep disorders.

The human melatonin 1a (hMella) receptor gene was screened for mutations using genomic DNA samples from patients with circadian rhythm sleep disorders and control subjects by single strand conformational polymorphism analysis (SSCP). We found seven mutations, two of which predict amino acid changes R54W and A157V, respectively. The prevalence of the R54W variant and that of the A157V variant were several times more common in non-24-h sleep-wake syndrome subjects than among control subjects, although the incidence was not significant in our study group. When expressed in COS-7 cells, the R54W mutant receptor exhibited significantly reduced B(max) and slightly enhanced affinity (reduced K(d)) compared to the wild type receptor, while the A157V variant receptor showed similar binding characteristics to the wild type. The identification of variants in the hMella receptor will provide a useful tool for analyzing genetic predisposition toward various diseases related to melatonin function and to clarify the physiological role of melatonin receptors in humans.

Alleles↗
PubMed1999

Genomic organization of the genes encoding dihydroflavonol 4-reductase for flower pigmentation in the Japanese and common morning glories.

Genomic DNA segments (approximately 17kb) containing three DFR genes in the Japanese and common morning glories were sequenced. The three DFR genes in both plants were found to be arranged in a tandem array, and all of them comprised six exons with identical intron positions. Their DFR-B genes carrying longer introns than the DFR-A and DFR-C genes were expressed extensively in the young buds of pigmented flowers, and the transcription starting site for the DFR-B mRNA of the Japanese morning glory was determined. The DFR-B gene of the common morning glory was expressed considerably in stems, moderately in sepals and leaves, whereas the DFR-A and DFR-C genes of the same plant were expressed scarcely but significantly in the young flower buds and stems. Several novel mobile element-like sequences of around 200bp were found in the genomic DFR regions. A phylogenetic tree indicated that each DFR gene in the Japanese morning glory is most closely related to the corresponding DFR gene in the common morning glory, and that the DFR-B gene is the most diversified gene among the three DFR genes. These structural and functional features of the DFR genes and their evolutionary implications are discussed.

Alcohol Oxidoreductases↗
PubMed1999

Multidrug resistance-associated protein (MRP) expression is correlated with expression of aberrant p53 protein in colorectal cancer.

Multidrug resistance-associated protein (MRP) is one of the major factors responsible for non-P-glycoprotein (Pgp)-mediated multidrug resistance of human tumour cells. In this study, we examined MRP and aberrant p53 expression in 54 colorectal cancers (CRC), 35 carcinoma in adenomas (CIA) and 40 adenomatous polyps by immunohistochemical procedures. 38 of 54 (70%) CRCs, 16 of 35 (46%) CIAs and 3 of 40 (8%) adenomatous polyps were MRP positive (chi 2 test, P < 0.0001). 36/54 (67%) CRCs, 10/35 (29%) CIAs and 0/40 adenomatous polyps were p53 positive. 30 of the 36 p53-positive CRCs were also MRP positive and 8/10 CIAs were both p53 and MRP positive. MRP overexpression correlated with aberrant p53 accumulation in CRCs and CIAs (chi 2 test, P < = or 0.01). Coexpression of MRP and p53 in the same cells was confirmed in the CRCs and CIAs by double staining procedures. These results suggested that MRP overexpression is related to aberrant p53 expression in CRC.

ATP-Binding Cassette Transporters↗
PubMed1999

Thrombospondin 2 expression is correlated with inhibition of angiogenesis and metastasis of colon cancer.

Two subtypes of thrombospondin (TSP-1 and TSP-2) have inhibitory roles in angiogenesis in vitro, although the biological significance of these TSP isoforms has not been determined in vivo. We examined TSP-1 and TSP-2 gene expression by reverse transcription polymerase chain reaction (RT-PCR) analysis in 61 colon cancers. Thirty-eight of these 61 colon cancers were positive for TSP-2 expression and showed hepatic metastasis at a significantly lower incidence than those without TSP-2 expression (P = 0.02). TSP-2 expression was significantly associated with M0 stage in these colon cancers (P = 0.03), whereas TSP-1 expression showed no apparent correlation with these factors. The colon cancer patients with TSP-2 expression showed a significantly low frequency of liver metastasis correlated with the cell-associated isoform of vascular endothelial growth factor (VEGF-189) (P = 0.0006). Vascularity was estimated by CD34 staining, and TSP-2(-)/VEGF-189(+) colon cancers showed significantly increased vessel counts and density in the stroma (P < 0.0001). TSP-2(-)/VEGF-189(+) colon cancer patients also showed significantly poorer prognosis compared with those with TSP-2(+)/VEGF-189(-) (P = 0.0014). These results suggest that colon cancer metastasis is critically determined by angiogenesis resulting from the balance between the angioinhibitory factor TSP-2 and angiogenic factor VEGF-189.

Analysis of Variance↗
PubMed1999

An anti-platelet agent, OPC-29030, inhibits translocation of 12-lipoxygenase and 12-hydroxyeicosatetraenoic acid production in human platelets.

1. In human platelets, arachidonic acid is mainly metabolized by the two enzyme systems; cyclo-oxygenase and 12-lipoxygenase. Cyclo-oxygenase produces prostaglandin H(2) which is further converted to thromboxane B(2). 12-Lipoxygenase synthesizes 12(S)-hydroperoxyeicosatetraenoic acid which is reduced to 12(S)-hydroxyeicosatetraenoic acid (12(S)-HETE). 2. An anti-platelet compound, OPC-29030, dose-dependently inhibited 12(S)-HETE production with an IC(50) of 0.06+/-0.01 microM, but not synthesis of thromboxane B(2) in human platelets. Although the compound suppressed 12(S)-HETE production in human platelets, cytosolic 12-lipoxygenase activity was not inhibited up to 10 microM. Essentially identical data were obtained with a 12-lipoxygenase of human erythroleukaemia cells which had megakaryocyte/platelet-like properties. 3. OPC-29030 also suppressed production of 5(S)-HETE, a 5-lipoxygenase product, in rat basophilic leukaemia cells without inhibiting enzyme activity. It has been shown that 5-lipoxygenase binds to membrane 5-lipoxygenase-activating protein (FLAP) to produce 5(S)-HETE, and thus FLAP inhibitor suppresses cellular 5(S)-HETE production. 4. A FLAP inhibitor, L-655,238, suppressed platelet 12(S)-HETE production, but had no effect on the 12-lipoxygenase activity. 5. Western blot analysis showed that platelet 12-lipoxygenase translocated from cytosol to membranes upon thrombin stimulation, and OPC-29030 suppressed this process in a dose-dependent manner. 6. These results suggest that the 12-lipoxygenase of human platelets binds to FLAP or a similar protein, and OPC-29030 suppresses 12(S)-HETE production by inhibiting a certain step of the 12-lipoxygenase translocation.

12-Hydroxy-5,8,10,14-eicosatetraenoic Acid↗
PubMed1999

A newly developed assay for melatonin using cells expressing human mel-1a receptor.

We have developed a radioreceptor binding assay (RRA) method for melatonin using membranes from Chinese hamster ovary cells that can stably express human mel-1a receptors. We measured melatonin levels in plasma samples collected every 4h for 24h using the RRA and radioimmunoassay (RIA) methods, simultaneously. There was a statistically significant correlation between the melatonin levels measured by the two methods, this newly developed method providing a sensitive bioassay. As it is possible to circumvent the cross-reactivity usually occurring in the RIA method, this method may be an important tool for detecting bioactive substances relative to the mel-1a receptor.

Animals↗
PubMed1999

Relation between the serum E-selectin level and the survival rate of patients with resected non-small cell lung cancers.

E-Selectin is an inducible adhesion molecule, which is expressed on cytokine-activated endothelial cells and is thought to interact with cancer cells to initiate metastases. The relationship between serum E-selectin levels and prognoses in 101 patients with resected non-small cell lung cancers (NSCLCs) was studied, and survival curves were compared in relation to E-selectin levels and expression of two carbohydrate antigens, Sialyl Lewisx (SLX) and Sialyl Lewisa (CA19-9), which were immunohistochemically detected in resected specimens in 65 of the 101 cases. The serum E-selectin level on admission was 48.9+/-25.7 ng/ml (mean+/-SD, n=101), and the E-selectin-positive rate was 22.7%, being correlated with the progression of T-factor. The high E-selectin group showed a significantly worse survival rate than the normal E-selectin group. Multivariate analysis confirmed the significant prognostic value of E-selectin. The mean postoperative E-selectin level in 52 cases (36.93 ng/ml) was significantly lower than the preoperative E-selectin level (43.57 ng/ml), indicating that certain NSCLCs might induce the expression of E-selectin. In cases expressing carbohydrate antigens (SLX, CA19-9), the high E-selectin group showed a significantly worse survival curve than the normal E-selectin group. On the other hand, there was no significant difference in the survival curve between the high and normal E-selectin groups when carbohydrate antigens were negative. These results suggest that patients who have high serum E-selectin levels, especially with carbohydrate antigen-positive NSCLC, might be expected to have poor prognoses.

Adult↗
PubMed1999

[Trans-sternal bilateral thoracotomy for bilateral metastatic lung tumors].

Trans-sternal bilateral thoracotomy has been performed in 4 patients with bilateral metastatic lung tumors. The primary lesion was rectum in one case and the colon in three cases. Two patients were male and 2 were female. Their ages ranged from 63 to 78 years old. An inframammary incision was made with the patient in the supine position, and the chest was entered via the 4th or 5th intercostal space. This procedure provided excellent exposure of the entire mediastinum. Postoperative pain was comparatively severe in the early postoperative period, but was controlled by administering an epidural block. This procedure allows good exposure of the entire thoracic space through a single operative field. We conclude that trans-sternal bilateral thoracotomy is an excellent approach in the patient with bilateral metastatic lung tumors.

Aged↗