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Biomedical subjects

T Yoshimura

Publications and source records attributed to T Yoshimura.

At least 559 records · Page 31Linked to original sources
PubMed1990

A multiwell cell settling and adherence chamber for morphology and differential counting.

A simple multiwell chamber is described that can be used to prepare randomly distributed cells on a microscope slide, suitable for morphological identification and differential counting. To the eight wells of the chamber are added 50-microliter volumes of cell suspension at concentrations of 10(3)-10(6) cells/ml. As the cells settle, fluid is slowly wicked away by a damp filter paper sandwiched between the microscope slide and the acrylic top plate of the multiwell chamber. Within 20-40 minutes, the cell monolayers on the slide are completely dry. The combined settling and bulk fluid removal results in a distribution of adherent cells that are sufficiently spread to exhibit excellent morphology after staining. If the chamber is centrifuged for 30 seconds at 50x g immediately after addition of cells, recovery of cells in the monolayer is virtually 100%, and as few as 50 input cells per 50 microliters can be detected. Agreement between predicted and observed differential counts of cell mixtures indicates that cells in the monolayer were distributed randomly.

Cell Adhesion↗
PubMed1990

Role of phosphatidylserine in membrane actions of tumor necrosis factor and interferons alpha and gamma.

For elucidation of the role of phosphatidylserine (PS) in the membrane actions of human recombinant tumor necrosis factor (TNF) and interferons (IFNs) alpha and gamma, the actions of these cytokines on liposome membranes containing PS were compared with those on liposome membranes containing two other anionic phospholipids, phosphatidylglycerol (PG) and phosphatidic acid (PA). At pH 4.0 or 4.5, where these three cytokines were bound almost completely to the liposome membranes, (1) the ratios of the rates of membrane action of TNF and IFN-alpha on PS-containing liposomes to those on PG- or PA-containing liposomes were similar, but always greater than the ratio of the former to the latter rates of IFN-gamma, and (2) progressive replacement of either PG or PA in membranes by PS at a constant anionic phospholipid concentration decreased or increased the rates of membrane action of TNF and IFN-alpha but only decreased that of IFN-gamma, suggesting that membrane PS modulates the membrane actions of these three cytokines. The possible role of PS in the cytotoxic actions of these cytokines on tumor cells is discussed.

Humans↗
PubMed1990

Cytokine-activated human endothelial cells synthesize and secrete a monocyte chemoattractant, MCP-1/JE.

We have demonstrated inducible expression of the mRNA encoding the monocyte chemoattractant MCP-1, the human homolog of the JE gene, in endothelial cells within 3 hours of treatment with IL-1 beta and tumor necrosis factor. IFN-gamma also induced expression of this mRNA after 24 hours, but to a lesser extent. MCP-1/JE protein steadily accumulated in the medium of endothelial cells during a 48-hour exposure to IL-1 beta. Medium conditioned by IL-1 beta-treated endothelial cells contained monocyte chemoattractant activity that was immunoadsorbed by anti-MCP-1 antibodies. These results suggest that endothelial cells secrete a monocyte chemoattractant, MCP-1/JE, in response to inflammatory mediators, and thus may contribute to the accumulation of monocytes at sites of inflammation.

Biological Factors↗
PubMed1990

[Possible involvement of leukotriene B4 on human luteal function: special reference to determination of leukotriene B4 produced by cultured luteal cells].

The present study was undertaken to determine the production of leukotriene B4 (LTB4) by cultured human luteal cells in the mid-luteal phase using a reverse phase column (C8). The luteal cells were cultured with or without hCG at 100 ng/ml for 8 days. In the preliminary experiment, methods for extracting culture media samples were assessed prior to radioimmunoassay. Reverse phase column C8, but not C18, made possible the determination of LTB4 produced by human luteal cells. Progesterone (P) production by cultured luteal cells reached its maximum on day 4 following exposure to hCG, and then declined gradually. The concentrations of LTB4 produced by luteal cells varied from 100 to 500 pg/10(5) cells/2 days. However, exposure to hCG did not affect LTB4 production by cultured luteal cells. The level of LTB4 in culture medium (115.0 +/- 37.8 pg/10(5) cells/2 days) was reduced on day 4, but increased thereafter. LTB4 production appeared to decrease concomitantly with increased P production of cultured luteal cells. In conclusion, cultured luteal cells produced considerable amounts of LTB4 throughout the entire culture period. These results suggest that lipoxygenase activity of luteal cells may be closely related to steroidogenic potential.

Adult↗
PubMed1990

[The effect of partial hepatectomy on glucose-stimulated insulin release from the isolated perfused pancreas in rats].

To investigate the functional changes of endocrine pancreas in the early stage after partial hepatectomy, we evaluated the changes in the secretion of insulin to glucose load in perfused rat pancreas at 4 and 7 days after about 70% hepatectomy. Insulin responses to glucose load were significantly augmented at 4 days compared with both the normal and sham-operation rats and at 7 days also significantly augmented compared with the normal rats. These results are in accordance with the morphological changes of islet and indicate that in the early stage after hepatectomy, the response of islet B-cells to glucose load are increased in order to support the glucose metabolism in this stage. Thus, insulin seems to play an important role in the regenerating liver after hepatectomy and these changes seem to be one of the adaptation of endocrine pancreas after hepatectomy to support the regenerating liver.

Animals↗
PubMed1990

[A dietary method for epidemiological study of cancer].

A semiquantitative frequency questionnaire was developed to estimate the average intake of foods and nutrients of individuals with the aim of utilization in cancer epidemiology. Validity of the questionnaire was evaluated in comparison with four 7-day dietary records carried on 25 spouses over one year. Absolute mean intakes estimated by the questionnaire and by the records were roughly comparable at the group level. However, the individual's estimated intakes generally showed poor agreements between the two methods in terms of rank correlation, except for a few foods such as fish and milk and for alcoholic beverages. Further efforts should be exercised to develop a more accurate dietary method.

Age Factors↗
PubMed1990

[Blastic crisis of primary myelofibrosis associated with multiple myeloblastomas ].

The patient is a 71-year-old female who underwent splenectomy due to splenomegaly 32 months after diagnosed as having primary myelofibrosis. On examination she was found to have massive skin nodules, lymph nodes swelling and an enlarged liver with an abnormal hematologic profile as follows: RBC count 3.68 x 10(6)/microliters; WBC count 151 x 10(3)/microliters with 11% blasts; and platelet count 42 x 10(3)/microliters. The bone marrow aspirate showed a hypocellular marrow with 19.2% blasts. Histological examination of the skin nodules revealed that they were myeloblastomas, thus suggesting leukemic transformation of primary myelofibrosis. Her WBC count dropped to about 20 x 10(3)/microliters through treatment with vindesine, cyclophosphamide, 6-mercaptopurine and prednisolone, but it did not drop further. Treatment with dexamethasone remarkably regressed the myeloblastomas, but she died of heart failure 4 months after diagnosis of leukemic transformation of primary myelofibrosis. The autopsy findings showed the formation of numbers of myeloblastomas in both the systemic fatty tissue and dura mater as well as extramedullary hematopoiesis in liver and lymph nodes. A rapid development of splenomegaly in a patient with primary myelofibrosis seems to be associated with leukemic transformation.

Aged↗
PubMed1989

Spectroscopic evidence for a copper-nitrosyl intermediate in nitrite reduction by blue copper-containing nitrite reductase.

The reactions of nitrogen monoxide (NO) with the blue copper-containing nitrite reductases from Alcaligenes sp. NCIB 11015 and Achromobacter cycloclastes IAM 1013 were investigated spectroscopically. The electron paramagnetic resonance (EPR) signals of the blue coppers vanished in the presence of NO at 77 K, being fully restored by the removal of NO. The additions of NO to the enzyme solutions resulted in the substantial bleaching of the visible absorption bands at room temperature. The reactions were also completely reversible. These results suggest the formation of a cuprous nitrosyl complex (Cu+-NO+), which is likely the intermediate in the enzymatic nitrite reduction.

Alcaligenes↗
PubMed1989

Rapid homogeneous enzyme immunoassay of plasma protein C.

A homogeneous EIA (H-EIA) technique for plasma protein C antigen using horseradish peroxidase (HRP) was developed. This technique, which avoids the bound/free separation and washing procedures and shortens the assay time to 30 min, has several advantages over the conventional EIA technique. The measurement was valid for 1.0% to 200% of the plasma protein C level and had intraassay coefficients of variation of 2.3-11.0%. The results of this assay correlated well with those of conventional sandwich EIA (x) with a regression equation of y = 0.782x + 6.637, r = 0.911 (n = 59). This method, which may also be applicable to automatic assay systems, is considered to be suited for clinical laboratory use.

Antigens↗
PubMed1989

Molecular cloning of cDNA for proteasomes (multicatalytic proteinase complexes) from rat liver: primary structure of the largest component (C2).

Proteasomes (multicatalytic proteinase complexes) from rat liver are composed of at least 13 nonidentical components [Tanaka, K., Yoshimura, T., Ichihara, A., Ikai, A., Nishigai, M., Morimoto, M., Sato, M., Tanaka, N., Katsube, Y., Kameyama, K., & Takagi, T. (1988) J. Mol. Biol. 203, 985-996]. The nucleotide sequence of one major component (C2) of the proteasomes has been determined from a recombinant cDNA clone isolated by screening a rat liver cDNA library with a mixture of synthetic deoxyribonucleotides as a probe. The sequence was composed of 1174 nucleotides including a coding region for the entire protein and noncoding regions of both the 5'- and 3'-sides. The polypeptide deduced from the open reading frame consisted of 263 amino acid residues, and its molecular weight was calculated to be 29,516. The partial amino acid sequences of several fragments (approximately 45% of the total residues), which were obtained by cleavage of C2 with lysyl endopeptidase and cyanogen bromide, were determined by automated Edman degradation and found to be in complete accordance with those deduced from the cDNA sequence. The amino acid composition of C2, determined by chemical analysis, was also consistent with that deduced from the cDNA sequence, indicating that the cloned cDNA actually encoded component C2. Computer analysis revealed little structural similarity of C2 to other proteins reported so far. Northern blot hybridization analyses showed that the mRNA encoding this novel protein C2 was expressed in all the rat tissues examined and in a variety of eukaryotic organisms such as amphibia, birds, and mammals with slight species-specific differences in size.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1989

Hormonal regulation of synthesis and secretion of pS2 protein relevant to growth of human breast cancer cells (MCF-7).

We have recently identified human epidermal growth factor-like immunoreactive factor synthesized and secreted by human breast cancer cells (MCF-7) as a secretory protein encoded by the pS2 gene, the transcription of which is directly induced by estrogen. We demonstrated in this paper that synthesis and secretion of pS2 protein as well as pS2 mRNA were induced about 5-fold specifically by physiological concentrations of estrogen, which stimulated growth of the cells about 5-fold. Stimulative effects of estrogen on both cell growth and synthesis/secretion of pS2 protein were inhibited completely by actinomycin D, cycloheximide, and antiestrogen. However, the increase in DNA synthesis from 6 h after the start of treatment of the cells with estrogen preceded the increase in the amount of pS2 protein in the culture medium from 12 h after that. Furthermore purified pS2 protein did not stimulate DNA synthesis of the cells. These results suggest that induction of pS2 protein by estrogen is not involved in the growth-stimulating effect of estrogen in MCF-7 cells.

Androgens↗
PubMed1989

Purification and amino acid analysis of two human glioma-derived monocyte chemoattractants.

Two chemoattractants for human monocytes were purified to apparent homogeneity from the culture supernatant of a glioma cell line (U-105MG) by sequential chromatography on Orange A-Sepharose, an HPLC cation exchanger, and a reverse phase HPLC column. On SDS-PAGE gels under reducing or nonreducing conditions, the molecular masses of the two peptides glioma-derived chemotactic factor 1 and 2 were 15 and 13 kD, respectively. Amino acid composition of these molecules was almost identical, and differed from other cytokines that have been reported. The NH2 terminus of each peptide was apparently blocked. When tested for chemotactic efficacy, the peptides attracted approximately 30% of the monocytes added to chemotaxis chambers, at the optimal concentration of 10(-9) M. Potency and efficacy were comparable with that of FMLP, which is often used as a reference attractant. The activity was chemotactic rather than chemokinetic. In contrast to their interaction with human monocytes, the pure peptides did not attract neutrophils. These pure tumor-derived chemoattractants can now be compared with attractants produced by normal cells and evaluated for their biological significance in human neoplastic disease.

Amino Acids↗
PubMed1989

Purification and amino acid analysis of two human monocyte chemoattractants produced by phytohemagglutinin-stimulated human blood mononuclear leukocytes.

Physicochemical characteristics of monocyte chemotactic activity in the culture fluid of PHA-stimulated human mononuclear leukocytes (MNL) were investigated. Among several chemotactic activity peaks eluted from a TSK-2000 gel filtration column, one peak, corresponding to a molecular mass of 17 kDa, accounted for about 40% of total chemotactic activity. On a chromatofocusing column, most of the 17-kDa activity eluted in a pH range of 9.4 to 7.9. It could bind to Orange-A Sepharose. These three characteristics--molecular mass, basic isoelectric point, and dye column binding--were similar to those of human glioma-derived monocyte chemotactic factor (GDCF), recently purified in our laboratory. Therefore, the MNL-derived chemoattractant was purified by the same procedures used for purification of GDCF, namely Orange-A Sepharose chromatography, carboxymethyl (CM)-HPLC, and reverse phase (RP) HPLC. About 50% of the culture fluid chemotactic activity bound to Orange-A Sepharose and was eluted in a single peak by a NaCl gradient. The active pool from the Orange-A column was separated into two sharp peaks by CM-HPLC, each of which eluted at identical acetonitrile concentrations from a RP HPLC column. By SDS-PAGE, the peptides had apparent molecular masses of 15 and 13 kDa and appeared homogeneous. Amino acid analysis showed that the composition of the two peptides was almost identical; and the N terminus of each peptide was apparently blocked. Shared characteristics of these peptides and the GDCF peptides include identical elution patterns from CM- and RP HPLC columns, identical SDS-PAGE migration, almost identical amino acid composition, and blocked N terminus. This suggests that the monocyte attractants isolated from culture fluid of PHA-stimulated MNL are identical to those derived from human glioma cells.

Amino Acids↗
PubMed1989

Production and characterization of human glioma cell-derived monocyte chemotactic factor.

Since infiltration of monocytes into tumors may be mediated by tumor-derived chemoattractants, we characterized the monocyte-chemotactic activity (MCA) produced by glioma cell lines. The amount of MCA in the culture fluid of five lines tested differed by a factor of 25. U-105MG, the best producer, was selected for further study. After cells reached confluence and the medium was changed, MCA was detected by day 3 and remained at comparable levels on days 4 and 5. The molecular mass of MCA was approximately 17 kilodaltons, and the estimated isoelectric point ranged between pI 7 and pI 9. Because of the high constitutive production of MCA by U-105MG, sufficient material can be obtained for complete chemical characterization of this mediator of inflammation.

Brain Neoplasms↗
PubMed1989

Human monocyte chemoattractant protein-1 (MCP-1). Full-length cDNA cloning, expression in mitogen-stimulated blood mononuclear leukocytes, and sequence similarity to mouse competence gene JE.

The purpose of this work was to analyze cDNA encoding human monocyte chemoattractant protein-1 (MCP-1), previously isolated from glioma cell line culture fluid. Screening of a cDNA library from total poly(A) RNA of glioma cell line U-105MG yielded a clone that coded for the entire MCP-1. Nucleotide sequence analysis and comparison with the amino acid sequence of purified MCP-1 showed that the cDNA clone comprises a 53-nucleotide 5'-non-coding region, an open reading frame coding for a 99-residue protein of which the last 76 residues correspond exactly to pure MCP-1, and a 389-nucleotide 3'-untranslated region. The hydrophobicity of the first 23 residues is typical of a signal peptide. Southern blot analysis of human and animal genomic DNA showed that there is a single MCP-1 gene, which is conserved in several primates. MCP-1 mRNA was induced in human peripheral blood mononuclear leukocytes (PBMNLs) by PHA, LPS and IL-1, but not by IL-2, TNF, or IFN-gamma. Among proteins with similar sequences, the coding regions of MCP-1 and mouse JE show 68% identity. This suggest that MCP-1 is the human homologue of the mouse competence gene JE.

Amino Acid Sequence↗
PubMed1989

Mechanism of protein-induced membrane fusion: fusion of phospholipid vesicles by clathrin associated with its membrane binding and conformational change.

The clathrin-induced fusion of liposome membranes, the membrane binding of clathrin, and the conformational states of clathrin were investigated over a wide pH range using large unilamellar and multilamellar vesicles composed of phosphatidylserine (PS), phosphatidylcholine (PC), PS/PC (2:1), PS/PC (1:1), or PS/PC (1:2). The pH profiles of clathrin-induced fusion of all types of liposomes containing PS showed biphasic patterns. Their pH thresholds were found in the pH range of 5-6 and shifted to lower pH values with decrease in the PS content. Similar shifts were observed in the pH range of 5-6 and shifted to lower pH values with decrease in the PS content. Similar shifts were observed in the pH profiles of clathrin binding to these vesicles, but the pH profiles of binding were different from the biphasic fusion patterns. With PC vesicles, only small degrees of fusion and clathrin binding were observed at pH 2-4. The pH dependences of the conformation and hydrophobicity of clathrin were determined by measuring the extent of the blue shift of the fluorescence maximum of 1-anilinonaphthalene-8-sulfonate in the presence of the protein, the fluorescence intensity of N-(1-anilinonaphthyl-4)maleimide bound to the clathrin molecule, the resonance energy transfer from its tryptophan to anilinonaphthyl residues, the partitioning of the protein in Triton X-114 solution, and the hydrophobicity index of clathrin using cis-parinaric acid. These measurements indicated that conformational change and exposure of hydrophobic regions occur below pH 6 and suggested that clathrin may adopt different conformational states in the pH region where it induced membrane fusion.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗