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T Yoshimura

Publications and source records attributed to T Yoshimura.

At least 541 records · Page 30Linked to original sources
PubMed1990

[Activities of enzymes converting 5-fluorouracil to 5-fluorouridine-5' monophosphate and 5-fluorodeoxyuridine-5' monophosphate in subcultured cell lines and solid tumor tissues].

The activities of five enzymes, orotate phosphoribosyltransferase (OPRTase), uridine kinase (UR kinase), thymidine kinase (TdR kinase), uridine phosphorylase (UR Prylase) and thymidine phosphorylase (TdR Prylase), were examined in subcultured human acute leukemia cell lines (HL-60, CCRF-CEM), subcultured human solid tumor cell lines (Colo-205, HeLa-S3) and human cancerous tissues with a view to compare the activation of 5-fluorouracil in them. There was no significant difference in the activity of any enzyme between HL-60 and CCRF-CEM, Colo-205 and HeLa-S3, and human lung cancerous tissue and human colon cancerous tissue. Compared between the acute leukemia cell lines and the solid tumor cell lines, the UR kinase activity was high in both cell lines. The OPRTase and UR Prylase activities were low in the solid tumor cell lines. In the cancerous tissues, both the UR kinase and TdR kinase activities were low, but the UR Prylase and TdR Prylase activities were markedly high. The results suggest that the intracellular activation of 5-fluorouracil varies with different human cancerous cells. When the anti-cancer activity of 5-fluorouracil is tested in vitro, the difference of fluoropyrimidine metabolism in subcultured cell lines from that in the cancerous tissue should be taken in account.

Fluorouracil↗
PubMed1990

Molecular cloning of cDNA for proteasomes from rat liver: primary structure of component C3 with a possible tyrosine phosphorylation site.

Proteasomes are multicatalytic proteinase complexes consisting of multiple components. Previously, we reported the cloning and sequencing of cDNA for the largest component, C2, of rat liver proteasomes [Fujiwara, T., Tanaka, K., Kumatori, A., Shin, S., Yoshimura, T., Ichihara, A., Tokunaga, F., Aruga, R., Iwanaga, S., Kakizuka, A., & Nakanishi, S. (1989) Biochemistry 28, 7332-7340]. In the present study, the nucleotide sequence of another component (C3) of proteasomes has been determined from a recombinant cDNA clone isolated by screening a rat liver cDNA library with synthetic oligodeoxynucleotide probes corresponding to partial amino acid sequences of the protein. The deduced sequence of component C3 consists of 234 amino acid residues with a calculated molecular weight of 25,925. These values are consistent with those obtained by protein chemical analyses. A single mRNA species hybridizing to the C3 cDNA of rat liver was expressed in all rat tissues examined and in a variety of other eukaryotic organisms, its distribution being similar to that of C2 mRNA. The wide distribution of the gene product, possibly C3, suggests that this protein functions similarly in most eukaryotes. C3 is an unmodified protein of a single gene and differs from any other known protein, but its overall amino acid sequence resembles those of other proteasomal components, including C2, suggesting that these components belong to a single family of proteins with the same evolutionary origin.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1990

Secretion by human fibroblasts of monocyte chemoattractant protein-1, the product of gene JE.

We recently purified human monocyte chemoattractant protein-1 (MCP-1) from culture fluids of either human glioma cell lines or mitogen-stimulated human peripheral blood mononuclear leukocytes. It has now been shown that MCP-1 is the product of the gene JE, which was first recognized by its expression in fibroblasts stimulated with platelet-derived growth factor (PDGF). We therefore studied secretion of MCP-1 by three human fibroblast cell lines. Monocyte chemotactic activity was found in culture fluids of all three lines after growth to confluence in DMEM-10% FCS, and the amounts secreted per cell were comparable for the three lines. The MRC-5 line was chosen for further study. Monocyte chemotactic activity secretion by confluent MRC-5 cultures continued after a switch to serum-free medium and was not inhibited by anti-PDGF antibody, indicating that secretion may not have been caused by autocrine release of PDGF. When concentrated serum-free MRC-5 culture fluid was injected into an HPLC gel filtration column, only one chemotactic activity peak was observed, which was in the same location as glioma-derived MCP-1. The activity was completely absorbed out by an anti-MCP-1 affinity column, which indicates that all the chemotactic activity in MRC-5 culture fluid was accounted for by MCP-1. PDGF caused a marked increase in chemotactic activity over that found in serum-free culture fluid of MRC-5 or 501T cells. Immunoprecipitation by anti-human MCP-1 showed two bands, corresponding to the two forms of MCP-1 previously described (MCP-1 alpha and beta); and the amounts increased in response to PDGF stimulation. Thus, the reported increase in human fibroblast JE mRNA in response to PDGF-containing serum stimulation is reflected in increased secretion of the MCP-1 gene product.

Biological Assay↗
PubMed1990

Determination of the regions of the clathrin molecule inducing membrane fusion.

Clathrin induces fusion of liposome membranes containing phosphatidylserine at acidic pH [Maezawa, S., Yoshimura, T., Hong, K., Düzgüneş, N., & Papahadjopoulos, D. (1989) Biochemistry 28, 1422-1428]. The regions of the clathrin molecule inducing membrane fusion were determined by examining the fusion abilities of clathrin fragments obtained by limited proteolysis of clathrin cages with thermolysin. Membrane fusion was assessed by resonance energy transfer assay in terms of the dilution of fluorescent phospholipids in liposome membranes. Proteolysis of clathrin decreased the fusion rate and the amount of protein but did not affect the specific fusion rate (i.e., the fusion rate per unit of protein), indicating that clathrin fragments retain the ability to induce fusion. Of the two proteolytic fragments of the clathrin heavy chain, the terminal domain and the residual proximal part, which were separated by ultracentrifugation or gel chromatography, only the proximal part showed fusion activity. Light chains seemed to have no role in membrane fusion, since they are susceptible to proteolytic digestion. The terminal domain induced reversible liposome membrane aggregation, which was also induced by the residual proximal part of the heavy chain and the whole molecule of clathrin. These results suggest that the terminal domain and the proximal portion of clathrin have critical roles in the steps of close apposition and fusion of membranes, respectively.

Animals↗
PubMed1990

Leukocyte specificity and binding of human neutrophil attractant/activation protein-1.

Neutrophil attractant/activation protein-1 (NAP-1) was previously shown to attract human neutrophils, but not monocytes. The purpose of this study was to determine if NAP-1 interacted with other types of blood leukocytes. In addition to its chemotactic activity for neutrophils, NAP-1 induced chemotactic responses by T lymphocytes and basophils. Chemotactic potency (10(-8) M for an optimal response) was the same for all three cell types. However, NAP-1 caused a chemotactic response in excess of random migration of 7% or 16% of basophils (depending on the medium used) and only 9% of T lymphocytes, in contrast to 30% of neutrophils. This agonist was not chemotactic for partially purified normal human eosinophils. The symmetrical histogram obtained by flow cytometry of neutrophils equilibrated at 0 degree C with fluoresceinated NAP-1 indicates that all neutrophils bound the ligand. A dose-response curve plateau, and inhibition of binding of NAP-1-FITC by unlabeled ligand are evidence for saturable binding to receptors, estimated to be 7000 per cell. Our results suggest that, for induction of an acute inflammatory response, the quantitatively significant action of NAP-1 is on neutrophils.

Basophils↗
PubMed1990

Multiple cDNA clones encoding nuclear proteins that bind to the tax-dependent enhancer of HTLV-1: all contain a leucine zipper structure and basic amino acid domain.

A trans-activator protein, p40tax, of human T cell leukemia virus type 1 (HTLV-1) activates its own promoter and cellular promoters of IL-2, IL-2 receptor alpha and GM-CSF genes. We isolated three cDNA clones encoding cellular proteins that bind to the p40tax-dependent enhancer of HTLV-1 by screening a lambda gt11 cDNA library of an HTLV-1 infected cell line. All three proteins, TREB5, TREB7 and TREB36, contained a leucine zipper structure and basic amino acid domain, which are conserved in FOS, JUN and CREB, and also had multiple potential phosphorylation sites. The proteins expressed in Escherichia coli bound to the p40tax-dependent enhancer of the 21 bp sequence, but not to an inactive mutant carrying a mutation in the CRE region. In DNase I footprint analysis, all three proteins protected the 21 bp sequences in the LTR; however, the patterns were not identical to each other. TREB7 and TREB36 protected all three repeats of the 21 bp, but TREB5 protected only the second repeat. TREB7 and TREB36 protected the 5' and middle portions of the 21 bp which are essential for p40tax-mediated trans-activation, whereas TREB5 and CREB1 protected a narrower part of the middle region of the second 21 bp repeat containing the CRE consensus sequence. These structural features and DNA binding properties suggest that TREB proteins are members of a CREB protein family and that some of them (i.e., TREB7 and TREB36) may be involved in p40tax-mediated trans-activation.

Amino Acid Sequence↗
PubMed1990

Accumulation of galactosylsphingosine (psychosine) does not interfere with phosphorylation and methylation of myelin basic protein in the twitcher mouse.

In attempts to elucidate mechanisms of demyelination in the twitcher mouse (Twi), phosphorylation and methylation of myelin basic protein (MBP) were examined in the brainstem and spinal cord of this species. Phosphorylation of MBP in isolated myelin by an endogenous kinase and an exogenous [32P]ATP was not impaired and protein kinase C activity in the brain cytosol was not reduced. When the methylation of an arginine residue of MBP was examined in slices of the brainstem and spinal cord, using [3H]methionine as a donor of the methyl groups, no difference was found between Twi and the controls. Radioactivity of the [3H] methionine residue of MBP of Twi was also similar to that of the controls. Thus, accumulation of psychosine in Twi does not interfere with the activity of endogenous kinase, methylation of MBP, and the synthesis and transport of MBP into myelin membrane.

Adenosine Triphosphate↗
PubMed1990

Difference between histidine and histamine in the mechanistic pathway of the fluorescence reaction with ortho-phthalaldehyde.

Histamine reacts with ortho-phthalaldehyde (OPA) in an alkaline medium to form an unstable fluorescent adduct (Fbase-Hm). Acidification of the solution to pH 2-4 gives a stable and highly fluorescent adduct (Facid-Hm). In contrast, histidine develops a relatively stable fluorescent adduct (Fbase-Hd) with OPA in an alkaline medium. Upon acidification of the solution, however, only trace amounts of the alternative fluorescent adduct (Facid-Hd) are produced although Fbase-Hd disappears similarly to Fbase-Hm. In this paper, the reaction pathway of histidine with OPA was clarified by the kinetic analysis of formation and degradation of Fbase-Hd. In comparing the results of this study with the previous ones for histamine (T. Yoshimura et al., 1987, Anal. Biochem., 164, 132-137), we elucidate the difference between histamine and histidine in the mechanism of fluorescence reaction with OPA. The presence of the carboxyl group in histidine not only stabilizes Fbase-Hd in an alkaline medium but also prevents the formation of a 1:2 adduct of histidine and OPA, the precursor of Facid-Hd.

Chemical Phenomena↗
PubMed1990

Human monocyte chemoattractant protein-1 (MCP-1).

During the past three years great advances have been made in the chemistry and biology of chemoattractants for human leukocytes. Two chemoattractant cytokines have been isolated, sequenced and cloned, each with distinctive leukocyte attractant specificity. Monocyte chemoattractant protein 1 (MCP-1), the subject of this review by Edward Leonard and Teizo Yoshimura, is secreted by PHA-stimulated mononuclear cells and can be identified by northern blotting in response to LPS or PHA. It attracts monocytes but not neutrophils. In contrast, neutrophil attractant/activation protein (NAP-1) (also known as interleukin 8 (IL-8)) attracts and activates human neutrophils but it is not a chemoattractant for human monocytes. Based on amino acid sequence analysis, each of these attractants has been assigned to one of two distinct families of cytokines that are thought to participate in host defense and inflammatory responses.

Amino Acid Sequence↗
PubMed1990

Stereospecific abstraction of epsilon-pro-R-hydrogen of L-lysine by L-lysine epsilon-dehydrogenase from Agrobacterium tumefaciens.

The stereochemical aspects of the L-lysine epsilon-dehydrogenase reaction were examined with (6R)-L-[6-3H]lysine and (6S)-DL-[6-3H]lysine. When (6S)-DL-[6-3H]lysine was used as a substrate, the tritium was found in the product, delta 1-piperideine-6-carboxylate. In contrast, the radioactivity from (6R)-L-[6-3H]lysine was not retained in the product. Thus, the pro-R hydrogen at the prochiral C-6 carbon of L-lysine is specifically abstracted by the enzyme: the enzyme behaves stereochemically as an amino acid D-dehydrogenase.

Amino Acid Oxidoreductases↗
PubMed1990

Substitution of S-(beta-aminoethyl)-cysteine for active-site lysine of thermostable aspartate aminotransferase.

The active site lysyl residue (K239) of the thermostable aspartate aminotransferase [EC 2.6.1.1] was replaced by cysteinyl residue by means of site-directed mutagenesis. The K239C mutant enzyme obtained was catalytically inactive. The reaction of the cysteinyl residue of the K239C mutant enzyme with ethylenimine led to the formation of S-(beta-aminoethylcysteinyl (SAEC) residue. The K239SAEC mutant enzyme obtained showed about 25% of the activity of wild-type enzyme, and absorbed at 375 nm, which suggested the internal Schiff base formation.

Amino Acids↗
PubMed1990

Angiotensin II and alpha-agonist. I. Responses of ovine fetoplacental vasculature.

During ovine pregnancy the uteroplacental vasculature is less responsive to angiotensin II (ANG II)-induced vasoconstriction than the systemic vasculature, whereas responses to alpha-agonists are just the opposite. Comparisons of fetal systemic and placental vascular responses to these agents are not well described, nor have they been compared with maternal responses. We determined steady-state responses to fetal infusions (5-7 min) of ANG II (0.023-5.73 micrograms/min) and phenylephrine (PHEN, 0.031-7.64 micrograms/min), continuously monitoring mean arterial pressure (MAP), heart rate (HR), and umbilical blood flow (UmBF). Although both vasoconstrictors caused dose-dependent increases in MAP and umbilical vascular resistance (UmVR), responsiveness (delta MAP and delta UmVR) to ANG II (mol/min) was 35- to 60-fold greater than to PHEN. ANG II caused dose-dependent decreases in UmBF (2-48%); PHEN had minimal effects except at the highest dose, UmBF decreasing only 18%. Although patterns of fetal responses of MAP, UmBF, and UmVR to ANG II resembled maternal responses of MAP and uterine blood flow and uterine vascular resistance, the former were greatly attenuated. Similar observations were made with PHEN for UmBF and UmVR but not MAP. ANG II is a more potent fetal systemic and placental vasoconstrictor than PHEN; however, compared with those of the mother the responses are attenuated. Moreover, the fetoplacental vascular bed appears unresponsive to alpha-adrenergic stimulation, possibly reflecting a mechanism for maintaining UmBF when plasma catecholamines are elevated.

Adrenergic alpha-Agonists↗
PubMed1990

Angiotensin II and alpha-agonist. II. Effects on ovine fetoplacental prostaglandins.

The fetoplacental vasculature is more sensitive to angiotensin II (ANG II) than to alpha-agonists, possibly reflecting their differing effects on vascular prostaglandin (PG) production. To examine this we studied in fetal sheep (123-138 days) the effects of ANG II (n = 7; 0.057, 1.15, and 5.73 micrograms/min) and phenylephrine (PHEN, n = 7; 0.306, 1.53, and 7.64 micrograms/min) on mean arterial pressure (MAP), heart rate (HR), and simultaneous measurements of umbilical arterial and venous PGE2, 6-keto-PGF1 alpha (PGI2) and thromboxane B2 (TxB2) concentrations. ANG II increased MAP and decreased HR dose dependently (P less than 0.05). Basal umbilical venous plasma PGE2 levels exceeded PGI2 (P less than 0.001) and increased during ANG II infusions from 502 +/- 63 to 516 +/- 113, 647 +/- 188, and 1,968 +/- 541 pg/ml (mean +/- SE, P less than 0.05), as did venous-arterial concentration differences (129 +/- 26 to 179 +/- 47, 244 +/- 58, and 1,287 +/- 507 pg/ml, respectively; P less than 0.05). ANG II also increased umbilical venous PGI2 levels from 110 +/- 13 to 116 +/- 24, 144 +/- 46, and 680 +/- 147 pg/ml (P less than 0.01) and the venous-arterial concentration difference from 3 +/- 6 to 20 +/- 16, 41 +/- 27, and 405 +/- 122 pg/ml (P less than 0.05), respectively. ANG II had no effect on plasma TxB2, and no umbilical venous-arterial concentration differences existed. Although PHEN increased MAP and decreased HR, plasma eicosanoid concentrations were unaltered. The fetus is more sensitive to ANG II than PHEN; however, only ANG II increased placental PGE2 and PGI2 production.(ABSTRACT TRUNCATED AT 250 WORDS)

Adrenergic alpha-Agonists↗
PubMed1990

Secretion of neutrophil attractant/activation protein by lipopolysaccharide-stimulated lung macrophages determined by both enzyme-linked immunosorbent assay and N-terminal sequence analysis.

Alveolar macrophages contribute to acute pulmonary inflammation by secretion of neutrophil chemoattractants. We determined if one of these attractants is neutrophil attractant/activating protein (NAP-1), which is secreted by blood monocytes stimulated by lipopolysaccharide (LPS). Alveolar macrophages were stimulated in tissue culture with 10 micrograms/ml LPS. Culture fluids collected at 24 h were assayed for both neutrophil chemotactic activity and the concentration of NAP-1 as determined by a sandwich ELISA. The concentration of NAP-1 in culture fluid to LPS-stimulated macrophages was 860 +/- 40 ng/ml (SEM for six normal subjects). NAP-1 in fluid of unstimulated macrophages was 40 +/- 15 ng/ml. We confirmed the presence of NAP-1 in culture fluid of LPS-stimulated lung macrophages by immunoaffinity and HPLC-CM column purification. The HPLC-CM elution profile of macrophage NAP-1 was identical to that of monocyte NAP-1, and the N-terminal sequence of the protein in one of the isolated peaks corresponded to that of monocyte-derived NAP-1 beta. Two lines of evidence show that NAP-1 does not account for all neutrophil chemotactic activity in culture fluid of 24-h, LPS-stimulated macrophages. At a dilution of culture fluid that elcited the same chemotactic response as a known concentration of pure NAP-1, the concentration of culture fluid NAP-1 was only one-tenth that of pure NAP-1. (ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1990

Neutrophil attractant/activation protein-1 (NAP-1 [interleukin-8]).

Neutrophil attractant/activation protein-1 (NAP-1 [interleukin-8]) is an 8,400 D protein that is a chemoattractant and granule release stimulus for neutrophils. NAP-1 was first purified from culture fluids of lipopolysaccharide-stimulated human blood mononuclear leukocytes. It was subsequently isolated from lipopolysaccharide-stimulated lung macrophages, mitogen-stimulated lymphocytes, and virus-infected fibroblasts. Interleukin-1 or tumor necrosis factor induces NAP-1 mRNA in many cells, including monocytes, fibroblasts, and endothelial cells. NAP-1 belongs in a family of host defense small proteins, which have a degree of sequence and structural similarity. Noteworthy are the four half-cystine residues in each protein, which are in register when the protein sequences are suitably aligned. Based on cloning data and N-terminal sequence analyses, NAP-1 is secreted as a 79 residue protein after cleavage of a 20 residue signal peptide. The commonly isolated 77 and 72 residue forms are probably extracellular cleavage products. NAP-1 has considerable charge heterogeneity. Charge and length variants all have chemotactic activity. In contrast to many chemoattractants, NAP-1 does not attract monocytes. Intradermal injection of NAP-1 causes neutrophil infiltration. The wide spectrum of cell sources and production stimuli suggests that NAP-1 mediates neutrophil recruitment in host defense and disease.

Chemotactic Factors↗
PubMed1990

Macrophages cultured in vitro release leukotriene B4 and neutrophil attractant/activation protein (interleukin 8) sequentially in response to stimulation with lipopolysaccharide and zymosan.

The capacity of lipopolysaccharide (LPS), zymosan, and calcium ionophore A23187 to induce neutrophil chemotactic activity (NCA), leukotriene B4 (LTB4), and neutrophil attractant/activation protein (NAP-1) release from human alveolar macrophages (AM) retrieved from normal nonsmokers was evaluated. LPS induced a dose-dependent release of LTB4 that began by 1 h, 4.0 +/- 3.2 ng/10(6) viable AM; peaked at 3 h, 24.7 +/- 13.5 ng/10(6) viable AM; and decreased by 24 h, 1.2 +/- 1.0 ng/10(6) viable AM (n = 8). Quantities of LTB4 in cell-free supernatants of AM stimulated with LPS were determined by reverse-phase high-performance liquid chromatography and corresponded well with results obtained by radioimmunoassay. By contrast, NAP-1 release began approximately 3-5 h after stimulation of AM with LPS, 197 +/- 192 ng/ml, and peaked at 24 h, 790 +/- 124 ng/ml. Release of NAP-1 was stimulus specific because A23187 evoked the release of LTB4 but not NAP-1, whereas LPS and zymosan induced the release of both LTB4 and NAP-1. The appearance of neutrophil chemotactic activity in supernatants of AM challenged with LPS for 3 h could be explained completely by the quantities of LTB4 present. After stimulation with LPS or zymosan for 24 h, AM had metabolized almost all generated LTB4. Preincubation of AM with nordihydroguiaretic acid (10(-4) M) completely abolished the appearance of NCA, LTB4, and NAP-1 in supernatants of AM challenged with LPS. Therefore, LPS and zymosan particles were potent stimuli of the sequential release of LTB4 and NAP-1 from AM.

Calcimycin↗
PubMed1990

Use of tumor diameter to estimate the growth kinetics of cancer and sensitivity of screening tests.

A statistical method has been developed that is useful for studying the relationship between the growth kinetics of malignant tumors and the detection probability either through symptoms or by screening. Mathematical models that describe the distribution of pathological variables in malignant tumors, detected after various histories of screening, are derived and parameters for detection probabilities and the growth kinetics are then estimated by the maximum likelihood procedure. By this method the probabilities of detection through symptoms as well as by screening can be estimated as functions of pathological variable(s) such as tumor size. The growth rate of tumor can also be estimated from the distribution of pathological variables. The present method was applied to gastric cancer in Japan, where an annual screening program for the disease exists. The detection probability for the indirect X-ray used as the screening test was estimated to be 0.323 x (diameter)2/[1 + 0.323 x (diameter)2]. The doubling time of gastric cancer was estimated to be 2.90 months.

Cell Cycle↗