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T Yoshimura

Publications and source records attributed to T Yoshimura.

At least 55 records · Page 3Linked to original sources
PubMed1991

Expression of monocyte chemoattractant protein 1 in macrophage-rich areas of human and rabbit atherosclerotic lesions.

The recruitment of monocyte-macrophages into the artery wall is one of the earliest events in the pathogenesis of atherosclerosis. Monocyte chemoattractant protein 1 (MCP-1) is a potent monocyte chemoattractant secreted by many cells in vitro, including vascular smooth muscle and endothelial cells. To test whether it is expressed in the artery in vivo, we used Northern blot analysis, in situ hybridization, and immunocytochemistry to study the expression of MCP-1 in normal and atherosclerotic human and rabbit arteries. Northern blot analysis showed that MCP-1 mRNA could be isolated from rabbit atherosclerotic lesions but not from the intima media of normal animals. Furthermore, MCP-1 mRNA was extracted from macrophage-derived foam cells isolated from arterial lesions of ballooned cholesterol-fed rabbits, whereas alveolar macrophages isolated simultaneously from the same rabbits did not express MCP-1 mRNA. MCP-1 mRNA was detected by in situ hybridization in macrophage-rich regions of both human and rabbit atherosclerotic lesions. No MCP-1 mRNA was found in sublesional medial smooth muscle cells or in normal arteries. By using immunocytochemistry, MCP-1 protein was demonstrated in human lesions, again only in macrophage-rich regions. Immunostaining of the serial sections with an antiserum against malondialdehyde-modified low density lipoprotein indicated the presence of oxidized low density lipoprotein indicated the presence of oxidized low density lipoprotein and/or other oxidation-specific lipid-protein adducts in the same areas that contained macrophages and MCP-1. We conclude that (i) MCP-1 is strongly expressed in a small subset of cells in macrophage-rich regions of human and rabbit atherosclerotic lesions and (ii) MCP-1 may, therefore, play an important role in the ongoing recruitment of monocyte-macrophages into developing lesions in vivo.

Adult↗
PubMed1991

[Morphometric analysis of normal human sural nerves for systematic clinical application].

To obtain normative data on the sural nerve for clinical evaluation of the pathologic process and severity of the various peripheral nerve disorders, a morphometric analysis was made on 30 control sural nerves from normal volunteers, and from patients and cadavers with no evidence of peripheral nerve involvement. The ages of volunteers, patients and cadavers ranged from 13 to 83 years. The data obtained included 1) the mean frequency of abnormal teased myelinated fibers and its upper limit value of 95% confidence interval, and 2) the mean densities of total, large and small myelinated fibers and of unmyelinated fibers and their lower limit value of 95% confidence interval for each decade. The linear correlation between the age and each of the frequencies of abnormal teased myelinated fibers and the densities of total, large and small myelinated fibers and of unmyelinated fibers were statistically significant (P less than 0.01). Therefore, the morphometric data obtained from the disease nerve should be compared with the normative data adjusted for the age of the subject described in this study.

Adolescent↗
PubMed1991

Spectral properties of Achromobacter xylosoxidans cytochromes c' and their NO complexes.

Cytochromes c' have been isolated from six strains of Achromobacter xylosoxidans: NCIB 11015 (formerly Alcaligenes sp. NCIB 11015), GIFU 543, 1048, 1051, 1055 and 1764. They are dimeric proteins with more positive redox potentials than those of cytochromes c' from phototrophic bacteria at neutral pH. The electronic absorption, EPR and MCD spectra on NO-ferrous cytochromes c' at physiological pH showed that the major part of the heme-iron of nitrosylheme was penta-coordinated. The EPR spectral results indicated that the ground state of the heme-iron of ferric cytochromes c' appears to be in an admixed spin states which consists of predominant high-spin with a slight intermediate-spin character at pH 7.2. These spectra were compared with those for cytochromes c' from phototrophic bacteria and the other hemoproteins.

Alcaligenes↗
PubMed1991

Neutrophil attractant/activation protein-1 and monocyte chemoattractant protein-1 in rabbit. cDNA cloning and their expression in spleen cells.

Rabbit neutrophil attractant/activation protein-1 (NAP-1) and monocyte chemoattractant protein-1 (MCP-1) were investigated. Rabbit spleen cells stimulated with 5 micrograms/ml of Con A produced both neutrophil and monocyte chemotactic activity. Physicochemical characteristics of those activities obtained by HPLC gel filtration and HPLC chromatofocusing were very similar to those of human NAP-1 and MCP-1, suggesting that rabbit spleen cells produce NAP-1 and MCP-1 after Con A stimulation. A cDNA library was constructed from mRNA purified from Con A-stimulated rabbit spleen cells and screened with oligonucleotide probes. By two rounds of screening, NAP-1 and MCP-1 cDNA were cloned. NAP-1 cDNA comprises 1500 bp with an open reading frame that encodes for a 101-amino acid protein highly similar to human NAP-1. MCP-1 cDNA comprises 607 bp with an open reading frame that encodes for a 124-amino acid protein highly similar to human MCP-1. Expression of NAP-1 and MCP-1 mRNA by rabbit spleen cells was studied. Both Con A- and LPS-stimulated spleen cells expressed NAP-1 and MCP-1 mRNA, but the kinetics of expression were different. Con A rapidly induced high NAP-1 and MCP-1 mRNA expression. LPS also rapidly induced NAP-1 mRNA expression, but high MCP-1 mRNA expression was not observed until 15 h after stimulation. Immunoprecipitation of metabolically labeled NAP-1 and MCP-1 with anti-human NAP-1 or MCP-1 polyclonal antibodies was attempted. Immunoprecipitated rabbit NAP-1 with a molecular mass of about 7 kDa was detected by SDS-PAGE and radioautography, but MCP-1 was not. Cloned rabbit NAP-1 and MCP-1 will give us opportunities to study the role of NAP-1 and MCP-1 in vivo.

Amino Acid Sequence↗
PubMed1991

Skeletal structure of clathrin triskelion in solution: experimental and theoretical approaches.

The physicochemical properties of the clathrin triskelion were determined by dynamic and static light-scattering and sedimentation analyses in Tris and triethanolamine (TEA) buffers of about pH 8, in which the clathrin triskelion has been found to be in different conformational states by electron microscopy [Heuser, J., & Kirchhausen, T. (1985) J. Ultrastruct. Res. 92, 1-27]. Dynamic light-scattering measurements provided diffusion coefficients (D0(20,w)) of 1.22 x 10(-7) and 1.23 x 10(-7) cm2/s, and ultracentrifugal analysis gave sedimentation coefficients (S0(20,w)) of 8.39 and 8.32 S in Tris and TEA buffer, respectively. The average Stokes radius of the protein was determined to be 175 A from its diffusion and sedimentation coefficients and its molecular weight. Static light-scattering analysis provided molecular weights of 6.58 x 10(5) and 6.41 x 10(5) and radii of gyration of 311 and 301 A in the respective buffers. These results indicate that the clathrin triskelion has a similar conformation in the two buffers. For clarification of the skeletal structure of the clathrin triskelion in solution, the physicochemical parameters were calculated by using two models in which the clathrin arms are bent at various angles in a plane, on the basis of the Bloomfield approximation and a formula derived to estimate the radius of gyration of proteins consisting of various structural units. Values for the Stokes radius, diffusion and sedimentation coefficients, and radius of gyration in the ranges of 178-170 A, (1.20-1.26) x 10(-7) cm2/s, 8.26-8.66 S, and 316-266 A, respectively, were obtained with these models with the arms bent in the range of 0-60 degrees.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1991

Macrophage stimulating protein: purification, partial amino acid sequence, and cellular activity.

Macrophage stimulating protein (MSP) was purified to homogeneity from human blood plasma by selection of biologically active fractions obtained by sequential immunoaffinity and high pressure liquid ion exchange chromatography. By sodium dodecyl sulfate-polyacrylamide gel electrophoresis the molecular mass of MSP was 70 kilodaltons (kD); under reducing conditions two gel bands were seen, at 47 and 22 kD. The disulfide-linked two-chain structure of MSP was confirmed by separation of reduced and alkylated MSP chains. A computer search comparison of six partial sequences of MSP digests showed that MSP has not been recorded in data banks of protein sequences. Two MSP fragments had greater than 80% identity in overlaps of 12-16 residues to sequences in the protein family that includes human prothrombin, plasminogen, and hepatocyte growth factor. The concentration of purified MSP required for half-maximal biological activity was the order of 10(-10) M. In addition to making mouse resident peritoneal macrophages responses to chemoattractants, MSP caused the appearance of long cytoplasmic processes and pinocytic vesicles in freshly plated macrophages. MSP also caused phagocytosis via the C3b receptor, CR1. Whereas resident peritoneal macrophages bind but do not ingest sheep erythrocytes opsonized with IgM anti-Forssman antibody and mouse C3b, addition of MSP caused ingestion. Thus, MSP causes direct or indirect activation of two receptors of the mouse resident peritoneal macrophage, CR1 and the C5a receptor.

Amino Acid Sequence↗
PubMed1991

Effect of substitution of a lysyl residue that binds pyridoxal phosphate in thermostable D-amino acid aminotransferase by arginine and alanine.

Lys-145 of the thermostable D-amino acid aminotransferase, which binds pyridoxal phosphate, was replaced by Ala or Arg by site-directed mutagenesis. Both mutant enzymes were purified to homogeneity; their absorption spectra indicated that both mutant enzymes contained pyridoxal phosphate bound non-covalently. Even though the standard assay method did not indicate any activity with either mutant, addition of an amino donor, D-alanine, to the Arg-145 mutant enzyme led to a slow decrease in absorption at 392 nm with a concomitant increase in absorption at 333 nm. This result suggests that the enzyme was converted into the pyridoxamine phosphate form. The amount of pyruvate formed was almost equivalent to that of the reactive pyridoxal phosphate in the mutant enzyme. Thus, the Arg-145 mutant enzyme is able to catalyze slowly the half-reaction of transamination. Exogenous amines, such as methylamine, had no effect on the half-reaction with the Arg-145 mutant enzyme. In contrast, the Ala-145 mutant enzyme neither underwent the spectral change by addition of D-alanine nor catalyzed pyruvate formation, in the absence of added amine. However, the Ala-145 mutant enzyme catalyzed the half-reaction significantly in the presence of added amine. These findings suggest that a basic amino acid residue, such as lysine or arginine, is required at position 145 for catalysis of the half-reaction. The role of the exogenous amines differs with various active-site mutant enzymes.

Alanine↗
PubMed1991

Growth regulation of a human mature B cell line, B104, by anti-IgM and anti-IgD antibodies.

An EBNA- human B lymphoma cell line, B104, was established. B104 cells express IgD as well as IgM on their surface, which is thought to be a basic characteristic of mature B cells. The growth of B104 cells was inhibited by treatment with a panel of anti-IgM antibodies. Cell cycle analyses revealed that the transition of B104 cells from the G2/M to the G0/G1 phase of the cell cycle was markedly inhibited by treatment with anti-IgM antibodies. Progression of B104 cells to the M phase of the cell cycle was found to be suppressed in the presence of anti-IgM antibodies. In contrast, both the entrance of G0/G1 phase cells into the S phase and the progression of S phase cells to the G2/M phase of the cell cycle did not seem to be inhibited significantly by treatment with anti-IgM antibodies. These results indicate that the mechanism of the inhibition of growth of B104 cells by anti-IgM antibodies is blockage of the transition from the G2 to the M phase of the cell cycle. In contrast to anti-IgM antibodies, anti-IgD antibodies could not cause growth inhibition of B104 cells at all. B cell growth factors such as IL-4 and IL-6 had no effect on the inhibition of growth of B104 cells by anti-IgM antibody. IFN-alpha and -beta, which have no B cell growth factor activity, did increase the number of cells that survived the treatment with anti-IgM antibodies. B104 is an excellent experimental model for the study of the mechanism of signal transduction through sIg as well as the functional difference between sIgM and sIgD.

Antibodies, Anti-Idiotypic↗
PubMed1991

Molecular cloning of rat monocyte chemoattractant protein-1 (MCP-1) and its expression in rat spleen cells and tumor cell lines.

Rat monocyte chemoattractant protein-1 (MCP-1) cDNA was cloned. A cDNA library was constructed from mRNA extracted from Con A-stimulated rat spleen cells. After 2 rounds of screening with a radiolabeled oligonucleotide probe and DNA sequencing from plasmid DNAs, one clone which encoded for MCP-1 was obtained. The cDNA clone comprises 665 base pairs with an open reading frame which encodes for 148 amino acid protein. Spleen cells expressed a significant amount of MCP-1 mRNA without stimulus. But higher expression was observed when cells were stimulated with Con A. MCP-1 mRNA was also expressed in tumor cell lines, although the amount of expressed MCP-1 mRNA was different among cell lines.

Amino Acid Sequence↗
PubMed1991

Chemotactic activity and receptor binding of neutrophil attractant/activation protein-1 (NAP-1) and structurally related host defense cytokines: interaction of NAP-2 with the NAP-1 receptor.

Neutrophil attractant/activation protein-1 (NAP-1) has sequence similarity to platelet factor-4 (PF-4) and to NAP-2 (a truncated from of connective tissue activating protein-III [CTAP-III(des 1-15)]. We compared chemotactic activity for neutrophils of these related proteins. We also included for comparison CTAP-III, CTAP-III(des 1-13), the C-terminal dodecapeptide of PF-4 [PF-4(59-70)], and C5a. Chemotactic potency (EC50) was highest for NAP-1 and C5a. Although chemotactic efficacy (peak percentage of neutrophils migrating) was comparable for C5a, NAP-1, and NAP-2, the NAP-2 response occurred only at concentrations 100-fold higher than the NAP-1 EC50 of 10(8) M. Data for the CTAP-III proteins confirmed that CTAP-III is not an attractant and that chemotactic activity appears as a result of cleavage of residues at the N-terminus to make CTAP-III(des 1-13) or NAP-2 [CTAP-III(des 1-15)]. Chemotactic activity of PF-4 was low and variable, with no significant response by neutrophils from six of nine subjects. In contrast, PF-4(59-70) regularly induced high chemotactic responses, although the EC50 of 1.6 x 10(5)M was 1,000-fold greater than that of NAP-1. The binding of fluoresceinated NAP-1 to neutrophils was inhibited by unlabeled NAP-1 or NAP-2 but not by PF-4 or PF-4 (59-70). This suggests that NAP-2 interacts with the neutrophil NAP-1 receptor. Despite the low chemotactic potency of NAP-2, it is a potential attractant at sites of injury because of the relatively large amounts of the parent CTAP-III released from platelets, as indicated by a serum concentration of approximately 10(-6) M.

Chemotaxis, Leukocyte↗
PubMed1991

Biological aspects of monocyte chemoattractant protein-1 (MCP-1).

In this communication, we have asked if MCP-1 is the mediator of cellular infiltration in DCH, outlining the criteria in Table 3. Preliminary data suggest that PHA-stimulated lymphocytes secrete MCP-1, and that MCP-1 can be produced in response to antigen stimulation. MCP-1 attracts monocytes and basophils, but not neutrophils. The question of a lymphocyte response to MCP-1 requires further study. We have emphasized that the discovery of leukocyte-specific NAP-1 and MCP-1 should now be followed by exploration of conditions in which one agonist is secreted without the other. This would be expected, for example, in DCH, which is characterized by mononuclear leukocyte infiltration without neutrophils.

Basophils↗
PubMed1991

Hormonal regulation of plasminogen activator and peroxidase activities in 7,12-dimethylbenz(a)anthracene-induced rat mammary tumors and the rat uterus.

Oophorectomy was found to decrease the plasminogen activator activity of rat mammary tumors induced by 7,12-dimethylbenz(a)anthracene (DMBA) to less than 7 per cent, while in vivo estradiol treatment restored its activity in a dose dependent fashion. The peroxidase activity was not changed either by oophorectomy or by the administration of estrogen. In the rat uterus, plasminogen activator activity was not changed by oophorectomy or by the administration of estrogen, however, its peroxidase activity decreased to less than 2 per cent following oophorectomy, while estrogen administration restored its activity. Estrogen regulated plasminogen activator activity in the DMBA-induced rat mammary tumors but not in the uterus and thus, the specific hormonal regulation of this enzyme may be an important factor for the hormonal dependent growth of such tumors.

9,10-Dimethyl-1,2-benzanthracene↗
PubMed1991

Production of monocyte chemoattractant protein-1 by malignant fibrous histiocytoma: relation to the origin of histiocyte-like cells.

Human malignant fibrous histiocytoma (MFH) comprise both fibroblast-like cells and histiocyte-like cells. We previously showed that the latter are not neoplastic cells, but are infiltrating macrophages. Since migration of blood monocytes into the tumor might be a response to a locally elaborated monocyte chemoattractant, we designed experiments to determine if the fibroblast-like tumor cells produced a chemoattractant for human monocytes. Malignant fibrous histiocytoma from three patients was put into culture. Cells of all three lines had a spindle shape, and showed no reactivity with antibodies against macrophages (MAC387), HLA-DR (LN3), or leukocyte common antigen. Immunohistochemically, they stained with antibody against human monocyte chemoattractant protein-1 (MCP-1). Culture supernatants of the three cell lines had chemotactic activity for monocytes. This activity was due to MCP-1, since it was absorbed by an anti-MCP-1 column. The production of MCP-1 by MFH tumor lines was confirmed by immunoprecipitation of metabolically labeled MCP-1. These results suggest that the histiocyte-like cells are the infiltrated macrophages that originate from blood monocytes attracted by tumor-derived MCP-1.

Chemokine CCL2↗
PubMed1991

Mechanism of protein folding. II. Lysozyme and phospholipase.

Refolding of hen egg-white lysozyme assuming the formation of secondary structures (alpha-helices and beta-sheets) is carried out by the method presented in the previous paper (N. Saitô et al., Proteins; Struct. Funct. Genet. 3 (1988) 199-208). To do this, the hydrophobic interactions between the hydrophobic residues which are located at the key positions for folding and can be identified without the knowledge of the native structure, and the nonbonded interactions between every pair of atoms (except hydrogen) or groups are introduced successively from short- to medium-distance pairs. The search for the energy minimum by these interactions can afford a conformation of especially the mutual arrangements between neighboring secondary structures. When these local structures are accomplished, some of the long-distance amino-acid pairs come close together and then the possible interactions (hydrophobic, nonbonded) are introduced. The three-dimensional structure of lysozyme thus obtained is shown to have locally correct arrangements of the secondary structures, but mutual relations between long-distance parts of the chain are not similar to the native structure. The introduction of disulfide bonds between appropriate cysteine residues is necessary to reach the native structure. The choice of cysteine pairs for disulfide bonding is made by the criterion given in the paper to follow (K. Watanabe, A. Nakamura, Y. Fukuda and N. Saitô. Biophys. Chem. 40 (1991) 293). The same treatment is applied to bovine pancreatic phospholipase with 7 disulfide bonds. The formation of the antiparallel beta-structures from neighboring beta-strands and the problem of the folding order are also discussed.

Amino Acid Sequence↗
PubMed1991

Overproduction of glutamate racemase of Pediococcus pentosaceus in Escherichia coli clone cells and its purification.

We previously isolated a 6.0-kb DNA fragment that specifies glutamate racemase activity from the chromosomal DNA of Pediococcus pentosaceus by digestion with HindIII (N. Nakajima, K. Tanizawa, H. Tanaka, and K. Soda, 1986), Agric. Biol. Chem. 50, 2823-2830). We digested it further with EcoRI to obtain a fragment of 1.8 kb, which was blunt-ended and ligated into the SmaI site of vector plasmid pKK223-3. The recombinant plasmid showed a high glutamate racemase activity upon transformation of Escherichia coli W3110 cells with it; the plasmid was named pICR223. Glutamate racemase was overproduced in the clone cells and occurred in inclusion bodies in the cells. The enzyme was solubilized with 6 M urea, renatured by dialysis to remove urea, and purified to homogeneity with an overall yield of about 70% after a single DEAE-cellulose column chromatography. The amount of enzyme produced by the clone cells corresponded to about 38% of the total insoluble protein.

Amino Acid Isomerases↗
PubMed1991

Thermostable phenylalanine dehydrogenase of Thermoactinomyces intermedius: cloning, expression, and sequencing of its gene.

The gene encoding the thermostable phenylalanine dehydrogenase [EC 1.4.1.-] of a thermophile, Thermoactinomyces intermedius, was cloned and its complete DNA sequence was determined. The phenylalanine dehydrogenase gene (pdh) consists of 1,098 nucleotides and encodes 366 amino acid residues corresponding to the subunit (Mr 41,000) of the hexameric enzyme. The amino acid sequence deduced from the nucleotide sequence of the pdh gene of T. intermedius was 56.0 and 42.1% homologous to those of the phenylalanine dehydrogenases of Bacillus sphaericus and Sporosarcina ureae, respectively. It shows 47.5% homology to that of the thermostable leucine dehydrogenase from B. stearothermophilus. The pdh gene was highly expressed in E. coli JM109, the amount of phenylalanine dehydrogenase produced amounting up to about 8.3% of that of the total soluble protein. We purified the enzyme to homogeneity from transformant cells in a day, with a 58% recovery.

Actinomycetales↗