Biomedical subjects

F Shibata

Publications and source records attributed to F Shibata.

At least 19 recordsLinked to original sources

PubMed2003

The effect of treatment with the oral growth hormone (GH) secretagogue MK-677 on GH isoforms.

Growth hormone (GH) consists of several isoforms. We have studied the proportion, expressed as percentage of total GH concentration, of non-22kDa (non-22K) GH isoforms and 20K GH during 8-week oral treatment with MK-677 25mg daily in 12 obese males. The proportion of non-22K GH isoforms in peak total GH samples after the initial MK-677 administration was higher than that after 2 and 8 weeks (p<0.01 and p<0.05, respectively). In selected non-peak total GH samples after the initial MK-677 administration, however, the proportion of non-22K GH isoforms was similar to that in the peak total GH samples after 2 and 8 weeks. The proportion of 20K GH in 2-h samples after the initial MK-677 administration was lower than that after 2 and 8 weeks (p<0.01 and p<0.05, respectively). We concluded that the proportion of non-22K GH isoforms was higher in peak, but not in non-peak, total GH samples after the initial MK-677 administration than that observed after multiple doses. The proportion of 20K GH in 2-h samples after the initial MK-677 administration was lower than that after 2 and 8 weeks. These moderate changes in the proportion non-22K GH isoforms are likely of small importance for the clinical response to MK-677 treatment.

Administration, Oral↗

PubMed2001

Cloning of DNA sequences localized on proximal fluorescent chromosome bands by microdissection in Pinus densiflora Sieb. & Zucc.

Japanese red pine, Pinus densiflora, has 2n=24 chromosomes, of which most carry chromomycin A3 (CMA) and 4',6-diamidino-2-phenylindole (DAPI) bands at their centromere-proximal regions. It was proposed that these regions contain highly repetitive DNA. The DNA localized in the proximal fluorescent bands was isolated and characterized. In P. densiflora, centromeric and neighboring segments of the somatic chromosomes were dissected with a manual micromanipulator. The centromeric DNA was amplified from the DNA contained in dissected centromeric segments by degenerate oligonucleotide primed-polymerase chain reaction (DOP-PCR) and a cloned DNA library was constructed. Thirty-one clones carrying highly repetitive DNA were selected by colony hybridization using Cot-1 DNA from this species as a probe, and their chromosomal localization was determined by fluorescent in situ hybridization (FISH). Clone PDCD501 was localized to the proximal CMA band of 20 chromosomes. This clone contained tandem repeats, comprising a 27 bp repeat unit, which was sufficient to provide the proximal FISH signal, with a 52.3% GC content. The repetitive sequence was named PCSR (proximal CMA band-specific repeat). Clone PDCD159 was 1700 bp in length, with a 61.7% AT content, and produced FISH signals at the proximal DAPI band of the remaining four chromosomes. Four clones hybridized strongly to the secondary constriction and gave weak signals at the centromeric region of several chromosomes. Clone PDCD537, one of the four clones, was homologous to the 26S rRNA gene. A PCR experiment using microdissected centromeric regions suggested that the centromeric region contains 18S and 26S rDNA. Another 24 clones hybridized to whole chromosome arms, with varying intensities and might represent dispersed repetitive DNA.

Base Sequence↗

PubMed2000

Identification of a common receptor for three types of rat cytokine-induced neutrophil chemoattractants (CINCs).

Rat cytokine-induced neutrophil chemoattractant-1 (CINC-1), CINC-2 and CINC-3/macrophage inflammatory protein-2 (MIP-2), members of the CXC chemokine family, are potent chemotactic factors for neutrophils. In order to identify the receptor for CINCs, rat CXC chemokine receptor 2 (CXCR2) was cloned and expressed in HEK293 cells. CINC-1, CINC-2 and CINC-3 induced calcium mobilizations dose-dependently in CXCR2-transfected cells, whereas formyl-methionyl-leucyl-phenylalanine (FMLP) did not. CINC-3 induced enhancement of cytoplasmic calcium concentration more potently than CINC-1 and CINC-2, and desensitized calcium transients induced by CINC-1 and CINC-2, which were essentially identical to those observed in rat neutrophils. In addition, anti-CXCR2 serum inhibited neutrophil chemotactic activities of three types of CINCs almost completely. The mutant CINC-3, whose amino-terminal amino acid sequence (SELR) was replaced to AAR, lost chemotactic activity of its own but inhibited that of CINC-1 and CINC-2 potently, and that of CINC-3 weakly. The results indicate that rat CXCR2 on neutrophils is the unique receptor for all three types of CINCs, and CINC-1/-2 and CINC-3 exert different biological activities through the common receptor.

Animals↗

PubMed2000

The expression of three types of CINCs by lipopolysaccharide-stimulated rat macrophages is inhibited similarly by anti-inflammatory steroids.

OBJECTIVE: Expression of CINCs was regulated differentially in lipopolysaccharide-stimulated rat macrophages. We examined whether the expression of CINCs in lipopolysaccharide-stimulated rat macrophages is similarly inhibited by anti-inflammatory drugs. MATERIALS AND METHODS: Rat peritoneal macrophages were stimulated by lipopolysaccharide in the presence of anti-inflammatory steroids (dexamethasone, prednisolone and hydrocortisone) or non-steroidal anti-inflammatory drugs (indomethacin and piroxicam). The production and mRNA expression of three types of CINCs were measured using enzyme-linked immunosorbent assay and northern hybridization. RESULTS: Anti-inflammatory steroids; dexamethasone, prednisolone and hydrocortisone, dose-dependently inhibited the production of CINC-1, -2 and -3, whose inhibitory patterns were similar to each other. Furthermore mRNA expression of each CINC was inhibited by dexamethasone in a dose-dependent manner. In contrast, non-steroidal anti-inflammatory drugs, indomethacin and piroxicam were without effect. Expression of each CINC was regulated differently; the production of CINC-1 reached a maximum at 12 h and then slightly decreased after lipopolysaccharide stimulation, whereas that of CINC-2 and CINC-3 increased up to 24 h. Dexamethasone inhibited the CINCs production and mRNA expression at 9 h after lipopolysaccharide stimulation. CONCLUSIONS: These results indicate no difference among CINC-1, -2 and -3 in the inhibition of production and mRNA expression of CINCs by anti-inflammatory steroids, although lipopolysaccharide differentially induces expression of each CINC expression in culture of rat macrophages.

Animals↗

PubMed2000

Differentiation and the polymorphic nature of the Y chromosomes revealed by repetitive sequences in the dioecious plant, Rumex acetosa.

The dioecious plant Rumex acetosa has a multiple sex chromosome system: females are 2n = XX + 12, males are 2n = XY1Y2 + 12, and the two Y chromosomes are heterochromatic. A DNA sequence abounded in the mare genome was isolated and analyzed. The sequence (RAE180) was a 180-bp-long tandemly arranged repetitive sequence, distributed in chromosomes Y1 and Y2, and two pairs of autosomes. Both Y chromosomes contained large amounts of RAE180 and the sequence formed many DAPI bands, while, on the two pairs of autosomes, RAE180 did not form DAPI bands. The internal structure and morphological changes of the Y chromosomes were analyzed by FISH, using RAE180 and the Y-chromosome-specific sequence RAYSI as probes. The pattern of the FISH signals caused by the accumulation of RAE180 and RAYSI suggested the structural change in the Y chromosomes during the process of sex chromosome evolution, and the morphological change in the Y chromosomes was explained by reciprocal translocation and inversion.

Chromosome Inversion↗

PubMed2000

Rat CXC chemokine GRO/CINC-1 paradoxically stimulates the growth of gastric epithelial cells.

BACKGROUND: CXC chemokines such as interleukin (IL)-8 are neutrophil chemoattractants, the levels of which increase in Helicobacter pylori-infected gastric mucosa. Many investigators have focused on the chemotactic aspects of IL-8: however, CXC chemokines are also reported to have angiogenic activity and to serve as remodelling factors. Rat GRO/CINC-1 is a rodent counterpart of human GROalpha, a member of the family of CXC chemokines. Gastric mucosa infected with H. pylori is in a state of hyperproliferation, with increases in the amounts of growth factors such as hepatocyte growth factor (HGF). AIM: To investigate whether rat GRO/CINC-1 had growth-stimulating activity for gastric epithelial cells. METHODS: The rat gastric epithelial cell line RGM-1 was incubated in serum-free medium for 12 h to adjust the cell cycle to the G0 phase, and GRO/CINC-1 was then added for 24 h. The total cell number was determined by fluorogenic analysis after propidium iodide staining, and cell proliferation was assessed by measuring 5-bromo-2'-deoxyuridine (BrdU) incorporation. The activity of p42/p44 mitogen-activated protein kinase (MAPK) was measured 5-20 min after the start of GRO/CINC-1 exposure. RESULTS: Cultures treated with GRO/CINC-1 showed a significant increase in cell number and BrdU incorporation in a concentration-dependent fashion. The MAPK activity increased within 5 min after GRO/CINC-1 application and returned to the control level at 20 min. CONCLUSION: The growth-stimulatory effect of GRO/CINC-1 on rat gastric epithelial cells suggests a dual function of this chemokine: proinflammatory action and induction of epithelial proliferation.

Animals↗

PubMed2000

Up-regulation of fas ligand at early stages and down-regulation of Fas at progressed stages of intrahepatic cholangiocarcinoma reflect evasion from immune surveillance.

We examined immunohistochemically the possible participation of the Fas/Fas ligand (FasL) system in intrahepatic cholangiocarcinoma (ICC) during the escape from immune surveillance, using 68 cases of ICC, 29 cases of normal intrahepatic large bile ducts, and 18 cases of biliary dysplasia. Apoptosis of tumor-infiltrating lymphocytes (TIL) was examined by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL). Fas was weakly expressed in normal intrahepatic bile ducts. Almost all biliary dysplasia and well-differentiated ICCs showed moderate to marked expression of Fas, while Fas expression was variable in moderately and poorly differentiated ICCs. Down-regulation of Fas expression was significantly correlated with histologic de-differentiation, vascular invasion, the size of ICCs, and short survival of ICC patients. By in situ hybridization, FasL mRNA were frequently and strongly expressed in biliary dysplasia compared with non-neoplastic intrahepatic bile duct. In well-differentiated ICCs, FasL mRNA expression was frequent and intense. But, the expression gradually decreased in moderately and poorly differentiated ICCs. Down-regulation of FasL mRNA expression in ICCs was correlated with perineural invasion and tumor size (over 4 cm) (P <.05). Apoptotic TIL were more frequent in ICC foci than in non-neoplastic foci remote from ICC foci. These findings suggest that a tumor evasion mechanism involving Fas/FasL exists in ICC; frequent and intense expression of FasL mRNA in well-differentiated ICCs enable them to escape immune surveillance by counterattacking Fas-bearing TIL. This counterattack becomes insensitive in poorly differentiated ICCs, in which the down-regulation of Fas gives them a resistance against the FasL-expressing TIL. These mechanisms may be involved in the tumor progression.

Adult↗

PubMed2000

Molecular cytogenetic analysis of supernumerary heterochromatic segments in Rumex acetosa.

The dioecious plant Rumex acetosa shows intraspecific karyotype variation, caused by supernumerary heterochromatic segments or DAPI (4',6-diamidino-2 phenylindole)-bands at the ends of the short arms of three pairs of autosomes. A DNA sequence (RAE730) specific to the supernumerary heterochromatic segments was cloned and sequenced. RAE730 was about 730 bp and AT-rich (71% AT-content). Using fluorescence in situ hybridization (FISH), RAE730 was localized in the supernumerary DAPI-positive heterochromatic segments on several mitotic chromosomes and chromocenters in interphase nuclei, but not in the DAPI-bands of Y or B chromosomes. RAE730 was tandemly arranged in the genome, and the copy number varied between plants from 40000 to 304000 copies per 2C, corresponding to the relative amount of supernumerary heterochromatic segments per genome. These results indicate that the karyotype variation caused by the supernumerary heterochromatic segment was generated by amplification or reduction of the tandem repeats of RAE730.

Base Sequence↗

PubMed1999

Physical interaction between interleukin-12 receptor beta 2 subunit and Jak2 tyrosine kinase: Jak2 associates with cytoplasmic membrane-proximal region of interleukin-12 receptor beta 2 via amino-terminus.

IL-12 is a heterodimeric cytokine, composed of p40 and p35 subunits, that exerts its biological effects by binding to specific cell surface receptors. Two human IL-12 receptor proteins, designated IL-12R beta 1 and IL-12R beta 2, have been previously identified. IL-12R beta 2 has box 1 motif, box 2 motif, and three tyrosine residues in its cytoplasmic domain. In response to IL-12, Jak2 and Tyk2, family members of Janus family protein tyrosine kinases, are phosphorylated in PHA-activated T lymphocytes. The present study demonstrates that Jak2 binds to the cytoplasmic membrane-proximal region of IL-12R beta 2, and box 2 motif and tyrosine residues in the cytoplasmic domain were not required for binding. The amino-terminus of Jak2 is necessary for association with IL-12R beta 2.

Amino Acid Substitution↗

PubMed1999

Chromosome painting of Y chromosomes and isolation of a Y chromosome-specific repetitive sequence in the dioecious plant Rumex acetosa.

The dioecious plant Rumex acetosa has a multiple sex chromosome system: XX in female and XY(1)Y(2) in male. Both types of Y chromosome were isolated from chromosome spreads of males by manual microdissection, and their chromosomal DNA was amplified using degenerate oligonucleotide primed-polymerase chain reaction (DOP-PCR). When the biotin-labeled DOP-PCR product was hybridized with competitor DNA in situ, the fluorescent signal painted the Y chromosomes. A library of Y chromosome DNA was constructed from the DOP-PCR product and screened for DNA sequences specific to the Y chromosome. One Y chromosome-specific DNA sequence was identified and designated RAYSI (R. acetosa Y chromosome-specific sequence I). RAYSI is a tandemly arranged repetitive DNA sequence that maps to the 4',6-diamidino-2-phenylindole bands of both Y chromosomes.

Base Sequence↗

PubMed1999

Rat macrophage inflammatory protein-1alpha, a CC chemokine, acts as a neutrophil chemoattractant in vitro and in vivo.

Recombinant rat macrophage inflammatory protein-1alpha (rMIP-1alpha) at a concentration of 3x10(-8) M had strong neutrophil chemotactic activity, though the potency of rMIP-1alpha was less than that of cytokine-induced neutrophil chemoattractant (CINC)-1 at lower concentrations. In addition, rMIP-1alpha induced neutrophil chemotaxis in vivo when rMIP-1alpha was injected into the preformed air-pouch on the back of rats. The adhesion of rMIP-1alpha-treated neutrophils to fibrinogen significantly increased, reaching a maximum adhesion at 10(-8) M. Stimulation of neutrophils with rMIP-1alpha induced a transient increase in intracellular free [Ca2+] dose-dependently. rMIP-1alpha still induced an increase in the intracellular [Ca2+] of rat neutrophils stimulated first with CINC-1, CINC-3 or C5a, suggesting that rat neutrophils have a specific receptor for rMIP-1alpha. Supporting these findings, an additive increase in chemotactic potency was found when both rMIP-1alpha and CINC-were added to the lower wells of Boyden chamber in vitro. In addition, high levels of rMIP-1alpha were detected in the inflammatory site of air-pouch/carrageenan-induced inflammation in rats. Our results suggest that rMIP-1alpha acts as a neutrophil chemoattractant and, together with CINCs, plays an important role in infiltration of neutrophils into inflammatory sites in rats.

Animals↗

PubMed1999

Unified projection operator formalism in nonequilibrium statistical mechanics.

The method of projection operators, which plays an important role in the field of nonequilibrium statistical mechanics, has been established with the use of the Liouville-von Neumann equation for a density matrix to eliminate irrelevant information from a whole system. We formulate a unified and general projection operator method for dynamical variables. The main features of our formalism parallel those for the Liouville-von Neumann equation. (1) Two types of basic equations, time-convolution and time-convolutionless decompositions, are systematically obtained without specifying a projection operator. (2) Expansion formulas for both decompositions are also obtained. (3) Problems incorporating a time-dependent Liouville operator can be flexibly treated. We apply the formulas to problems in random frequency modulation and low field resonance. In conclusion, our formalism yields a more direct and easier means of determining the average time evolution of an operator than the one for the Liouville-von Neumann equation.

Journal Article↗

PubMed1998

Gene structure, cDNA cloning, and expression of the rat cytokine-induced neutrophil chemoattractant-2 (CINC-2) gene.

Cytokine-induced neutrophil chemoattractant-2 (CINC-2) belongs to the CXC chemokine family and consists of two isoforms, CINC-2 alpha and CINC-2 beta. We have studied the genomic organization and expression of the CINC-2 gene. The gene spans approximately 14 kb and is composed of three common exons, one CINC-2 alpha-specific exon and two CINC-2 beta specific exons. This finding suggests that two isoforms of CINC-2 are encoded by mRNAs produced by alternative splicing. Each isoform is encoded in four exons, and exon-intron boundaries are placed identically within the aligned sequences of CXC chemokines. The CINC-2 alpha-specific exon encodes an extra C-terminal serine residue, in addition to three amino acid residues (DKS) which were determined from amino acid sequence analysis of CINC-2 alpha previously. The 5' flanking region of the gene contains a TATA box and putative binding sites for NF-kappa B and AP-1. Northern blot analyses showed that the mRNA level for CINC-2 was very low in rat peritoneal macrophages without stimulation and increased up to 4 h after lipopolysaccharide stimulation, similar to that for CINC-1 or CINC-3. Thereafter, the mRNA expression decreased gradually. However, the mRNA level of CINC-2 remained high 24 h after stimulation, in contrast to that of CINC-1 or CINC-3. These data indicate the expression of CINC-2 is regulated differently among the CINCs.

Animals↗

PubMed1998

Differential production of chemokines by phagocytosing rat neutrophils and macrophages.

In this study, rat neutrophils and macrophages produced cytokine-induced neutrophil chemoattractants (CINCs) and rat macrophage inflammatory protein-1 alpha (MIP-1 alpha) in different patterns during phagocytosis of heat-killed yeast cells in vitro. The cultured supernatants of the phagocytosing rat neutrophils and macrophages had chemotactic activities toward neutrophils, and the chemotactic potencies were markedly inhibited by anti-CINCs IgGs or/and anti-MIP-1 alpha IgG, suggesting that CINCs and MIP-1 alpha are major neutrophil chemoattractants produced by the phagocytosing neutrophils and macrophages. Dexamethasone suppressed the production of CINCs and MIP-1 alpha by the phagocytosing cells in a dose-dependent manner. Our results demonstrate significant differences in the production of CINCs and MIP-1 alpha by neutrophils and macrophages during phagocytosis of yeast cells and thus may suggest the different contribution of each chemokine to neutrophil recruitment in the processes of inflammation in rats.

Animals↗

PubMed1998

Reynosin from Sassurea lappa as inhibitor on CINC-1 induction in LPS-stimulated NRK-52E cells.

An inhibitor on CINC-1 (cytokine-induced neutrophil chemoattractant-1) induction in LPS-stimulated rat kidney epithelioid NRK-52E cells was purified from the roots of Sassurea lappa Clarke, a herbal medicine used in Korean traditional prescriptions for gastric intestinal diseases by a variety of column chromatographic procedures. The inhibitor was identified as reynosin, a sesquiterpene lactone isolated and characterized previously from Ambrosia confertiflora DC., and Magnolia grandiflora L. Reynosin exhibited a dose-dependent inhibition on CINC-1 induction in LPS-stimulated NRK-52E cells, where 50% of inhibitory effect was shown at the concentration of about 1 microM.

Animals↗

PubMed1997

Identification and characterization of receptor for cytokine-induced neutrophil chemoattractant-3 on rat neutrophils.

Cytokine-induced neutrophil chemoattractants (CINCs), members of alpha-chemokines, are considered to be major neutrophil chemotactic factors in rats. Recombinant CINC-3/rat macrophage inflammatory protein-2 (MIP-2) having an additional tyrosine residue at the carboxyl terminus (CINC-3-Tyr) was constructed, purified, radiolabeled with 125I, and used for binding studies. The specific binding of 125I-labeled CINC-3-Tyr (125I-CINC-3-Tyr) to rat neutrophils reached a plateau after approximately 60 min at 4 degrees C. This binding of 125I-CINC-3-Tyr could be reversed by adding an excess amount of unlabeled CINC-3-Tyr. Scatchard analysis revealed approximately 12,000 binding sites per cell on rat neutrophils with an apparent Kd value of 120 pM. Chemical cross-linking experiments suggested that the rat neutrophil CINC-3 receptor is a mass of approximately 69 kDa. Taken together, our results strongly suggest that rat neutrophils express a high affinity receptor for CINC-3.

Animals↗

PubMed1997

Effects of cytokine-induced neutrophil chemoattractants (CINCs) on shape change, adhesiveness and phagocytosis of rat neutrophils.

Rat cytokine-induced neutrophil chemoattractants (CINC)-1, -2 alpha, -2 beta and -3 belong to alpha-chemokines and are known to be potent chemoattractants for rat neutrophils in vivo and in vitro. In this study, the abilities of CINCs to affect several functions of rat neutrophils are estimated. All CINCs at a concentration of 10(-8) M induced the polarization of rat neutrophils. The adhesion of each CINC-treated neutrophil to fibrinogen significantly increased, reaching a maximal adhesion at 10(-8) M of each CINC. The CINCs enhanced the phagocytosis of heat-killed yeast cells by neutrophils in a dose-dependent manner. In contrast, CINCs did not induce the production of reactive nitrogen intermediates. The results suggest that CINCs play a significant role in neutrophil infiltration and phagocytosis at inflammatory sites in rats.

Animals↗

PubMed1996

Cytokine-induced neutrophil chemoattractant (CINC)-2 alpha, a novel member of rat GRO/CINCs, is a predominant chemokine produced by lipopolysaccharide-stimulated rat macrophages in culture.

Neutrophil chemotactic factors (chemokines) have been purified from conditioned medium of lipopolysaccharide (LPS)-stimulated rat macrophages in culture. The LPS-stimulated macrophages produced one acidic chemokine, rat macrophage inflammatory protein (MIP)-1alpha, and four basic chemokines, cytokine-induced neutrophil chemoattractant (CINC)-1. CINC-2alpha, CINC-2beta and CINC-3/rat MIP-2. CINC-2alpha, a novel chemokine recently isolated from conditioned medium of rat granulation-tissue culture, was the major chemoattractant among these four basic chemokines. The results suggest that CINC-2alpha is produced by activated macrophages in vivo and plays an important role in the infiltration of neutrophils into inflammatory sites in rats

Amino Acid Sequence↗