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Biomedical subjects

H Kawashima

Publications and source records attributed to H Kawashima.

At least 199 records · Page 11Linked to original sources
PubMed1995

Brain uptake and metabolism of [1-11C]octanoate in rats: pharmacokinetic basis for its application as a radiopharmaceutical for studying brain fatty acid metabolism.

The uptake of octanoate in rat brain and its metabolism were investigated by means of intravenously injecting [1-11C] or [1-14C]octanoate as a tracer. The radioactivity in the cerebrum was increased by an injection of [1-11C]octanoate, and reached its peak level (0.33% ID/g) in about 2 to 5 min, and then decreased slowly. The cerebrum-to-blood ratio of the radioactivity increased with time over a period of 30 min. At 30 sec, [1-11C]octanoate that remained unchanged in the cerebrum accounted for only 8% of the total radioactivity, in spite of there being about 90% in the blood. By means of an injection of [1-14C]octanoate, more than 70% of the total radioactivity in the cerebrum was found to be attributable to radiolabeled glutamate and glutamine at each time point measured between 30 sec and 30 min. The results show that [1-11C]octanoate enters rat brain easily and is trapped in the cerebrum, probably in the form of glutamate and glutamine, and the usefulness of [1-11C]octanoate as a radiopharmaceutical for studying brain fatty acid metabolism by positron emission tomography is therefore suggested.

Animals↗
PubMed1995

Expression of multiple forms of brain cytochrome P450.

Multiple forms of cytochrome P450 (P450) in brain tissue have been demonstrated to be expressible in brain tissue using polymerase chain reaction (PCR) techniques, Northern blotting, hydroxylation activity assessment and cloning approaches. The antidepressant drug imipramine is metabolized by brain microsomes to multiple products by pathways inhibitable by quinidine, 7,8-benzoflavone, and ketoconazole, well-known inhibitors of P450-catalyzed reactions. Moreover, PCR studies revealed that a number of P450s are expressible in brain tissue and in glioma C6 cells. Quantitative PCR studies further demonstrated the response of many of these forms to induction in agreement with hydroxylation activity results.

Animals↗
PubMed1995

Anti-inflammatory effects of sulfatides in selectin-dependent acute lung injury.

Selectins promote adhesive interactions between leukocytes and activated endothelial cells, the adhesion being mediated by 'counter-receptors' on endothelial cells and consisting of oligosaccharide conjugates containing sialic acid and fucose. There are also suggestions that selectins bind sulfated compounds, including sulfatides. Intravenous infusion of selectin-reactive oligosaccharides has been found to prevent selectin-dependent inflammatory lung injury. In the current studies using two models of neutrophil and selectin-dependent acute lung injury in rats, sulfatide and its modified versions were infused i.v. and the protective effects determined. Naturally occurring sulfatide, synthetic sulfatides and sulfated ganglioside were highly protective against lung injury following systemic activation of complement. Desulfated sulfatide was inactive. The protective effects of synthetic sulfatides required sulfation of galactose in position 3. Sulfatide was also protective in the IgG immune complex model of lung injury. The protective effects of sulfatides were associated with reduced content of myeloperoxidase (derived from neutrophils) in lung tissue. These data indicate that sulfatides have significant in vivo protective effects in neutrophil and selectin-dependent models of lung injury.

Animals↗
PubMed1995

Role of transforming growth factor beta 1 on hepatic regeneration and apoptosis in liver diseases.

AIMS: To investigate the effects of transforming growth factor beta 1 (TGF-beta 1) on regeneration and induction of apoptosis of liver cell and bile duct in various liver diseases. METHODS: Formalin fixed paraffin wax sections of 18 liver tissue samples were obtained by needle biopsy, surgery, or necropsy; these included six liver cirrhosis, three obstructive jaundice; five fulminant hepatitis, one subacute hepatitis, and three normal liver. Expression of TGF-beta 1, apoptosis related Le(y) antigen, Fas antigen, a receptor for tumour necrosis factor, and biotin nick end labelling with terminal deoxynucleotidyl transferase mediated dUTP (TUNEL) for locating DNA fragmentation, was investigated histochemically. RESULTS: TGF-beta 1 was expressed in areas of atypical bile duct proliferation, where bile duct continuously proliferated from liver cells. In occlusive jaundice and fulminant hepatitis, TUNEL was positive in nuclei and cytoplasm of metaplastic cells which formed incomplete bile ducts, and these cells appeared to extend from TGF-beta 1 expressing liver cells. Fas antigen was found only on the cell membrane of proliferated bile duct in fulminant hepatitis, which differed from TGF-beta 1 and TUNEL positive areas. Le(y) antigen was expressed in liver cell and bile duct at the areas with atypical bile duct proliferation, but its coexpression with TUNEL was rare. CONCLUSIONS: TGF-beta 1 plays a role in the arrest of liver cell regeneration and atypical bile duct proliferation, and in areas of rapidly progressing atypical bile duct proliferation, such as in fulminant hepatitis or bile retention. Apoptosis appears to be induced by TGF-beta 1. This phenomenon may account for the inadequate hepatic regeneration that occurs with liver disease.

Apoptosis↗
PubMed1995

[Quality control of fine needle aspiration cytology of breast lesions].

From 1988 to 1993 we treated 1,310 cases of fine needle aspiration cytology of breast lesions. The results were positive for malignancy in 218, suspicious in 134, negative in 857, and inadequate specimen in 101. Histological examinations were performed in 461 cases; 243 malignancies and 218 benign lesions. The cytological diagnosis of these cases was positive in 211, suspicious in 91, negative in 142 and inadequate specimen in 17. The cytologic diagnosis of the cases of histological malignancy was positive in 198, suspicious in 24, negative in 17 and inadequate specimen in 4. Therefore we have 17 false negative cases. The histological diagnoses in these cases were scirrhous carcinoma (7), papillotubular carcinoma (6), invasive lobular carcinoma (2), mucinous carcinoma (1) and angiosarcoma (1). There were 13 cases of cytologically false positive cases; the histological diagnoses were fibroadenoma (4), mastopathy (4), papilloma (3), adenosis (1) and xanthogranuloma (1). For over six years, we have used a computer system incorporating both cytologic and histologic records. This system is very useful in quickly review the correlations of cytologic-histologic diagnoses. In addition to this system, a close relationship between clinical to pathological department is necessary in the quality control of diagnostic cytology.

Biopsy, Needle↗
PubMed1994

Molecular cloning and functional expression of a cDNA encoding the human V1b vasopressin receptor.

Arginine vasopressin modulates the release of adrenocorticotropic hormone, beta-endorphin, and prolactin from the anterior pituitary. Release is mediated by the V1b receptor through the mobilization of intracellular Ca2+ by phosphatidylinositol hydrolysis. In contrast to its well characterized peripheral actions, such as antidiuresis, contraction of vascular smooth muscle, and stimulation of hepatic glycogenolysis, the exact site and mechanism of vasopressin action in the pituitary remain unclear. This is largely due to a lack of information on the molecular identity and exact localization of the V1b receptor. This lack prompted us to try to isolate this receptor subtype. Here we report the molecular cloning and functional expression of a complementary DNA encoding the human V1b receptor. The deduced 424-amino acid sequence of the receptor has highest overall homology with the V1a, V2, and oxytocin receptors, with homologies of 45, 39, and 45%, respectively. The receptor expressed in COS-1 cells has a single binding site for arginine vasopressin with a Kd of 0.17 +/- 0.04 nM. It binds various agonists and antagonists of vasopressin with affinities distinct from those of V1a and V2 receptors but consistent with those anticipated for the V1b receptor on the basis of the pharmacological studies. Furthermore, arginine vasopressin evoked calcium-dependent chloride current in Xenopus oocytes transfected with the receptor, which was not affected by a V1a/V2 antagonist. In contrast, the current evoked in oocytes transfected with V1a receptor was abolished by the antagonist. Northern blot analysis revealed that the receptor expression is restricted to the pituitary. These data clearly indicate that the cloned cDNA encodes the V1b receptor.

Amino Acid Sequence↗
PubMed1994

Corneal endothelial cells inhibit T cell proliferation by blocking IL-2 production.

We have reported that Ia+ LEW rat corneal endothelial (CE) cells could not induce Ag-specific proliferation of MHC compatible T cell lines. Instead, the presence of CE cell monolayers inhibited T cell proliferation assays. We now report on the effect of CE cells on IL-2 production, IL-2R expression, and IL-2 responsiveness of T cells. Ag-specific growth inhibition of the 1C3.4 T cell hybridoma was unaffected by the presence of CE cells but IL-2 production was suppressed by CE cells. Similar CE cell-mediated inhibition of IL-2 secretion was found with fresh splenic lymphocytes stimulated by Con A. Controls showed that neither CE cells nor their supernatant affected the CTLL cell response to rIL-2, allowing reliable measurement of IL-2 production. Further evidence that the block is at the level of IL-2 production was shown by the observation that IL-2R expression was up-regulated after TCR occupancy whether or not CE cells were present. Also, T cells expressing IL-2R were responsive to exogenous rIL-2 whether or not CE cells were present. CE cells themselves do not induce IL-2R expression or IL-2 responsiveness. Under normal culture conditions, the R1170 rat T cell line continually expresses a significant level of IL-2R and dies before IL-2R is down-regulated to background levels. However, in the presence of CE cells, these R1170 cells down-regulate IL-2R and lose IL-2 responsiveness while remaining viable.

Animals↗
PubMed1994

Identification of matrix metalloproteinase 9 in rabbit osteoclasts.

Osteoclasts are multinucleate giant cells that play key roles in bone resorption. To identify genes predominantly expressed in osteoclasts, we screened a cDNA library of osteoclasts with cDNA probes of osteoclasts and alveolar macrophages. Clones specifically hybridizing to the osteoclast probe were isolated and sequenced. The nucleotide sequence of one such clone, F17, was found to share significant similarity with the sequences of human and mouse matrix metalloproteinase 9 (MMP-9) cDNA. By isolation and sequencing of the full-length cDNA, F17 was revealed to encode the rabbit counterpart of MMP-9. By Northern blotting, messenger RNA for MMP-9 was found to be highly and predominantly expressed in isolated osteoclasts when compared with its level in alveolar macrophages and other tissues. By gelatin zymography, gelatinase activity was detected in conditioned medium of isolated osteoclasts, suggesting that MMP-9 is secreted by isolated osteoclasts. Expression of MMP-9 was also observed in in vivo osteoclasts in metacarpal bones of newborn rabbits by in situ hybridization. These facts suggest that MMP-9 is one of the major proteases produced by osteoclasts and possibly plays a role in osteoclastic bone resorption.

Amino Acid Sequence↗
PubMed1994

Characterization of cloned human cholecystokinin-B receptor as a gastrin receptor.

The cholecystokinin (CCK)-B receptor cloned from human brain was characterized as a gastrin receptor by using heterologous expression systems of COS-7 cells and Xenopus oocytes. 125I-gastrin binding to human CCK-B receptor expressed in COS-7 was time-dependent, saturable and also specific, as well as 125-I-CCK-8. The binding of 125I-gastrin was inhibited by CCK-8 about 10-fold more potently than by gastrin. The rank order of potency of several antagonists to 125I-gastrin binding was YM022 > CI-988 > L-365,260 > L-364,718. Addition of GTP gamma S, a nonhydrolysable analog of GTP, dose-dependently inhibited 125I-gastrin binding, and lowered the gastrin binding affinity, Gastrin (10(-9)-10(-7) M) also evoked a Ca(2+)-dependent Cl- current in Xenopus oocytes expressing CCK-B receptors. These results suggest that the pharmacological profile of the cloned human CCK-B receptor using 125I-gastrin is closely parallel to that reported in gastric mucosa, and that the receptor transduces cellular signals of gastrin as well as those of CCK-8.

Amino Acid Sequence↗
PubMed1994

Exogenous group II phospholipase A2 induces prostaglandin E2 production in mouse peritoneal macrophages.

Cultures of mouse peritoneal resident macrophages produced prostaglandin E2 when exposed to extracellular group II phospholipase A2. The response to group II phospholipase A2 was concentration dependent, and prostaglandin E2 production in response to 1 microgram/ml purified group II enzyme was comparable to the maximal response elicited by lipopolysaccharide. Group II phospholipase A2 required millimolar concentrations of extracellular Ca2+ for the induction of prostaglandin E2 production, as well as for phospholipase A2 activity. YM-26734 (4-(3,5-didodecanoyl-2,4,6-trihydroxyphenyl)-7-hydroxy-2-(4-hydroxyph eny l) chroman), a selective inhibitor of group II phospholipase A2, inhibited not only the enzyme activity but also the prostaglandin E2 production-inducing activity of group II phospholipase A2 in a concentration-dependent manner. These findings suggest that group II phospholipase A2 released into the extracellular space may induce prostaglandin E2 production through hydrolysis of plasma membrane phospholipids. Taken together with the previous finding that YM-26734 suppressed inflammatory responses in vivo, these results suggest that group II phospholipase A2 may play a role in the excitation and/or progression of inflammatory processes through the production of eicosanoids.

Animals↗
PubMed1994

Purification and cDNA cloning of human liver CYP4A fatty acid omega-hydroxylase.

Laurate omega-hydroxylase activity of human liver microsomes was strongly inhibited by an antibody against rabbit fatty acid omega-hydroxylase P450 4A5, and Western blot analysis with this antibody showed the presence of two immunochemically related proteins with apparent molecular weights of approximately 50 and 52 kDa in all of 14 human liver specimens examined. A fatty acid omega-hydroxylase (designated P450HL omega) was purified to a specific content of 15 nmol of P450/mg of protein from microsomes of a single human liver on the basis of its laurate omega-hydroxylase activity and its reactivity with the P450 4A5 antibody. This P450HL omega showed an apparent molecular weight of 52 kDa on SDS-PAGE. Furthermore, a cDNA clone (designated HL24) has been isolated from a human liver cDNA library by using the cDNA for P450 4A5 as a probe. The sequence of residues 5 through 25 deduced from cDNA HL24 was identical to the NH2-terminal amino acid sequence of P450HL omega except for one undetermined residue. This cDNA encoded a protein of 519 amino acids with a molecular weight of 59,347. The amino acid sequence predicted from the cDNA showed 82% identity with that of P450 4A5. Northern blot analysis showed that the mRNA hybridized to the cDNA is expressed in the human liver and kidney. (ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1994

Factors improving L-threonine production by a three L-threonine biosynthetic genes-amplified recombinant strain of Brevibacterium lactofermentum.

When a Brevibacterium lactofermentum L-threonine producer that accumulated L-lysine as well, was transformed with a recombinant plasmid carrying the indigenous hom, thrB, and thrC genes, delayed growth and plasmid instability were observed. Addition of organic nutrients, vitamins (thiamine.HCl and d-biotin), and NaCl under the optimal culture conditions solved these problems and further increased threonine production in a small jar fermentor.

Brevibacterium↗
PubMed1994

Corneal endothelial cells block T cell proliferation, but not T cell activation or responsiveness to exogenous IL-2.

We previously reported that Ia+ monolayers of LEW rat corneal endothelial (CE) cells were unable to stimulate proliferation of MHC compatible T cell lines or IL-2 release from hybridomas, and inhibited [3H]-thymidine incorporation when added to conventional lymphocyte proliferation assays. Our purpose was to further analyze the mechanism of the inhibitory activity of CE cells on T lymphocyte activation. Mitogen-induced proliferative responses of splenocytes were found to be as susceptible to inhibition by CE cells as previously reported for antigen-specific activation of T cell lines. Antigen presenting cell (APC) antigen pulsing experiments showed that CE cells did not inhibit antigen processing. Flow cytometry and microscopic observation of the co-cultures revealed that T cells became activated in the presence of antigen, APC and CE cells, exhibiting morphologic changes of blast cell formation, although they did not divide unless given exogenous IL-2. However, if T cells were preactivated in the absence of CE cells, they were no longer susceptible to inhibition if subsequently transferred into CE cell-conditioned medium or onto CE cells. Evidence for an inhibitory factor in CE cell culture supernatant was revealed by two approaches: 1) addition of conditioned medium from CE cell cultures led to inhibition of lymphocyte proliferation assays, and 2) split-well assays also demonstrated the existence of a cell-free immunosuppressive factor produced by the CE cells. However, the inhibition mediated by supernatant alone was less potent than that by direct T cell contact with CE cells, implying that cell-cell interaction contributed to the inhibition. Indomethacin, a prostaglandin synthetase inhibitor, did not reverse CE cell-mediated inhibition. Neutralizing antibodies to TGF-beta 1 and 2 did not reverse the inhibition by CE cells. In summary, T cells received activation signals from APC in the presence of CE cells, but proliferation was inhibited unless exogenous lymphokine was added.

Animals↗
PubMed1993

Purification and characterization of UDP-GlcNAc:Gal beta 1-4Glc(NAc) beta-1,3-N-acetylglucosaminyltransferase (poly-N-acetyllactosamine extension enzyme) from calf serum.

UDP-GlcNAc:Gal beta 1-4Glc(NAc) beta-1,3-N-acetylglucosaminyltransferase is involved in the initiation and the extension of poly-N-acetyllactosamine biosynthesis. This enzyme has been purified to about 125,000-fold with a 0.2% yield from calf serum. The purification was achieved by ammonium sulfate precipitation and chromatography on concanavalin A-Sepharose, DEAE-Toyopearl, SP-Toyopearl, Sephacryl S-200, AF-Blue-Toyopearl, and Mono Q columns. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed a single band corresponding to an apparent M(r) of 70,000. This component was specifically photoaffinity-labeled with 4-thiouridine diphosphate. Exoglycosidase digestion and methylation analysis of the reaction products demonstrated that the enzyme catalyzed the transfer of one N-acetylglucosamine to position C-3 of the terminal galactosyl residue of lactose or N-acetyllactosamine in the beta linkage. The enzyme required Mn2+ ions for its activity and showed a broad pH optimum around 7.0. Apparent Km values for lactose, N-acetyllactosamine, and UDP-GlcNAc were 18.2, 19.6, and 0.129 mM, respectively. Acceptor specificity was tested using several oligosaccharides. The results indicated that terminal Gal beta 1-4Glc(NAc) sequences (type II chains) were preferred substrates for the enzyme. Terminal Gal beta 1-3GlcNAc sequences (type I chains), Lewis X trisaccharides (Gal beta 1-4(Fuc alpha 1-3)GlcNAc), and monosaccharides (galactose) did not serve as substrates.

Animals↗
PubMed1993

Transient recruitment of osteoclasts and expression of their function in osteopetrotic (op/op) mice by a single injection of macrophage colony-stimulating factor.

Severe deficiency of osteoclasts in op/op mice, caused by the absence of functional macrophage colony-stimulating factor (M-CSF), is cured by daily injections of purified recombinant human M-CSF (rhM-CSF). In this study, we found that a single injection of 5 micrograms rhM-CSF is enough for recruitment of osteoclasts in mutant mice. Osteoclast number increased during the period between 2 and 4 days after the single rhM-CSF injection. When YM175, a new derivative of bisphosphonate, was administered to the mice 4 days after rhM-CSF injection or later, osteoclasts disappeared by 3 days after YM175 administration. However, a significant number of osteoclasts were detected even at 3 days after YM175 administration when YM175 was administered 3 days after rhM-CSF injection or earlier. These results indicate that YM175 is cytotoxic only to functioning osteoclasts and that recruitment of osteoclasts is finished 4 days after a single rhM-CSF injection. The osteoclasts actively resorbed bone trabeculae for a prolonged period, demonstrating that M-CSF is not requisite for the functioning of mature osteoclasts.

Animals↗