Biomedical subjects

T Osawa

Publications and source records attributed to T Osawa.

At least 127 records · Page 7Linked to original sources

PubMed1996

Immunochemical detection of carboxymethylated Apo B-100 in copper-oxidized LDL.

The objective of this study was to detect carboxymethyllysine (CML) moieties in biological materials using immunological methods. We prepared a polyclonal antibody specific to the carboxymethylated protein. The antibody recognizes the CML moiety in protein. Protein-bound carboxymethylcysteine (CMS) is scarcely recognized by the antibody. By use of the antibody, the formation of carboxymethylated apo B in copper-oxidized low density lipoprotein (ox-LDL) was immunochemically confirmed. To clarify the source compounds of carboxymethylation, various aldehyde-modified BSAs were prepared, and their reactivities with the antibody were investigated. Among the aldehyde-modified BSAs used, the glyoxal-BSA adduct showed a strong antigenicity. As far as we know, this is the first report of the detection of the CML moiety in ox-LDL using immunological methods.

Antibodies↗

PubMed1996

4-Hydroxy-2-nonenal cytotoxicity in renal proximal tubular cells: protein modification and redox alteration.

4-Hydroxy-2-nonenal (HNE), one of the major products of membrane lipid peroxidation, has been shown recently to be present in a form covalently attached to proteins in the renal proximal tubules of rats treated with a renal carcinogen, ferric nitrilotriacetate (Toyokuni, S., et al. (1994) Proc. Natl. Acad. Sci. USA 91, 2616-2620; Uchida, K., et al. (1995) Arch. Biochem. Biophys. 317, 405-411). In the present study, the mechanism of HNE cytotoxicity was studied using the renal tubular epithelial cells (LLC-PK1), focusing on the protein modification and alteration of cellular redox status induced by HNE. Upon treatment with HNE for 2 h, the LLC-PK1 cells were found to be resistant to the low concentration (10 microM) of HNE, while HNE at higher concentrations (> or = 50 microM) mediated cell death. The cytotoxicity of HNE appeared to be correlated with the HNE modification of cellular proteins. Among a number of proteins modified by HNE, a glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase was detected as one of the major targets of HNE in the cells. On the other hand, exposure of LLC-PK1 cells to HNE resulted in rapid reduction of cellular glutathione (GSH) levels, suggesting that HNE influenced primarily the redox status of the cells. Depletion of GSH with buthionine sulfoximine, a potent suppressor of GSH biosynthesis, before HNE treatment caused the cells to be sensitive to HNE cytotoxicity and to HNE modification of cellular proteins, whereas the increase in intracellular GSH levels by treatment with N-acetylcysteine before HNE treatment resulted in a dose-dependent inhibition of HNE-mediated protein modification. These results suggest that intracellular GSH is a determinant on cellular resistance against the HNE-mediated cytotoxicity.

Acetylcysteine↗

PubMed1996

Involvement of the beta-diketone moiety in the antioxidative mechanism of tetrahydrocurcumin.

We examined the inhibitory effects of curcumin and tetrahydrocurcumin (THC), one of the major metabolites of curcumin, on the lipid peroxidation of erythrocyte membrane ghosts induced by tertbutylhydroperoxide. The results demonstrated that THC showed a greater inhibitory effect than curcumin. To investigate the mechanism of antioxidative activity, we examined the effects of several inhibitors, such as antioxidant enzymes, hydroxyl radical scavengers, 1O2 quencher, and chelating agents for metal ions. Given that all inhibitors failed to inhibit membrane peroxidation, THC must scavenge radicals such as tert-butoxyl radical and peroxyl radical. To clarify the antioxidative mechanism of THC, in particular the role of the beta-diketone moiety, dimethylated THC was incubated with peroxyl radicals generated by thermolysis of 2,2'-azobis(2,4-dimethylvaleronitrile). Four oxidation products were detected, three of which were identified as 3,4-dimethoxybenzoic acid, 3',4'-dimethoxyacetophenone, and 3-(3,4-dimethoxyphenyl)-propionic acid. The fourth oxidation product seems to be an unstable intermediate, and its detailed structure has not been determined. These results suggest that the beta-diketone moiety of THC must exhibit antioxidative activity by cleavage of the C-C bond at the active methylene carbon between two carbonyls in the beta-diketone moiety. Because THC is one of the major metabolites of curcumin, it may also exhibit the same physiological and pharmacological properties as the active form of curcumin in vivo by means of the beta-diketone moiety as well as phenolic hydroxy groups.

Animals↗

PubMed1996

Oxidative stress response in iron-induced renal carcinogenesis: acute nephrotoxicity mediates the enhanced expression of glutathione S-transferase Yp isozyme.

An iron chelate, ferric nitrilotriacetate (Fe-NTA), induces acute renal proximal tubular necrosis, a consequence of free radical-mediated oxidative tissue damage, that eventually leads to a high incidence of renal adenocarcinoma in rodents. In the present study, we investigated the free radical-induced oxidative stress response in this carcinogenesis model, focusing on the expression of glutathione S-transferases (GSTs) which catalyze the conjugation of reactive chemicals with glutathione and play an important role in protecting cells. A single intraperitoneal Fe-NTA treatment (15 mg Fe/kg body weight) induced a rapid oxidative stress, which was monitored by the accumulation of lipid peroxidation products and the loss of sulfhydryl contents in the kidneys, resulting in a 30% reduction of GST activity 1 h after an Fe-NTA treatment. The enzyme activity returned to the control level after 16 h. The immunoblot analysis of GST isozymes demonstrated that the level of alpha-class GSTs (GST-Ya and GST-Yc) and pi-class GST (GST-Yp), major GST isozymes constitutively produced in the kidney, decreased immediately within 1 h of the Fe-NTA treatment. The onset of the recovery of GST-Yp protein levels was detected 3 h after the Fe-NTA treatment. The enhanced production of GST-Yp in gene expression was evident in the drastic elevation of mRNA levels and these increases coincided with a substantial rise in the GST activity and protein levels. The alpha-class GSTs were not inducible by treatment with Fe-NTA. The immunohistochemical analysis demonstrated that the expression of GST-Yp was strongly induced in the regenerating proximal tubular cells. A steady accumulation of GST-Yp protein was observed in the subacute toxicity experiments with multiple injections of Fe-NTA. These results suggest that the enhanced expression of GST-Yp is important in mediating cell repairs or increasing the resistance to subsequent injury.

Adenocarcinoma↗

PubMed1996

Characterization of carbohydrate-binding protein p33/41: relation with annexin IV, molecular basis of the doublet forms (p33 and p41), and modulation of the carbohydrate binding activity by phospholipids.

A protein, p33/41, expressed in bovine kidney and many other tissues was identified as a lectin which binds to sialoglycoproteins and glycosaminoglycans in a calcium-dependent manner. Partial amino acid sequences of p33/41 are highly homologous to those of calcium/phospholipid-binding annexin protein, annexin IV (endonexin), p33/41 exhibited similar calcium/phospholipid-binding activity (Kojima, K., Ogawa, H., Seno, N., Yamamoto, K., Irimura, T., Osawa, T., and Matsumoto, I. (1993) J. Biol. Chem. 267, 20536-20539). To further characterize p33/41, we cloned the p33/41 cDNA and characterized the recombinant protein encoded by this cDNA. Oligonucleotide probes were synthesized based on partial amino acid sequences of p33/41 and used for screening. A p33/41 cDNA clone was isolated encoding a protein of 319 amino acids with a calculated molecular mass of 35,769 Da. The deduced amino acid sequence was identical to that of bovine annexin IV except for one amino acid substitution. The recombinant protein gave two 33-kDa (p33) and 41-kDa (p41) bands on SDS-polyacrylamide gel electrophoresis under non-reducing conditions, and only one 33-kDa band under reducing conditions, as did the native protein. Mass spectrometric analysis combined with site-directed mutagenesis of each of the four cysteine residues of the recombinant protein revealed that p41 is a dimer of p33 cross-linked at Cys-198 via a disulfide bond. The recombinant protein bound to columns of heparin and fetuin glycopeptides in a calcium dependent manner and to phospholipid vesicles composed of phosphatidylserine (PS)/phosphatidylcholine (PC), phosphatidylethanolamine (PE)/PC or phosphatidylinositol (PI)/PC. Furthermore, concurrent binding assays showed that the binding of the recombinant protein to phospholipid vesicles was not affected by heparin, whereas that to heparin was influenced by the phospholipid composition of the vesicles; the highest binding was observed with vesicles composed of PE/PC. These results suggest that p33/41 binds two types of ligands via different sites and that phospholipids modulate the carbohydrate binding activity of p33/41.

Amino Acid Sequence↗

PubMed1996

Oxidative stress response in iron-induced acute nephrotoxicity: enhanced expression of heat shock protein 90.

Iron overload with ferric nitrilotriacetate (Fe-NTA) induces acute renal proximal tubular necrosis, a consequence of oxidative tissue damage, that leads to a high incidence of renal adenocarcinoma in rodents. In the present study, we determined the proteins preferentially produced in response to the Fe-NTA-induced oxidative injury. A single intraperitoneal Fe-NTA treatment led to the enhanced production of a number of proteins with molecular masses of 85-95 kDa. These included heat shock protein 90 (HSP90) as determined by immunoprecipitation. The enhanced production of HSP90 was prominent in the renal tubular cells. Steady accumulation of HSP90 was observed in the subacute toxicity experiments with multiple injections of Fe-NTA, suggesting that the enhanced production of HSP90 is important in increasing resistance to subsequent injury caused by the Fe-NTA-induced oxidative stress.

Animals↗

PubMed1996

Molecular cloning and expression of cDNA encoding human macrophage C-type lectin. Its unique carbohydrate binding specificity for Tn antigen.

A human macrophage calcium-dependent (C-type) lectin cDNA clone was obtained from a library derived from IL-2-treated peripheral blood monocytes. The cDNA cloning was based on the structural homology to hepatic asialoglycoprotein receptors. The nucleotide sequence of this cDNA clone was homologous to those of the galactose- and N-acetylgalactosamine-specific C-type macrophage lectins of rodents. In the putative carbohydrate recognition domain, deduced amino acid sequence revealed 60 and 63% homology to galactose- and N-acetylgalactosamine-specific C-type macrophage lectins of mice and rats, respectively. The cDNA clone was ligated into a mammalian expression vector and transfected into COS-1 cells. In the lysates of these cells, an MR 38,000 component, which bound to galactose-Sepharose, was identified after electrophoretic separation by its interaction with polyclonal antisera against synthetic polypeptides representing a portion of the carbohydrate recognition domain. The carbohydrate-binding specificity of the recombinant macrophage lectin was investigated by comparing elution profiles of various glycopeptides having defined carbohydrate structures from immobilized macrophage lectins. When N-terminal octapeptides from human glycophorin A that bore NeuAc alpha 2-3 Gal beta 1-3 (NeuAc alpha 2-6) GalNAc and its sequentially deglycosylated derivatives were compared, glycopeptides carrying three constitutive GalNAc-Ser/Thr (Tn Ag) strongly bound to the recombinant human macrophage lectin. This is the first study to demonstrate that human macrophage cell surface lectin recognizes Tn Ag, a well-known human carcinoma-associated epitope.

Amino Acid Sequence↗

PubMed1996

Oxidation products of cyanidin 3-O-beta-D-glucoside with a free radical initiator.

Recently, we have reported that anthocyanins show strong antioxidative activity, but no attention has been paid to anthocyanins from the viewpoint of the reaction mechanism of alkylperoxyl radicals; therefore, we investigated the reaction products of antioxidative anthocyanins (cyanidin 3-O-beta-D-glucoside). Cyanidin 3-O-beta-D-glucoside was reacted with 2,2'-azobis(2,4-dimet hylvaleronitrile) to generate the alkylperoxyl radicals, and the reaction products were isolated by high-performance liquid chromatography. The products were identified as 4,6-dihydroxy-2-O-beta-D-glucosyl-3-oxo-2,3-dihydroben-zofuran+ ++ and protocatechuic acid. Based on reaction products, the antioxidative mechanism of cyanidin 3-O-beta-D-glucoside may be different from that of alpha-tocopherol; cyanidin 3-O-beta-D-glucoside would produce another radical scavenger, as it would break down the structure and scavenge the radicals.

Anthocyanins↗

PubMed1996

Subcloning of three osteoblastic cell lines with distinct differentiation phenotypes from the mouse osteoblastic cell line KS-4.

Three distinct osteoblastic cell lines (KS418, KS460, and KS483) were subcloned from the mouse osteoblastic KS-4 cells, which possessed the abilities not only to differentiate into mature osteoblasts, but also to support osteoclast differentiation in coculture with spleen cells. The order of the magnitude of the basal alkaline phosphatase (ALP) activity was KS483 > KS418 > KS460. KS483 cells were also more differentiated than KS418 and KS460 in terms of ALP activity and osteocalcin production, when cultured in growth medium containing 10% fetal bovine serum. In long-term culture, KS418 and KS483 apparently differentiated into mature osteoblasts and formed calcified nodules without addition of beta-glycerophosphate. Electron microscopic analysis demonstrated that calcification occurring in the nodules was initiated in the matrix vesicles as observed in bone formation in vivo. Nodule formation and mineral deposition occurred simultaneously in the presence of beta-glycerophosphate, but the former always preceded the latter without addition of beta-glycerophosphate. In contrast, KS460 cells did not show time-dependent increases of ALP activity, type I collagen expression and osteocalcin production, which were induced by treatment with recombinant osteogenic protein-1 (OP-1). The three cell lines similarly supported osteoclast differentiation in coculture with spleen cells in response to 1,25-dihydroxyvitamin D3. These results indicate that the three cell lines subcloned from the original KS-4 cells represent phenotypically distinct osteoblasts during osteoblast differentiation, but are equipped similarly with the capacity to support osteoclast differentiation. The subcloned cells of the KS-4 series may provide useful systems in which to study osteoblast differentiation and function.

Actins↗

PubMed1996

Transdominant inhibition of Moloney murine leukemia virus proliferation by defective mutants of reverse transcriptase.

To investigate the dominant-negative inhibition of Moloney murine leukemia virus (Mo-MuLV) proliferation by polymerization-defective mutants of reverse transcriptase (RT), we constructed several plasmids harboring the Mo-MuLV RT gene in which the YVDD sequence, one of the conserved sequences in RNA-dependent polymerases, was altered, and then transformed mouse NIH3T3 cells and Escherichia coli with the mutant plasmids. Mouse NIH3T3 cells expressing these mutant RT genes were highly resistant to Mo-MuLV proliferation. The mutant RT expressed in E. coli exhibited no polymerization activity, but it retained its binding activity to the template RNA and inhibited in vitro poly(dG) synthesis occurring with the wild-type RT. These results suggest that the competition for binding of the two types of enzymes to the template is responsible for the resistance to Mo-MuLV proliferation and that the YVDD sequence of Mo-MuLV may be a good target for dominant-negative inhibition of retroviral proliferation.

3T3 Cells↗

PubMed1996

8-hydroxy-2'-deoxyguanosine is increased in epidermal cells of hairless mice after chronic ultraviolet B exposure.

8-Hydroxy-2'-deoxyguanosine (8-OHdG) is a mutation-prone (G:C to T:A transversion) DNA base-modified product generated by reactive oxygen species or photodynamic action. G:C to T:A transversions are observed in the p53 and ras genes of UVB-induced skin cancers of mice and in squamous and basal cell carcinomas of human skin exposed to sunlight. In the current study, 8-OHdG formation was evaluated in the epidermis of hairless mice after repeated exposure to UVB, and possible mechanisms involved were studied. Exposure of hairless mice to either 3.4 [2 minimal erythema dose (MED)] or 16.8 (10 MED) kJ/m2 of UVB three times a week for 2 wk induced a 2.5- or 6.1-fold increase, respectively, in the levels of 8-OHdG in DNA, compared to the unexposed controls. An immunohistochemical method using a monoclonal antibody specific for 8-OHdG showed stronger and more extensive staining in the nuclei of UV-irradiated epidermal cells than in those of nonirradiated cells. Western blots probed with antibodies against 4-hydroxy-2-nonenal-modified proteins confirmed the involvement of reactive oxygen species in the epidermal damage induced by chronic UVB exposure. 3-Nitro-L-tyrosine was detected in western blots in a concentration-dependent manner, suggesting that peroxynitrite derived from the reaction of nitric oxide and superoxide, both of which were probably released from inflammatory cells, was involved in modifying the DNA bases. Therefore, the formation of 8-OHdG after UVB exposure appears to be regulated by at least three pathways: photodynamic action, lipid peroxidation, and inflammation and may play a role in sunlight-induced skin carcinogenesis.

8-Hydroxy-2'-Deoxyguanosine↗

PubMed1996

Characterization by Western blotting of mouse intestinal glycoproteins bound by Escherichia coli heat-labile enterotoxin type I.

Escherichia coli heat-labile enterotoxin type I (LT-I)-binding galactoproteins, which were not recognized by cholera toxin, were detected in intestinal epithelial cells of BALB/c mouse by Western blotting. Inhibitory studies using lectins and modifications of sugar chain suggest that LT-I recognizes certain mucin-type sugar chains containing the terminal Galbeta1-3GalNAc sugar sequence in the galactoproteins. The terminal sugar sequence is identical to that of GM1 ganglioside, the well-documented functional receptor for LT-I.

Animals↗

PubMed1996

Preparation of controlled release granules of TA-5707F using enteric polymers and ethylcellulose, and their in vivo evaluation.

Two types of multiple controlled release dosage forms, hydroxypropylmethyl cellulose acetyl succinate (HPMC-AS) coated granules and double layer coated granules with HPMC-AS and ethyl cellulose (EC), were prepared for the newly developed antihistaminergic drug, TA-5707F, using a centrifugal fluidizing granulator. The in vitro dissolution rate of TA-5707F in the second fluid from the former granules could be altered by changing the grade (HPMC-AS-M, AS-H, AS-V) or coating amount. However, the plasma concentration determined in fasted dogs was not markedly affected by the HPMC-AS grades. From the granules coated with 30% (w/w) AS-V (the polymer with the highest pH required for dissolution), little TA-5707F was released into the second fluid, but a fairly high plasma concentration was detected although a constant plasma level was not maintained in the fasted dogs. The dissolution rates from the double layer coated granules were changed by the coating percent of EC. The plasma concentration of TA-5707F in dogs correlated well with the in vitro dissolution rate and was not markedly affected by food. Thus, two kinds of double layer coated granules (coated 0.75% EC and 1% EC) were administered to humans, and showed sustained-releasing plasma profiles; that is, the plasma concentration increased gradually and reached a Cmax 5 and 8 h after administration, respectively. The amount of urinary excretion of TA-5707F and its metabolite was 70% of the dose. This value was almost equal to that of the rapid release tablet. In dogs the in vivo absorption rate was similar to the in vitro dissolution rate, but in humans it was only about half.

Adult↗

PubMed1996

Enhancement of oral bioavailability and pharmacological effect of 1-(3,4-dimethoxyphenyl)-2,3-bis(methoxycarbonyl)-4-hydroxy-6,7,8- trimethoxynaphthalene (TA-7552), a new hypocholesterolemic agent, by micronization in co-ground mixture with D-mannitol.

To improve the bioavailability of the sparingly water-soluble drug, 1-(3,4-dimethoxyphenyl)-2,3-bis(methoxycarbonyl)-4-hydroxy-6,7,8- trimethoxynaphthalene (TA-7552), the usefulness of the co-grinding method with D-mannitol was investigated. The co-grinding was performed at various weight ratios of TA-7552 and D-mannitol using a ball mill. The particle size was markedly reduced with increasing amount of D-mannitol. A mixture ratio greater than or equal to 1:3 of the drug and D-mannitol produced submicron-sized particles. In dogs, bioavailability increased with increasing amount of D-mannitol. The 1:9 co-ground mixture gave complete absorption, as did a lecithin solution of the drug. Even co-ground powders with lower amounts of D-mannitol provided relatively high bioavailability in comparison with ground drug powder alone of a similar particle size. Further, pharmacological examination using rats indicated that the inhibition of cholesterol absorption was intestified with the reduction of particle size. These findings suggest that the co-grinding method with D-mannitol is useful for enhancing the bioavailability and pharmacological effectiveness of this sparingly water-soluble drug.

Animals↗

PubMed1996

Inhibition of Escherichia coli heat-labile enterotoxin by an amino carbonyl product, lactose-alpha-lactalbumin.

The inhibition by lactose-alpha-lactalbumin amino carbonyl product of Escherichia coli heat-labile enterotoxin was studied by GM1-ELISA and by assay with CHO-K1 cells. The product dose-dependently inhibited the binding of the enterotoxin to GM1 ganglioside and decreased the morphological change of CHO-K1 cells caused by this toxin. The results suggest that this product may be a receptor analogue in the intestine.

Animals↗

PubMed1996

Inhibitory effects of caffeic acid ethyl ester on H2O2-induced cytotoxicity and DNA single-strand breaks in Chinese hamster V79 cells.

Cytotoxicity and DNA single-strand breaks caused by H2O2 were assessed by a colony formation assay and a DNA precipitation assay, respectively, with Chinese hamster V79 cells. In both assays, caffeic acid ethyl ester showed protective effects. The structure-activity relationship showed that the o-dihydroxy structure of caffeic acid ethyl ester was essential for the protective effects.

Animals↗

PubMed1995

Fertirelin and buserelin compared by LH release, milk progesterone and subsequent reproductive performance in dairy cows treated for follicular cysts.

This field study compared the efficacy of a single injection of 200 microg fertirelin with that of 20 microg buserelin in shortening the recovery period of 68 laotating Holstein-Friesian cows with ovarian follicular cysts 65 d post partum or later. Differential diagnosis was based not only on palpation per rectum but also on skim milk progesterone profiles ( or= 1.0 ng/ml) 1 wk after treatment, was evidenced in 75% of the fertirelin group and 72% of the buserelin group. A 74% conception rate was achieved in fertirelin-treated cows, with a mean interval of 71 d from treatment to conception. In the buserelin-treated cows, 65% conceived and the treatment-to-conception interval was 63 d. Differences were insignificant. At the doses used the two GnRH analogs were deemed equally effective in managing particularly stubborn follicular cysts.

Journal Article↗

PubMed1995

4-Hydroxy-2-nonenal-trapping ELISA: direct evidence for the release of a cytotoxic aldehyde from oxidized low density lipoproteins.

Among the aldehydes that originate from the peroxidation of cellular membrane lipids, 4-hydroxy-2-nonenal (HNE) is thought to be largely responsible for cytopathological effects observed during oxidative stress. Taking advantage of the fact that HNE is very reactive with proteins and forms stable Michael addition-type adducts, a novel immunochemical procedure for quantifying "free" HNE has been developed. The method designated as "HNE-trapping ELISA" is based on the detection of HNE trapped by a protein that has been coated in the immunoplate. The HNE-derived epitopes generated in the coating protein are then detected by the ELISA using a monoclonal antibody (mAbHNEJ-2) specific to the haptenic groups of the HNE-protein conjugates. Using this method, we determined that a considerable amount of HNE was released from human plasma low density lipoproteins (LDL) treated with copper ions or endothelial cells.

Aldehydes↗