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Biomedical subjects

T Osawa

Publications and source records attributed to T Osawa.

At least 73 records · Page 4Linked to original sources
PubMed1999

Activation of stress signaling pathways by the end product of lipid peroxidation. 4-hydroxy-2-nonenal is a potential inducer of intracellular peroxide production.

In the present study, we studied the signal transduction mechanism that is involved in the expression of c-Jun protein evident after exposure of rat liver epithelial RL34 cells to the major end product of oxidized fatty acid metabolism, 4-hydroxy-2-nonenal (HNE). HNE treatment of the cells resulted in depletion of intracellular glutathione (GSH) and in the formation of protein-bound HNE in plasma membrane. In addition, HNE strongly induced intracellular peroxide production, suggesting that HNE exerted oxidative stress on the cells. Potent expression of c-Jun occurred within 30 min of HNE treatment, which was accompanied by a time-dependent increase in activator protein-1 (AP-1) DNA binding activity. We found that HNE caused an immediate increase in tyrosine phosphorylation in RL34 cells. In addition, HNE strongly induced phosphorylation of c-Jun N-terminal kinases (JNK) and p38 mitogen-activated protein kinases and also moderately induced phosphorylation of extracellular signal-regulated kinases. The phosphorylation of JNK was accompanied by a rapid and transient increase in JNK and p38 activities, whereas changes in the activity of extracellular signal-regulated kinase were scarcely observed. GSH depletion by L-buthionine-S, R-sulfoximine, a specific inhibitor of GSH biosynthesis, only slightly enhanced peroxide production and JNK activation, suggesting that HNE exerted these effects independent of GSH depletion. This and the findings that (i) HNE strongly induced intracellular peroxide production, (ii) HNE-induced JNK activation was inhibited by pretreatment of the cells with a thiol antioxidant, N-acetylcysteine, and (iii) H2O2 significantly activated JNK support the hypothesis that pro-oxidants play a crucial role in the HNE-induced activation of stress signaling pathways. In addition, we found that, among the inhibitors of tyrosine kinases, cyclooxygenase, and Ca2+ influx, only quercetin exerted a significant inhibitory effect on HNE-induced JNK activation. In light of the JNK-dependent induction of c-jun transcription and the AP-1-induced transcription of xenobiotic-metabolizing enzymes, these data may show a potential critical role for JNK in the induction of a cellular defense program against toxic products generated from lipid peroxidation.

Aldehydes↗
PubMed1999

The structure of the interstitial surfaces of the epithelial basement membranes of mouse oral mucosa, gingiva and tongue.

It is known that the gaps between epithelium and the underlying connective tissue usually occur between the epithelial cells and the basement membrane, resulting in exposure of the cellular surface of the lamina densa. After dithiothreitol separation, the epithelia of oral mucosa, gingiva, and tongue were mechanically peeled off from the underlying connective tissues. This treatment severed the connections between the basement membrane and the underlying connective tissue and the anchoring fibrils were pulled off from the collagen layer. In contrast, connections between the epithelial cells and basement membrane were preserved, resulting in exposure of the interstitial surfaces of the laminae densae. Scanning electron-microscopic observations of those interstitial surfaces were possible using the specimens prepared as above. The basement membranes of these three oral epithelia were morphologically the same not only by transmission but also by scanning electron microscopy. Scanning electron-microscopic observations revealed that their laminae densae were composed of fine fibrils and demonstrated three-dimensionally the projection of the anchoring fibrils from the laminae densae to the interstitial side. These findings coincide with those for epidermal basement membrane, which had already been observed with the same method.

Animals↗
PubMed1999

Protective role of dietary polyphenols in oxidative stress.

The oxidative break down of the membrane polyunsaturated fatty acids is known to be accompanied by the formation of a complex mixture of lipidhydroperoxides and secondary products. These compounds are highly reactive and are capable of rapid reaction with cellular nucleophiles such as phospholipids and proteins, and it was found that these reaction products are candidates as impotant biomarkers to evaluate antioxidative activity of dietary antioxidants. The author has been involved in developing immunochemical detection methods for oxidative stress by application of polyclonal and monoclonal antibodies. From the hypothesis that endogenous antioxidants in plants must play an important role for antioxidative defense systems from oxidative stress, an intensive search for novel type of natural antioxidants has been carried out from numerous plant materials, including those used as foods, and we have isolated and identified a number of lipid-soluble and water-soluble dietary antioxidants from crop seeds, sesame seeds and some spices. In this paper, the recent progress of research on functions of dietary antioxidants is reviewed.

Antioxidants↗
PubMed1999

Chemoprevention by curcumin during the promotion stage of tumorigenesis of mammary gland in rats irradiated with gamma-rays.

We have evaluated the chemopreventive effects of curcumin on diethylstilbestrol (DES)-induced tumor promotion of rat mammary glands initiated with radiation. Sixty-four pregnant rats received whole body irradiation with 2.6 Gy gamma-rays from a 60Co source at day 20 of pregnancy and were divided into two groups after weaning. In the control group of 39 rats fed a basal diet and then implanted with a DES pellet for 1 year, 33 (84.6%) developed mammary tumors. Twenty-five rats were fed diet containing 1% curcumin immediately after weaning and received a DES pellet, as for the control. The administration of dietary curcumin significantly reduced the incidence (28.0%) of mammary tumors. Multiplicity and Iball's index of mammary tumors were also decreased by curcumin. Rats fed the curcumin diet showed a reduced incidence of the development of both mammary adenocarcinoma and ER(+)PgR(+) tumors in comparison with the control group. On long-term treatment with curcumin, body weight and ovarian weight were reduced, but liver weight was increased. Compared with the control rats, the curcumin-fed rats showed a significant reduction in serum prolactin, whereas estradiol-17beta and progesterone concentrations were not significantly different between the two groups. Curcumin did not have any effect on the concentration of free cholesterol, cholesterol ester and triglyceride. Feeding of the curcumin diet caused a significant increase in the concentrations of tetrahydrocurcumin, arachidonic acid and eicosapentaenoic acid and a significant decrease in thiobarbituric acid-reactive substance concentration in serum. Whole mounts of the mammary glands showed that curcumin yielded morphologically indistinguishable proliferation and differentiation from the glands of the control rats. These findings suggest that curcumin has a potent preventive activity during the DES-dependent promotion stage of radiation-induced mammary tumorigenesis.

Animals↗
PubMed1999

Dietary defatted sesame flour decreases susceptibility to oxidative stress in hypercholesterolemic rabbits.

Plant glucosides possess antioxidative properties due to their ability to scavenge free radicals. Sesame seeds contain a class of these compounds, the sesaminol glucosides. To evaluate their antioxidative activity in vivo, we fed rabbits diets containing 1% cholesterol (Chol) with or without 10% defatted sesame flour (DSF) (containing 1% sesaminol glucosides) for 90 d. We determined the susceptibility of their tissues to oxidation ex vivo as well as serum total cholesterol (TC), phospholipid (PL), triglyceride (TG) and HDL cholesterol (HDL-C) concentrations. Serum TC, HDL-C, PL and TG levels were unaffected by the addition of DSF. The HDL-C in the Chol + DSF group was greater than in the Chol group at 45 d. Both were greater than in the groups that did not consume cholesterol. Liver TC and TG were significantly lower in rabbits fed the diet containing DSF plus 1% cholesterol than in those fed 1% cholesterol alone. Lipid peroxidation activity, measured as 2-thiobarbituric acid reactive substances (TBARS), was lower in the liver (P < 0.05) and serum (P = 0.06) of rabbits fed DSF plus cholesterol than in rabbits fed the cholesterol diet. Although we did not detect sesaminol glucosides in peripheral tissues, we observed abundant quantities of sesaminol in rabbits fed DSF, the principal metabolite. Our findings suggest that feeding DSF to rabbits does not protect cholesterol-induced hypercholesterolemia, but may decrease susceptibility to oxidative stress in rabbits fed cholesterol, perhaps due to the antioxidative activity of sesaminol.

Analysis of Variance↗
PubMed1999

The advantage of the osmium conductive metal coating for the detection of the colloidal gold-conjugated antibody by SEM.

For the detection of the colloidal gold-conjugated antibody by scanning electron microscopy, the comparison between the secondary and the back-scattered electron images is necessary. After the various durations of osmium conductive metal coatings, enlargements of the diameters of the antibodies were observed. The thick coating reduced the contrast of the backscattered electron images. By the optimal thickness of the coating, the secondary electron image showed a minimum reduction in the resolution, and the backscattered electron image showed not only the gold particles with high contrast, but also the outline of the basic morphology.

Animals↗
PubMed1999

High levels of 8-hydroxy-2'-deoxyguanosine appear in normal human epidermis after a single dose of ultraviolet radiation.

Major photoproducts induced by carcinogenic ultraviolet (UV) radiation are the cylobutane pyrimidine dimers (CPDs) and pyrimidine-pyrimidone (6-4) photoproducts (6-4 PPs). 8-Hydroxy-2 -deoxyguanosine (8-OHdG) is also a DNA base-modified product generated by reactive oxygen species in conditions of ultraviolet stress, Although UVB-induced CPDs and 6-4 PPs have been investigated in animal and human skin, little is known about the role of 8-OHdG in UVB-induced human skin damage or carcinogenesis. Normal human skin from three volunteers was exposed to UV radiation, and the time course of induction and removal of 8-OHdG was examined by immunohistochemical analysis with catalysed signal amplification on formalin-fixed paraffin sections. Formation of CPDs and 6-4 PPs was also examined by immunostaining on the same skin specimens. Control epidermis with no exposure to UV radiation showed little nuclear staining of 8-OHdG, but an increased level of 8-OHdG was clearly observed in epidermis biopsied after irradiation. Induced 8-OHdG can rapidly be removed from nucleus during the first 24-48 h, as the staining intensity diminished gradually, almost reaching the control level by 72-96 h after irradiation. Staining for CPDs or 6-4 PPs revealed induction of these photoproducts in human skin, although 6-4 PP-positive cells disappeared more rapidly than those that stained for CPDs or 8-OHdG. Together with protective effect of antioxidants, our results indicate that not only CPDs and 6-4 PPs but also 8-OHdG may play a significant part in UV carcinogenesis.

8-Hydroxy-2'-Deoxyguanosine↗
PubMed1999

Digestible energy expenditure in grazing activity of growing horses.

Ten Thoroughbred yearlings (5 females and 5 males) were used to examine the effect of time of grazing on pasture forage and digestible energy (DE) intake, bodyweight gain and DE expenditure in grazing activity. Five females were grazed for 17 h/day (LTG), 5 males were grazed for 7 h/day (STG) and they were fed differently. As a result, DE intake from pasture forage of LTG horses and STG horses was 27.3 and 12.7-13.9 Mcal/day, respectively. The average daily gain (ADG) of LTG and STG horses was 0.37 and 0.39-0.61 kg/day, respectively. The regression lines between DE intake and ADG of both groups were parallel and the difference in DE intake between them was 10 Mcal of DE. It was suggested that DE expenditure in grazing activity (entirely at night) of growing horses was 1 Mcal/h under the conditions of this study.

Animal Feed↗
PubMed1999

Phytate and phosphorus movements in the digestive tract of horses.

This experiment was conducted to study phytate degradation and the effect of dietary phytate level on phosphorus absorption in the gastrointestinal tract of horses. Six Thoroughbred horses were fed diets containing low-phytate diet or high-phytate diet for 5 days. The diets were supplemented with Cr2O3 as an unabsorbable marker. The horses were killed 3 h after the last feeding and digesta in some segments of the intestine were collected. In both dietary groups, the daily passage of phytate phosphorus was decreased in the upper small intestine and in the lower large intestine. The daily passage of phytate phosphorus in the lower large intestine did not differ between these groups. Although the passage of total phosphorus increased in the upper small intestine and in the upper large intestine of both groups, its passage decreased in the lower large intestine. These results suggest that phytate phosphorus is degraded in the upper small intestine and in the lower large intestine of both groups. Phosphorus is suggested to be mainly absorbed in the lower large intestine. Therefore, phytate phosphorus is considered to be easily absorbed in horses because the major site of phosphorus is the lower large intestine where most of phytate is already degraded.

Animal Feed↗
PubMed1999

New potent antioxidative o-dihydroxyisoflavones in fermented Japanese soybean products.

A potent antioxidative 6-hydroxydaidzein (6-OHD) was newly isolated from soybean koji fermented with Aspergillus oryzae. 6-OHD, in addition to 8-hydroxydaidzein and 8-hydroxygenistein, were found to be present in various fermented soybean products, including their koji. Considering that these o-dihydroxyisoflavones had strong antioxidative activities, they may contribute to protecting from oxidative deterioration during the processing of fermented soybean products.

Antioxidants↗
PubMed1999

Occurrence of stereoisomers of 1-(2'-pyrrolidinethione-3'-yl)- 1,2,3,4-tetrahydro-beta-carboline-3-carboxylic acid in fermented radish roots and their different mutagenic properties.

Stereoisomers of the tetrahydro-beta-carboline derivative, 1-(2-pyrrolidinethione)-3-yl)-1,2,3,4-tetrahydro-beta-carboline- 3-carboxylic acid (PTCC), were formed from L-tryptophan with 4-methylthio-3-butenyl isothiocyanate, and their mutagenic properties and contents in different types of the radish products were studied. The isomers were identified as (1S*, 3S*, 3R*)- and (1R*, 3S*, 3R*)-PTCCs; the former was found as the major compound but had no mutagenic activity, while the latter was mutagenic toward Salmonella typhimurium TA 98 in the presence of a rat microsomal fraction. Both (1S*, 3S*, 3R*)- and (1R*, 3S*, 3R*)-PTCC were detected in a ratio of about 4:1 in a product fermented for 8 months, but only a trace was apparent in products manufactured within a few weeks.

Animals↗
PubMed1998

Immunohistochemical detection of dityrosine in lipofuscin pigments in the aged human brain.

Lipofuscin is a yellowish brown fluorescent pigment which is sequestered within cytoplasmic granules during aging. To examine the contribution of protein oxidation to lipofuscin accumulation, we performed immunohistochemical detection of dityrosine, which is considered one of the specific markers for protein oxidation, in lipofuscin in the aged human brain using an antibody specific to dityrosine. By characterization using competitive enzyme-linked immunosorbent assay, the specificity of the antibody to dityrosine was confirmed. None of the other tyrosine-related compounds such as L-tyrosine, 3-nitrotyrosine, 3-chlorotyrosine, or 3,4-dihydroxyphenylalanine cross-reacted with the antibody. The anti-dityrosine antibody reacted with lipofuscin granules in the pyramidal neurons of the aged human brain. The results suggest that protein oxidation by free radicals and/or peroxidases may play an important role in lipofuscin accumulation.

Aged↗
PubMed1998

Plasma beta-endorphin around parturition and its relationship to cortisol level and resumption of pituitary and ovarian functions in dairy cows.

The objectives were to evaluate the concentrations of beta-endorphin in peripheral circulation around parturition and to investigate their relationship to the concentrations of cortisol and postpartum resumption of pituitary and ovarian functions in dairy cows. Subjects were 21 Holstein-Friesian cows in late pregnancy. Blood samples were collected from these animals from day 270 in pregnancy until the first ovulation after calving. Average immunoreactive (IR) beta-endorphin concentrations in cows with dystocia (n = 8) in periparturient period (from day 270 of pregnancy until 24 h after calving) were slightly higher than those in cows with normal calving (n = 13) in the same period although the difference was not significant. During the periparturient period, the peak level of IR beta-endorphin was seen at the time of rupture in cows with normal calving and it was observed at the time of parturition in cows with dystocia. The trend of IR beta-endorphin secretion appeared to be concomitant with cortisol secretion in the periparturient period but not in postpartum period (from 24 h after calving until the first ovulation). Average IR beta-endorphin concentrations in cows with dystocia in the postpartum period were not significantly different from those in cows with normal calving. There was no significant association between average IR beta-endorphin concentrations in early postpartum period (from 24 h until 7 days after calving) and the responsiveness of luteinising hormone (LH) to exogenous GnRH administered on day 7 postpartum. However, a negative correlation (r = -0.593; n = 21; P = 0.004) was observed between average IR beta-endorphin concentrations and LH concentrations during the period from day 1 until the first ovulation in the 21 cows. In addition, a positive correlation (r = 0.498; n = 21; P = 0.020) was seen between the beta-endorphin concentrations and time to the first ovulation after calving. The results from this study suggest that beta-endorphin may release into peripheral blood differently from cortisol and that it may be involved in regulating LH secretion and thus the resumption of ovarian cyclicity in postpartum dairy cows.

Animals↗
PubMed1998

Acrolein is a product of lipid peroxidation reaction. Formation of free acrolein and its conjugate with lysine residues in oxidized low density lipoproteins.

Lipoprotein peroxidation, especially the modification of apolipoprotein B-100, has been implicated to play an important role in the pathogenesis of atherosclerosis. However, there have been few detailed insights into the chemical mechanism of derivatization of apolipoproteins during oxidation. In the present study, we provide evidence that the formation of the toxic pollutant acrolein (CH2=CH-CHO) and its conjugate with lysine residues is involved in the oxidative modification of human low density lipoprotein (LDL). Upon incubation with LDL, acrolein preferentially reacted with lysine residues. To determine the structure of acrolein-lysine adduct in protein, the reaction of acrolein with a lysine derivative was carried out. Employing Nalpha-acetyllysine, we detected a single product, which was identified to be a novel acrolein-lysine adduct, Nalpha-acetyl-Nepsilon-(3-formyl-3,4-dehydropiperidino )lysine. The acid hydrolysis of the adduct led to the derivative that was detectable with amino acid analysis. It was revealed that, upon in vitro incubation of LDL with acrolein, the lysine residues that had disappeared were partially recovered by Nepsilon-(3-formyl-3, 4-dehydropiperidino)lysine. In addition, we found that the same derivative was detected in the oxidatively modified LDL with Cu2+ and that the adduct formation was correlated with LDL peroxidation assessed by the consumption of alpha-tocopherol and cholesteryl ester and the concomitant formation of cholesteryl ester hydroperoxide. Enzyme-linked immunosorbent assay that measures free acrolein revealed that a considerable amount of acrolein was released from the Cu2+-oxidized LDL. Furthermore, metal-catalyzed oxidation of arachidonate was associated with the formation of acrolein, indicating that polyunsaturated fatty acids including arachidonate represent potential sources of acrolein generated during the peroxidation of LDL. These results indicate that acrolein is not just a pollutant but also a lipid peroxidation product that could be ubiquitously generated in biological systems.

Acrolein↗
PubMed1998

A novel biomarker for hyperglycemia, MRX isolated from hydrolysate of glycated proteins.

Long-lived proteins can undergo non-enzymatic glycation to form highly crosslinked structures with characteristic fluorescence during aging and diabetes processes. In this paper, a typical fluorophore, named Maillard reaction product X (MRX), was isolated from the hydrolysate of glycated proteins. MRX could be formed by incubation of bovine serum albumin with glucose, followed by acid hydrolysis. The structure of MRX was determined to be 8-hydroxy-5-methyldihydrothiazolo[3,2-alpha] pyridinium-3-carboxylate. MRX was also found to be formed by the incubation of cysteine and arginine with glucose, followed by hydrolysis. We found the formation of MRX in the recently developed genetically diabetic Otsuka Long-Evans Tokushima Fatty (OLETF) rats and compared them with that in the control Long-Evans Tokushima Otsuka (LETO) rats. Significantly higher levels of MRX were observed from the serum (p < 0.005) and urinary protein (p < 0.001) of OLETF rats in comparison with those of LETO rats. MRX must be a potential candidate as a biomarker for hyperglycemia.

Animals↗
PubMed1998

Protein-bound acrolein: potential markers for oxidative stress.

Acrolein (CH2==CH---CHO) is known as a ubiquitous pollutant in the environment. Here we show that this notorious aldehyde is not just a pollutant, but also a lipid peroxidation product that could be ubiquitously generated in biological systems. Upon incubation with BSA, acrolein was rapidly incorporated into the protein and generated the protein-linked carbonyl derivative, a putative marker of oxidatively modified proteins under oxidative stress. To verify the presence of protein-bound acrolein in vivo, the mAb (mAb5F6) against the acrolein-modified keyhole limpet hemocyanin was raised. It was found that the acrolein-lysine adduct, Nepsilon-(3-formyl-3, 4-dehydropiperidino)lysine, constitutes an epitope of the antibody. Immunohistochemical analysis of atherosclerotic lesions from a human aorta demonstrated that antigenic materials recognized by mAb5F6 indeed constituted the lesions, in which intense positivity was associated primarily with macrophage-derived foam cells and the thickening neointima of arterial walls. The observations that (i) oxidative modification of low-density lipoprotein with Cu2+ generated the acrolein-low-density lipoprotein adducts and (ii) the iron-catalyzed oxidation of arachidonate in the presence of protein resulted in the formation of antigenic materials suggested that polyunsaturated fatty acids are sources of acrolein that cause the production of protein-bound acrolein. These data suggest that the protein-bound acrolein represents potential markers of oxidative stress and long-term damage to protein in aging, atherosclerosis, and diabetes.

Acrolein↗
PubMed1998

Detection of oxidized phospholipid-protein adducts using anti-15-hydroperoxyeicosatetraenoic acid-modified protein antibody: contribution of esterified fatty acid-protein adduct to oxidative modification of LDL.

The reaction of lipid hydroperoxide with protein was investigated using an antibody, which was prepared using 15-hydroperoxyeicosatetraenoic acid (15-HPETE)-modified keyhole limpet hemocyanin as an immunogen. The obtained antibody recognized not only 15-HPETE-modified bovine serum albumin (BSA) but also 13-hydroperoxyoctadecadienoic acid (13-HPODE)-modified BSA. Glutaroyl-BSA adduct, which was prepared by the reaction of glutaric anhydride with protein, was also recognized by the antibody. The results revealed that the carboxyl terminus of lipid moiety in adducts was required for an appearance of the antigenicity. The cross-reactivity of phosphatidylcholine hydroperoxide-modified BSA (PCAOOH-BSA) with the antibody was examined. The antibody could not recognize the intact PCAOOH-BSA, whereas alkaline-treated modified BSA revealed the antigenicity. Furthermore, stearic acid at the 1 position in the phospholipid was liberated from the PCAOOH-BSA following treatment with 0.25 N NaOH. The result showed that the phospholipid moiety could be covalently bound to the protein molecule. The formation of esterified fatty acid-protein adduct during oxidation was confirmed using low-density lipoprotein (LDL). During oxidation of LDL by copper ion or 2,2'-azo-bis(2-amidinopropane)dihydrochloride, the formation of antigenic materials was observed in a time- or dose-dependent fashion. The antigenicity was significantly enhanced by the alkaline treatment on the oxidized LDL, suggesting that considerable amounts of oxidized esterified fatty acids can covalently react with apoprotein B-100 in oxidatively modified LDL.

Amidines↗