Biomedical subjects
Publications and source records attributed to T Osawa.
Papaver orientale, Dicentra spectabilis and Chelidonium majus, three Papaveraceae collected from Hokkaido area, were studied for their fungitoxic alkaloids. Ten alkaloids were isolated, identified and tested for their fungitoxic activity. Two of these isolates were found to be new compounds.
Reactive oxygen species (ROS) have been shown to be responsible for inducing DNA damage after ultraviolet radiation (UV). Antioxidant, vitamin E and epigallocatechin gallate extracted from green tea, applied topically to the skin, delayed the onset of UV-induced skin cancer in mice. Since olive oil is reported to have a potent antioxidative effect in in vitro system, we asked whether, topical use of olive oil reduces the number and delays the onset of UV-induced skin cancer in mice. We found that super virgin olive oil painted immediately after UVB radiation significantly delayed the onset and reduced the number of skin cancer, but pretreatment of super virgin olive oil and pre- and/or post treatment by regular olive oil neither retarded nor reduced skin cancer formation in UV-irradiated mice. Further, 8-hydroxy-deoxyguanosine (8-OHdG) formation in mice epidermis was apparently reduced by super virgin olive oil painted immediately after UV radiation, although cyclobutane pyrimidine dimers and (6-4) photoproducts were not reduced by olive oil treatment. Our results suggest that daily topical use of super virgin olive oil after sun bathing may delay and reduce UV-induced skin cancer development in human skin, possibly by decreasing ROS-induced 8-OHdG which is responsible for gene mutation.
We investigated the effect of polyphenols derived from cacao liquor on the mutagenic action of heterocyclic amines (HCAs) in vitro and ex vivo. In the Ames test, the cacao liquor polyphenols showed antimutagenic effects in bacteria treated with HCA in the presence of an S-9 mixture; however, they showed less efficacy than quercetin. On the other hand, the cacao liquor polyphenols showed potent antimutagenic activity in bacteria treated with activated forms of HCA, compared with quercetin. We also evaluated the effect of these compounds on enzymatic activation of HCA. They weakly suppressed the production of activated HCA. In the host-mediated assay in mice, a method used to estimate the potential carcinogenicity of chemicals ex vivo, oral administration of the cacao liquor polyphenols, reduced the number of colonies of revertant bacteria recovered from the liver. These data suggest that the cacao liquor polyphenols have an antimutagenic effect not only in vitro, but also ex vivo.
Eriocitrin, a flavonoid glycoside present in lemon fruit, is metabolized in vivo to a series of eriodictyol, methylated eriodictyol, 3,4-dihydroxyhydrocinnamic acid, and their conjugates. Plasma antioxidant activity increased following oral administration of aqueous eriocitrin solutions to rats. Eriocitrin metabolites were found in plasma and renal excreted urine through HPLC and LC-MS analyses. Eriocitrin was not detected in plasma and urine, but eriodictyol, homoeriodictyol, and hesperetin in their conjugated forms were detected in plasma of 4.0 h following administration of eriocitrin. In urine for 24 h, both nonconjugates and conjugates of these metabolites were detected. 3,4-Dihydroxyhydrocinnamic acid, which is metabolized from eriodictyol by intestinal bacteria, was detected in slight amounts with each form in 4.0-h plasma and 24-h urine. Eriocitrin was suggested to be metabolized by intestinal bacteria, and then eriodictyol and 3,4-dihydroxyhydrocinnamic of its metabolite were absorbed. Following administration of eriocitrin, plasma exhibited an elevated resistance effect to lipid peroxidation. Eriocitrin metabolites functioning as antioxidant agents are discussed.
This investigation evaluated the preventive effect of curcumin on radiation-induced tumor initiation in rat mammary glands. Fifty-four female rats were mated and then divided into two groups at day 11 of pregnancy. As the control group, 27 rats were fed a basal diet during the experimental period. As the experimental group, 27 rats were fed a diet containing 1% curcumin between day 11 of pregnancy and parturition (day 23 of pregnancy). All rats of both groups received whole body irradiation with 1.5 Gy gamma-rays from a (60)Co source at day 20 of pregnancy and were then implanted with a diethylstilbestrol pellet 1 month after weaning. A high incidence (70.3%) of mammary tumorigenesis was observed in the control group. The tumor incidence (18.5%) was significantly reduced in the rats fed curcumin during the initiation stage. The appearance of the first palpable tumor was delayed by 6 months in the curcumin-fed group and the average latent period until the appearance of mammary tumors was 2.5 months longer in the curcumin-fed group than in the control group. By histological examination, the proportion of adenocarcinoma (16.7%) in total tumors in the curcumin-fed rats was found to be decreased to half that (32.1%) in the control group. Compared with the control rats, the body weight of rats in the experimental group was decreased slightly by administration of the curcumin diet from day 11 of pregnancy, in spite of a similar intake of diet, but had recovered to the level of the control by the end of the experiment. At the time of irradiation, curcumin did not have any effect on organ weight or on the development and differentiation of mammary glands of pregnant rats. In addition, the serum concentrations of fatty acids, thiobarbituric acid-reactive substances and ovarian and pituitary hormones, except LH, remained at the control level. Also, no change in litter size and body weight of pups born from curcumin-fed rats indicated no toxicity of curcumin. These results suggest that curcumin does not have any side-effects and is an effective agent for chemoprevention acting at the radiation-induced initiation stage of mammary tumorigenesis.
Reactive oxygen species have been shown to play a role in ultraviolet light (UV)-induced skin carcinogenesis. Vitamin E and green tea polyphenols reduce experimental skin cancers in mice mainly because of their antioxidant properties. Since olive oil has also been reported to be a potent antioxidant, we examined its effect on UVB-induced skin carcinogenesis in hairless mice. Extra-virgin olive oil was applied topically before or after repeated exposure of mice to UVB. The onset of UVB-induced skin tumors was delayed in mice painted with olive oil compared with UVB control mice. However, with increasing numbers of UVB exposures, differences in the mean number of tumors between UVB control mice and mice pretreated with olive oil before UVB exposure (pre-UVB group) were lost. In contrast, mice that received olive oil after UVB exposure (post-UVB group) showed significantly lower numbers of tumors per mouse than those in the UVB control group throughout the experimental period. The mean number of tumors per mouse in the UVB control, pre-UVB and post-UVB groups was 7.33, 6.69 and 2.64, respectively, in the first experiment, and 8.53, 9.53 and 3.36 in the second experiment. Camellia oil was also applied, using the same experimental protocol, but did not have a suppressive effect. Immunohistochemical analysis of DNA damage in the form of cyclobutane pyrimidine dimers (CPD), (6-4) photoproducts and 8-hydroxy-2'-deoxyguanosine (8-OHdG) in samples taken 30 min after a single exposure of UVB showed no significant difference between UVB-irradiated control mice and the pre-UVB group. In the post-UVB group, there were lower levels of 8-OHdG in epidermal nuclei, but the formation of CPD and (6-4) photoproducts did not differ. Exposure of olive oil to UVB before application abrogated the protective effect on 8-OHdG formation. These results indicate that olive oil topically applied after UVB exposure can effectively reduce UVB-induced murine skin tumors, possibly via its antioxidant effects in reducing DNA damage by reactive oxygen species, and that the effective component may be labile to UVB.
Lectins are carbohydrate-binding proteins widely used in biochemical, immunochemical, and histochemical studies. Bauhinia purpurea lectin (BPA) is a leguminous lectin with an affinity for galactose and lactose. Nine amino acids, DTWPNTEWS, corresponding to the amino acid sequence from aspartic acid-135 to serine-143 in the primary structure of BPA were replaced with the corresponding amino acid residues from the mannose-binding Lens culinaris lectin (LCA), and the chimeric lectin obtained was expressed in Escherichia coli cells. The carbohydrate-binding specificity of the recombinant chimeric lectin was investigated in detail by comparing the elution profiles of various glycopeptides and oligosaccharides with defined carbohydate structures from immobilized lectin columns. Glycopeptides carrying three constitutive carbohydrate sequences of Galbeta1-3GalNAc-Ser/Thr and a complex-type biantennary glycopeptide, which show a high affinity for BPA or LCA, were shown to have no affinity for the chimeric lectin. In contrast, hybrid-type and high mannose-type glycopeptides with a Manalpha1-6(Manalpha1-3)Manalpha1-6Man sequence were found to have a moderate affinity for the chimeric lectin. This result demonstrates that a novel type of lectin with a unique carbohydrate-binding specificity can be constructed from BPA by substituting several amino acid residues in its metal-binding region with other amino acid residues. Additional lectin(s) with distinctly different carbohydrate-binding specificities will provide a powerful tool for many studies.
Bauhinia purpurea lectin (BPA) is one of the beta-galactose-binding leguminous lectins. Leguminous lectins contain a long metal-binding loop, part of which determines their carbohydrate-binding specificities. Random mutations were introduced into a portion of the cDNA coding BPA that corresponds to the carbohydrate-binding loop of the lectin. An library of the mutant lectin expressed on the surface of lambda foo phages was screened by the panning method. Several phage clones with an affinity for mannose or N-acetylglucosamine were isolated. These results indicate the possibility of making artificial lectins (so-called "cyborg lectins") with distinct and desired carbohydrate-binding specificities.
Enzyme-histochemical demonstration of lymphatic vessels in the golden hamster periodontium was performed on cryostat sections using the 5'-nucleotidase (5'-Nase) staining method by light microscopy and backscattered electron imaging of scanning electron microscopy. The inhibition of the 5'-Nase activity by decalcification was cancelled by the Mg++ ion supply. The reaction products of 5'-Nase activity were produced on the lymphatic endothelial cells and the tubular structures of lymphatic vessels were seen as highlights by backscattered electron imaging. The invasion of 5'-Nase-positive lymphatic vessels into the alveolar bone from the periodontium was found in the present study.
The processes of degeneration and the regeneration of the lip epidermal cells was observed by electron microscopy, focussing on the substance and the structure of the lamina lucida, on which regenerating cells migrated. After the repetitive freezing and thawing treatment, epidermal cells degenerated and detached from the dermis. The separation occurred between the epidermal cells and the basement membrane, leaving a small amount of cell debris on the lamina densa. After the separation of the epidermis, there were some thick parts in the lamina densa which appeared to be the part below hemidesmosomes. Regenerating epidermal cells migrated from the nondegenerated area along the cellular surface of the old lamina densa. They migrated over the cell debris which was gradually phagocitized, and formed new hemidesmosomes with the old lamina densa. Regenerating epidermal cells did not make close contact with the old lamina densa during their migration, but there was a clear space in between, indicating that some of the materials and the structure of the lamina lucida of the old basement membrane was preserved. By immunoelectron microscopy using anti-HSPG (heparan sulfate proteoglycan) antibody, it became clear that after the epidermal separation, HSPG was preserved in the basement membrane to some extent, especially in the thick parts of the lamina densa located below. The immunoelectron micrographs support the view that hemidesmosomes may reform at the previous locations at the old lamina densa.
The peroxynitrite-scavenging ability of some phenolic antioxidants, p-coumaric acid, caffeic acid and sinapinic acid, was examined and compared with ascorbic acid and tocopherol using 3-nitrotyrosine formation as a marker. Among these, caffeic acid and sinapinic acid strongly inhibited the formation of 3-nitrotyrosine in protein. The treatment of protein with peroxynitrite in the presence of sinapinic acid, but not caffeic acid, produced a novel product determined by reversed-phase high performance liquid chromatography (HPLC). The product formed was purified and then identified as a mono-lactone type dimer (ML) of sinapinic acid by nuclear magnetic resonance (NMR) and liquid chromatography-mass spectrometry (LC-MS). This ML was converted from a di-lactone type dimer, obtained from sinapinic acid with peroxidase/hydrogen peroxide, in neutral buffer. In this report, we have proposed that the ML of sinapinic acid is generated via one-electron oxidation by peroxynitrite treatment.
Oxidative stress is associated with important pathophysiological events in a variety of diseases. It has been postulated that free radicals and lipid peroxidation products generated during the process may be responsible for these effects because of their ability to damage cellular components such as membranes, proteins, and DNA. In the present study, we provide evidence that oxidative stress causes a transient impairment of intracellular proteolysis via covalent binding of 4-hydroxy-2-nonenal (HNE), a major end product of lipid peroxidation, to proteasomes. A single intraperitoneal treatment with the renal carcinogen, ferric nitrilotriacetate, caused oxidative stress, as monitored by accumulation of lipid peroxidation products and 8-hydroxy-2'-deoxyguanosine, in the kidney of mice. In addition, transient accumulation of HNE-modified proteins in the kidney was also found by competitive enzyme-linked immunosorbent assay and immunohistochemical analyses. This and the observation that the HNE-modified proteins were significantly ubiquitinated suggested a crucial role of proteasomes in the metabolism of HNE-modified proteins. In vitro incubation of the kidney homogenates with HNE indeed resulted in a transient accumulation of HNE-modified proteins, whereas the proteasome inhibitor significantly suppressed the time-dependent elimination of HNE-modified proteins. We found that, among three proteolytic activities (trypsin, chymotrypsin, and peptidylglutamyl peptide hydrolase activities) of proteasomes, both trypsin and peptidylglutamyl peptide hydrolase activities in the kidney were transiently diminished in accordance with the accumulation of HNE-modified proteins during oxidative stress. The loss of proteasome activities was partially ascribed to the direct attachment of HNE to the protein, based on the detection of HNE-proteasome conjugates by an immunoprecipitation technique. These results suggest that HNE may contribute to the enhanced accumulation of oxidatively modified proteins via an impairment of ubiquitin/proteasome-dependent intracellular proteolysis.
We recently reported that feeding cyanidin 3-O-beta-d-glucoside (C3G), a typical anthocyanin pigment, lowered the serum thiobarbituric acid-reactive substance (TBARS) concentration and increased the oxidation resistance of the serum to lipid peroxidation in rats. These results suggest that C3G acts as a potent antioxidant in vivo when acute oxidative stress is encountered. In the present study, we evaluated whether feeding C3G suppresses oxidative injury to the liver caused by hepatic ischemia-reperfusion (I/R), which was used as a model for oxidative stress. Rats were fed a diet containing C3G (2 g/kg diet) for 14 days and then subjected to hepatic I/R. I/R treatment elevated the liver TBARS concentration and the serum activities of marker enzymes (glutamic oxaloacetic transaminase, glutamic pyruvic transaminase, and lactate dehydrogenase) for liver injury and lowered the liver reduced glutathione concentration. Feeding C3G significantly suppressed these changes caused by hepatic I/R. Although the liver ascorbic acid concentration was also lowered by hepatic I/R, feeding C3G restored this concentration more quickly compared to the control rats. These results indicate that orally administered C3G suppresses I/R-induced oxidative damage and suggest that C3G functions as a potent antioxidant in vivo under oxidative stress.
CD44 is a transmembrane glycoprotein involved in various cell adhesion events, including lymphocyte migration, early hemopoiesis, and tumor metastasis. To examine the requirements of CD44 for signal delivery through the extracellular domain, we constructed a chimeric CD44 protein fused to the intracellular domain of Fas on its C-terminus. In cells expressing the CD44-Fas fusion protein, apoptosis could be induced by treatment with certain anti-CD44 mAbs alone, especially those recognizing the epitope group d, which has been previously shown to play a role in ligand binding, indicating that ligation of a specific region of the CD44 extracellular domain results in signal delivery. Of note was that appropriate ligation of the epitope h also resulted in the generation of apoptotic signal, although this region was not thought to be involved in ligand binding. In contrast, the so-called blocking anti-CD44 mAbs (epitope group f) that can abrogate the binding of hyaluronate (HA) failed to induce apoptosis even after further cross-linking with the secondary Ab, indicating that a mere mAb-induced oligomerization of the chimeric proteins is insufficient for signal generation. However, these blocking mAbs were instead capable of inhibiting apoptosis induced by nonblocking mAb (epitope group h). Furthermore, a chimeric protein bearing a mutation in the HA binding domain and hence lacking the ability to recognize HA was incapable of mediating the mAb-induced apoptosis, suggesting that the functional integrity of the HA binding domain is crucial to the signal generation in CD44.
Sulochrin, a metabolite of fungi, has been shown to have an inhibitory activity to eosinophil degranulation. A series of sulochrin derivatives substituted at ortho-positions to the 10-carbonyl group was examined the activity. The importance of alkylester at C-6 position and several chemical properties of substituted groups at ortho-positions to exhibit activity are described.
The objectives of this study were to estimate the structure of the lipid hydroperoxide-modified lysine residue and to prove the presence of the adducts in vivo. The reaction of lipid hydroperoxide toward the lysine moiety was investigated employing N-benzoyl-glycyl-L-lysine (Bz-Gly-Lys) as a model compound of Lys residues in protein and 13-hydroperoxyoctadecadienoic acid (13-HPODE) as a model of the lipid hydroperoxides. One of the products, compound X, was isolated from the reaction mixture of 13-HPODE and Bz-Gly-Lys and was then identified as N-benzoyl-glycyl-Nepsilon-(hexanonyl)lysine. To prove the formation of Nepsilon-(hexanonyl)lysine, named HEL, in protein exposed to the lipid hydroperoxide, the antibody to the synthetic hexanonyl protein was prepared and then characterized in detail. Using the anti-HEL antibody, the presence of HEL in the lipid hydroperoxide-modified proteins and oxidized LDL was confirmed. Furthermore, the positive staining by anti-HEL antibody was observed in human atherosclerotic lesions using an immunohistochemical technique. The amide-type adduct may be a useful marker for the lipid hydroperoxide-derived modification of biomolecules.
Methylglyoxal (MG), an endogenous metabolite that increases in diabetes and is a common intermediate in the Maillard reaction (glycation), reacts with proteins and forms advanced glycation end products. In the present study, we identify a novel MG-arginine adduct and also characterize the structure of a major fluorescent adduct. In addition, we describe the immunochemical study on the MG-arginine adducts using monoclonal antibody directed to MG-modified protein. Upon incubation of Nalpha-acetyl-L-arginine with MG at 37 degrees C, two nonfluorescent products and one fluorescent product were detected as the major products. The nonfluorescent products were identified as the Ndelta-(5-hydro-5-methyl-4-imidazolon-2-yl)-L-ornithine derivatives (5-hydro-5-methylimidazolone) and a novel MG-arginine adduct having a tetrahydropyrimidine moiety (Ndelta-(4-carboxy-4,6-dimethyl-5, 6-dihydroxy-1,4,5,6-tetrahydropyrimidine-2-yl)-L-ornithine). On the basis of the following chemical and spectroscopic evidence, the major fluorescent product, putatively identified as Ndelta-(5-methylimidazolon-2-yl)-L-ornithine (5-methylimidazolone), was found to be identical to Ndelta-(5-hydroxy-4, 6-dimethylpyrimidine-2-yl)-L-ornithine (argpyrimidine): (i) the low and high resolution fast atom bombardment-mass spectrometry gave a molecular ion peak at m/z of 297 (M+H) and a molecular formula of C10H25O6N4, respectively, which coincided with argpyrimidine; (ii) the 1H NMR spectrum of this product in d6-Me2SO showed a singlet at 2.10 ppm corresponding to six protons; (iii) the peak corresponding to the 5-methylimidazolone derivative was not detected by the liquid chromatography-mass spectrometry with the mode of selected ion monitoring; (iv) incubation of 5-hydro-5-methylimidazolone, a putative precursor of 5-methylimidazolone, at 37 degrees C for 14 days scarcely generated 5-methylimidazolone. On the other hand, as an immunochemical approach to the detection of these MG adducts, we raised the monoclonal antibodies (mAb3C and mAb6B) directed to the MG-modified protein and found that they specifically recognized the major fluorescent product, argpyrimidine, as the dominant epitope. The immunohistochemical analysis of the kidneys from diabetic patients revealed the localization of argpyrimidine in intima and media of small artery walls. Furthermore, the accumulation of argpyrimidine was also observed in some arterial walls of the rat brain after middle cerebral artery occlusion followed by reperfusion. These results suggest that argpyrimidine may contribute to the progression of not only long term diabetic complications, such as nephropathy and atherosclerosis, but also the tissue injury caused by ischemia/reperfusion.
We have clarified for the first time how cyanidin 3-O-beta-D-glucoside (C3G), which is a potent antioxidant anthocyanin, is absorbed and metabolized in vivo. Rats were orally administered C3G (0.9 mmol/kg body weight), and C3G rapidly appeared in the plasma. However, the aglycon of C3G (cyanidin; Cy) was not detected, although it was present in the jejunum. Protocatechuic acid (PC), which may be produced by degradation of Cy, was present in the plasma and the concentration was 8-fold higher than that of C3G. These results suggest that plasma PC and C3G may contribute to the antioxidant activity of the plasma. In the liver and kidney, C3G was metabolized to methylated C3G (methyl-C3G), suggesting that C3G and/or methyl-C3G act as antioxidants in the tissues.