Detection of in vivo DNA damage induced by ethanol in multiple organs of pregnant mice using the alkaline single cell gel electrophoresis (Comet) assay

Ethanol is principal ingredient of alcohol beverage, but considered as human carcinogen, and has neurotoxicity. Alcohol consumption during pregnancy often causes fetal alcohol syndrome. The DNA damage is one of the important factors in carcinogenicity or teratogenicity. To detect the DNA damage induced by ethanol, we used an in vivo alkaline single cell gel electrophoresis (Comet) assay in pregnant mice organs and embryos. Pregnant ICR mice on Day 7 of gestation were treated with 2, 4 or 8 g/kg ethanol, and maternal organs/tissues and embryos were subjected to the Comet assay at 4, 8, 12 and 24 hr after ethanol treatment. Four and 8 g/kg ethanol induced DNA damage in brain, lung and embryos at 4 or 8 hr after the treatment. Two g/kg ethanol did not cause any DNA damage, and 8 g/kg ethanol only increased the duration of DNA damage without distinct increase in the degree of the damage. No significant DNA damage was observed in the liver. To detect the effect of acetaldehyde, disulfiram, acetaldehyde dehydrogenase inhibitor, was administered before 4 g/kg ethanol treatment. No significant increase of DNA damage was observed in the disulfiram pre-treated group. These data indicate that ethanol induces DNA damage, which might be related to ethanol toxicity. Since pre-treatment of disulfiram did not increase DNA damage, DNA damage observed in this study might not be the effect of acetaldehyde.     

Antiinflammatory effect of Japanese horse chestnut (Aesculus turbinata) seeds

The antiinflammatory effects of Japanese horse chestnut (Aesculus turbinata) seeds were examined in vivo and in vitro. The extract of this seed (HCSE) inhibited croton oil-induced swelling of the mouse concha. HCSE inhibited cyclooxygenase (COX) -1 and -2 activities, but had no effect on 15-lipoxygenase and phospholipase A2 activities. Inhibition of COX-2 occurred at a lower concentration of HCSE than for COX-1. Japanese horse chestnut seeds contain coumarins and saponins, but these chemicals did not inhibit COX activities. These results suggest that the antiinflammatory effect of Japanese horse chestnut seeds is caused, at least partly, by the inhibition of COX. The inhibitor of COX in this seed may be a chemical(s) other than coumarins and saponins.     

Matrix metalloproteinases (MMPs) activity in cultured canine bone marrow stromal cells (BMSCs)

Autologous bone marrow stromal cells (BMSCs) infusion therapy improves the hepatic fibrosis. To investigate the mechanism of remission, we evaluated the matrix metalloproteinase (MMP)-2 and -9 activity in canine BMSCs and the effect of pro-inflammatory cytokines on their expression. The activity and the gene expression of MMPs were analyzed by gelatin zymography and quantitative RT-PCR, respectively. The specific gelatinase bands were indicative effect of MMP-2 and -9 in canine BMSCs. MMP-2 expression seemed to be increased by TNF-α and IL-1β while MMP-9 was enhanced by TNF-α and IL-6. These results suggested that remissive effect on liver fibrosis might be partly attributable to the MMP-2 and -9 activity in BMSCs under the inflammatory condition.     

Normal calves produced after transfer of embryos cultured in a chemically defined medium supplemented with epidermal growth factor and insulin-like growth factor I following ovum pick up and in vitro fertilization in Japanese black cows

The objective of this study was to examine whether high concentrations of epidermal growth factor (EGF) and/or insulin-like growth factor I (IGF-I) would have a beneficial effect on bovine embryo development in vitro and to obtain normal calves by using an ovum pick up method and embryo culture in a chemically defined medium. When compared with controls, EGF (100 or 200 ng/ml) or IGF-I (50 or 100 ng/ml) significantly increased the rate of embryos that developed into blastocysts during an 8-day culture after the in vitro fertilization of oocytes obtained from ovaries from a slaughterhouse. IGF-I induced a dose-dependent increase in cell number in both the inner cell mass and the trophectoderm, whereas EGF stimulated proliferation only in the inner cell mass. A combination of EGF (100 ng/ml) and IGF-I (50 ng/ml) produced an additive effect, and embryos developed into blastocysts at a comparatively high rate (27.9%) compared with controls (12.0%). A similar rate of development was achieved using a combination of EGF and IGF-I in the culture of embryos following ovum pick up by ultrasound-guided transvaginal follicular aspiration and in vitro fertilization, and 5 blastocysts that developed after the culture were transferred into uteri; two embryos implanted, and normal calves were born. These results suggest that the combined use of EGF and IGF-I makes bovine embryo culture in a chemically defined medium a practical and useful procedure for producing blastocysts, and its application to embryo culture following ovum pick up and in vitro fertilization could be useful for producing normal calves.     

Effects of warming basal ends of Carolina poplar (Populus × canadensis Moench.) softwood cuttings at controlled low-air-temperature on their root growth and leaf damage after planting

We investigated the effects of warming the basal ends of Carolina poplar (Populus × canadensis Moench.) softwood cuttings at controlled low-air-temperature on their root growth and leaf damage after planting. The warming treatment was applied to the cuttings by soaking 10 mm beyond the cut end in warmed water maintained at 30 °C in a cold chamber maintained at an air temperature of 10 °C and a photosynthetic photon flux density (PPFD) of 10 μmol m^?2 s^?1 (near the light compensation point at 10 °C) until rooting was observed. The warmed cuttings were then grown in a growth chamber at an air temperature of 30 °C, relative humidity 85–90 %, and a PPFD of 100 μmol m^?2 s^?1. Control cuttings were grown in the growth chamber throughout the experiment. Rooting occurred simultaneously for both warmed and control cuttings, irrespective of air temperature. Root development was greater and leaf damage, evaluated on the basis of extent of necrosis, was less for warmed cuttings than for control cuttings. The reduction of leaf damage for warmed cuttings probably resulted from reduced post-planting water stress and leaf senescence, because of improved root development as a result of the pre-planting warming treatment. This technique could improve the propagation of cuttings of woody plants, because it would ensure that the cuttings are ready to develop roots with minimum loss of carbohydrates, irrespective of weather conditions.     

Heat shock-derived reactive oxygen species induce embryonic mortality in in vitro early stage bovine embryos

Heat shock is known to increase the mortality of early stage embryos, but the exact mechanism is unclear. In the present study, we investigated the possibility that the increased mortality is caused by heat shock-generated reactive oxygen species (ROS). The level of ROS was controlled by using beta-mercaptoethanol (beta-ME), a scavenger of ROS. In vitro-produced 8-cell stage embryos were cultured at 38.5 C or heat-shocked by exposure to 41 C for 6 h with 0, 10 and 50 microM beta-ME. Intracellular ROS levels were measured by a fluorescent dye, 2',7'-dichlorodihydrofluorescein diacetate (DCHFDA), and intracellular reduced form of glutathione (GSH) contents were estimated by another fluorescent dye, 4-chloromethyl-6,8-difluoro-7-hydroxycoumarin. Total glutathione content was estimated by the glutathione recycling assay. On day 8 after insemination, heat shock decreased the percentage of embryos that developed to the blastocyst stage and increased intracellular ROS levels, but there was no significant effect on the GSH and total glutathione contents. In contrast, beta-ME significantly decreased ROS levels in heat-shocked embryos and increased the GSH and total glutathione concentrations. Ten microM beta-ME significantly improved the viability of heat-shocked embryos. beta-ME caused no detrimental effects when it was added at normal culture temperature (38.5 C). These results indicate that ROS is the primary cause of increased embryonic mortality in heat-shocked early stage embryos.     

Effects of resveratrol treatment on mitochondria and subsequent embryonic development of bovine blastocysts cryopreserved by slow freezing

This study evaluated the effects of cryopreservation by slow freezing on the mitochondrial function, DNA integrity, and developmental ability of bovine embryos and examined whether resveratrol treatment of the frozen‐thawed blastocysts improved embryonic viability. In vitro produced bovine embryos were subjected to slow freezing. After thawing, the ATP content and mitochondrial DNA integrity (mtDNA), determined by real‐time PCR targeting short and long mitochondrial sequences, was found to be lower in frozen‐thawed embryos than in fresh embryos, and mtDNA copy number was significantly reduced during the 24‐hr incubation post warming. Furthermore, immunostaining against double‐strand DNA revealed DNA damage in frozen‐thawed embryos. When frozen‐thawed embryos were incubated in the medium containing 0.5 µM resveratrol, SIRT1 expression, and survival rate of the embryos significantly improved compared with the vehicle‐treated embryos. In addition, cell‐free mtDNA content in medium was higher in case of resveratrol‐treated embryos than of vehicle‐treated embryos. In conclusion, slow freezing affects mitochondrial integrity and function in the blastocysts. In the frozen‐thawed embryos, mitochondria were removed during post‐thawing incubation and resveratrol enhanced the process, resulting in improved survivability of the embryos.     

Storage of plug seedlings of tomato under limited fertilisation,and growth,flowering and yield after planting

We stored plug seedlings of tomato (Solanum lycopersicum L. cv. Momotaro) under limited fertilisation (LF) for 12 weeks, and evaluated growth and development during storage and after planting. Seedlings in the LF treatment were grown in substrate containing starter fertiliser and irrigated with water. Controls (CT) were irrigated with nutrient solution. Stem growth, leaf growth, and biomass accumulation slowed or ceased during storage of LF seedlings, while CT seedlings showed increases in these parameters. At 2 weeks after planting, the stem length of LF seedlings was shorter than that of CT seedlings, but the two seedling types had similar numbers of leaves. At harvest, LF and CT plants had similar numbers of leaves under the fruit trusses, but the trusses formed at lower heights in LF than in CT seedlings. LF and CT plants produced similar numbers of fruits and similar yields from the first to third trusses. These results indicate that tomato plug seedlings can be stored for more than 2 months under LF.     

Sulfuric acid bleaching of kraft pulp III: reactivity of kraft pulping-resistant structures under acidic conditions

To investigate the bleaching mechanism, a lignincarbohydrate complex (LCC) model compound, a vinyl ether-type lignin model dimer, and a hexeneuronic acid model compound were treated with dilute sulfuric acid of different pHs. Beech kraft pulp and red pine kraft pulp were also treated with dilute sulfuric acid and then extracted with aqueous alkali. The amount of hexeneuronic acid degradation products in acid effluents and lignin dissolved in alkali effluents were determined. It was found that the benzyl ether-type LCC bond and the vinyl ether bond in lignin were effectively cleaved under the pH where sulfuric acid bleaching of kraft pulp was effective. Hexeneuronic acid group was also effectively degraded during sulfuric acid bleaching. In beech kraft pulp bleaching, both lignin removal and hexeneuronic acid removal contributed to the kappa number reduction. In red pine bleaching, the contribution of hexeneuronic acid removal was negligible, and most of the kappa number reduction was achieved by the lignin removal.Part of this report was presented at the 9th International Symposium on Wood and Pulping Chemistry, Montreal, July 1997     

百香果树莓复合果酒的酿造工艺优化及其体外抗氧化性研究

为生产优质果酒,采用单因素结合响应面的方法,以酒体感官评价为指标开展百香果树莓复合果酒的酿造工艺优化研究并测定其多酚含量,之后通过考查复合果酒对DPPH自由基、羟基自由基、超氧阴离子自由基的清除率及其还原力开展百香果树莓复合果酒的体外抗氧化性研究。结果表明：复合果酒的最佳酿造工艺为百香果树莓果浆配比1:2（g·g^-1）、初始糖度22.4 Brix、料水比1:1（g·mL^-1）、发酵温度19 ℃、发酵时间11 d;与同多酚浓度的Vc相比,此果酒具有更高的羟基自由基和超氧阴离子清除能力,DPPH自由基、羟基自由基、超氧阴离子自由基清除率可达87.8%、66.9%和47.4%,还原力可达29.2%。按照优化的工艺酿造的复合果酒酒度为12.3%(vol),多酚含量为353.9 mg·L^-1,呈红宝石色,清澈透明无悬浮物、有典型的百香果和树莓香气、丰满爽口有新鲜感,具有良好的抗氧化性。