Global gene expression profiling of androgen disruption in Qurt strain medaka

Androgen disrupting chemicals (ADCs) are endocrine disrupting chemicals (EDCs) that mimic or antagonize the effect of physiological androgens. Microarray-based detection of altered gene expression can be used as a biomarker of EDC exposure. Therefore, the purpose of this study was to identify and compare gene expression profiles of the androgen 11-ketotestosterone (11-KT), the antiandrogen flutamide (FLU), and the antiandrogenic fungicide vinclozolin (VIN), on Qurt medaka (Oryzias latipes). Biologically effective concentrations for 11-KT (100 microg/L), VIN (100 microg/L), and FLU (1000 microg/L) determined in range-finding studies were used for exposures. The oligonucleotide microarray included 9379 probes for EDC-affected genes, medaka cDNAs, sequences from the medaka genome project, and the UniGene database. We found that treatment with FLU, VIN, and 11-KT caused significant (false discovery rate = 0.01) differential expression of at least 87, 82, and 578 genes, respectively. Two sets of responsive genes are associated to vertebrate sex differentiation and growth, and 50 genes were useful in discriminating between ADC classes. The discriminating capacity was confirmed by a remarkable similarity of the antiandrogenic expression profiles of VIN and FLU, which were distinct from the androgenic profile of 11-KT. Gene expression profiles characterized in this study allow for reliable screening of ADC activity.     

棉织物一浴法在位生成银抗菌整理

采用在位还原法对棉织物进行银抗菌整理。通过还原剂和还原处理工艺的优化选择,制定了棉织物在位生成银一浴法抗菌整理浸轧和浸渍工艺。经优化的浸轧法或浸渍法都能使整理品获得较高的银含量,耐洗牢度较好,整理品白度仅稍有下降,手感几乎没有影响。采用浸渍工艺,当整理品银含量为103μg/g时,对金黄色葡萄球菌的抑菌率可以达到99.99%;当整理液银含量约为50 mg/L,浸渍-汽蒸工艺整理品经50次洗涤后银含量为112μg/g,对大肠埃希菌和金黄葡萄球菌的抑菌率仍能达到99.99%。     

黑米对高脂膳食饲喂雄性小鼠抗氧化相关基因的调控

目的:研究黑米对高脂喂养小鼠血清、肝脏和脑组织抗氧化水平以及肝脏抗氧化相关基因表达的影响。方法以喂养高脂高胆固醇膳食的雄性C57BL/6J小鼠为动物模型随机分为四组,每组12只,以高脂高胆固醇膳食组为对照组(C组,52.6 g玉米淀粉/100 g粮)、三个实验组分别在小鼠高脂饲料中补充白米(C+W组,52.6 g白米/100 g粮)和黑米(C+BL,26.3 g黑米/100 g粮和C+BH,52.6 g黑米/100 g粮)。8周后收集血液及组织进行检测,测定血清、肝脏和脑组织中总超氧化物歧化酶(SOD)、谷胱甘肽过氧化物酶(GSH-PX)、过氧化氢酶(CAT)和丙二醛含量(MDA)水平以及肝脏抗氧化相关基因SOD、CAT、GSH-Px、HO-1、NF2L2等的mRNA表达。结果:白米组(C+W)血清、肝脏及脑中SOD、GSH-Px、CAT活性和MDA含量较高脂对照组无统计学差异,C+BL组(P<0.05)和C+BH组(P<0.01)血清中SOD酶活力较对照组升高,且C+BH组MDA含量显著降低(P<0.05);与高脂组相比,黑米实验组(C+BL和C+BH)肝脏中SOD酶活性显著升高(P<0.05),C+BH组MDA含量显著降低(P<0.05);C+BH组脑中GSH-Px酶活性与高脂组相比显著升高(P<0.05),C+BL组(P<0.05)和C+BH组(P<0.01)脑中SOD酶活较对照组升高,C+BH组MDA含量显著降低(P<0.05)。RT-PCR结果显示,与高脂组相比,黑米添加组(C+BL和C+BH)SOD2、CAT mRNA表达极显著上调(P<0.01),GSH-Px mRNA表达显著上调(P<0.01),C+BH组HO-1 mRNA表达水平显著升高(P<0.05)。结论:高脂膳食造成氧化应激,黑米膳食干预抗氧化可能是增加抗氧化酶的表达来减少氧化损伤。     

Sulfide oxidation by gene expressions of sulfide-quinone oxidoreductase and ubiquinone-8 biosynthase in Escherichia coli

Sulfides (S2),SH-) such as hydrogen sulfide belong to a class of sulfur compounds with unpleasant odors. In order to confer sulfide-oxidizing ability on the intestine-inhabiting bacteria, the sulfide-quinone oxidoreductase gene (sqr) in Rhodobacter capsulatus DSM-155 and genes for quinone biosynthesis (ubiC, ubiA and ispB) in Escherichia coli XL1 Blue-MRF' were transduced into E. coli BL21(DE3). Plasmids pT7-7 and pSTV were used as vectors of sqr, and ubiCA and ispB, respectively. The recombinants sqr-BL21(DE3) and ubiCA,ispB-sqr-BL21(DE3) were successfully constructed. The maximal sulfide-removing activities of the whole cells and membrane fractions of sqr-BL21(DE3) attained at pH 8.0 and 7.8, were 267 nmol/mg cells (dry weight)/min and 1250 nmol/mg membrane fraction (protein)/min, respectively. The molecular ratio of sulfide (S2-) oxidized and oxygen (O2) consumed was 2:1. SQR activity in the recombinant cells was positively restricted under anaerobic conditions and also by the addition of electron transfer inhibitors. Ubiquinone-8 (UQ-8) biosynthesis in the cells of ubiCA,ispB-sqr-BL21(DE3) increased as much as 2.2-fold compared with that of (pSTV)-sqr-BL21(DE3) during the 12-16 h incubation period. The maximal sulfide removal in the quinone-raised E. coli was attained slightly earlier, however, SQR activities thereafter were lower than those in (pSTV)-sqr-BL21(DE3).     

过度煎炸油对大鼠氧化应激及相关基因表达的影响

采用定量灌胃的方式研究过度煎炸油(DFEO)的摄入对大鼠氧化应激的影响,同时探讨其引起氧化损伤可能的机理。研究发现:干预6周后,DFEO组血清GSH–Px、T–AOC水平显著降低(ρ0.05),MDA水平显著升高(ρ0.05);肝组织切片显示出现脂肪空泡,肝窦扩张,肝细胞界限不清;同时RT–PCR结果表明,过度煎炸油会导致大鼠肝脏组织HMOX1、VEGF基因显著下调(ρ0.01)。这说明DFEO富含大量毒性物质,会影响正常的基因表达,使机体清除氧自由基能力下降,甚至破坏正常的组织细胞形态。     

Pine needle extract suppresses adiposity via mediating adipogenic gene expressions of white adipose tissue in high fat diet-fed rats

Effect of pine needle extract (PNE) on adiposity was examined through morphologic examination and adipogenic gene expression of white adipose tissue (WAT) in high fat diet (HFD)-fed rats. Four type diets; normal diet (ND), HFD, and diets respectively supplemented with PNE in both ND and HFD, were fed for 8 weeks. The adipose fat mass and the sizes in WAT were significantly lower in PNE-supplemented HFD group than in HFD group. The serum levels of PNE-supplemented HFD group were similar to those of ND group. The supplementation of PNE to HFD markedly increased adiponectin mRNA, but decreased those of leptin and tumor necrosis factor (TNF)-α in WAT, respectively. PNE enhanced glucose uptake of epididymal WAT under insulin stimulation. The results suggest that the beneficial effect of PNE supplementation on adipose fat and serum profiles in HFD-fed rats is partly modulated by adipogenic genes, such as adiponectin, leptin, and TNF-α mRNA in WAT.     

高原雪菊与平原雪菊中黄酮类成分及其关键酶基因表达的差异分析

为了分析比较高原雪菊与平原雪菊类黄酮化合物成分差异，采用超高液相色谱-串联四级杆飞行时间质谱（UPLC-QTOF-MS）技术对雪菊头状花序的类黄酮化合物进行定性定量分析。依据UPLC-QTOF-MS 矩阵中的保留时间、质荷比及碎片离子对雪菊中的黄酮类代谢产物进行分析鉴定，比较高原雪菊与平原雪菊代谢产物的相对含量。结果表明，从昆仑雪菊中共分析鉴定出204个黄酮类成分，PCA 分析结果显示两个产地样本的黄酮类化合物差异明显，通过 OPLS-DA 筛选出4,4""-二甲氧基查耳酮、棕矢车菊素、7,4""-二羟基黄酮和槲皮素等15种差异表达的黄酮类代谢物，其中6种黄酮类化合物在平原雪菊中含量较高，9种黄酮类化合物在高原雪菊中含量较高。通过对差异代谢物通路分析，发现类黄酮生物合成（Flavonoid biosynthesis）、次生代谢物的生物合成（Biosynthesis of secondary metabolites）、黄酮和黄酮醇生物合成（Flavone and flavonol biosynthesis）、代谢途径（Metabolic pathways）、异黄酮生物合成（Isoflavonoid biosynthesis）和苯丙酸类化合物的生物合成（Biosynthesis of phenylpropanoids）等6条代谢途径显著差异。结论：昆仑雪菊的黄酮类化合物种类多样且含量丰富，其含量丰富的黄酮类化合物可能是昆仑雪菊具有显著药用价值的物质基础，且不同产地雪菊含有特有的黄酮类成分，可通过检测特定的黄酮类化合物进行雪菊产地的鉴定。     

Chronic prepartum light-dark phase shifts in cattle disrupt circadian clocks,decrease insulin sensitivity and mammary development,and are associated with lower milk yield through 60 days postpartum

Circadian and metabolic systems are interlocked and reciprocally regulated. To determine if the circadian system regulates glucose homeostasis and mammary development, the function of the circadian system was disrupted by exposing cattle to chronic light-dark cycle phase shifts from 5 wk before expected calving (BEC) to parturition. Multiparous Holstein cows were exposed to 16 h of light and 8 h of dark (CON, n = 8) or phase shifting (PS, n = 8) the light cycle 6 h every 3 d beginning 35 d BEC. After calving, both treatments were exposed to CON lighting. Mammary biopsies were taken at 21 d BEC and 21 d in milk (DIM), and histological analysis indicated PS treatment decreased the ratio of lumen to alveolar area and percentage of proliferating epithelial cells in the prepartum period. Intravenous glucose tolerance test was performed at 14 d BEC and 7 DIM by administering 50% dextrose. Blood glucose, β-hydroxybutyrate, insulin, and nonesterified fatty acids were consequently measured over 3 h. At 14 d BEC no treatment differences were observed in baseline glucose or insulin. Treatment had no effect on blood glucose or glucose area under the curve at 14 d BEC and 7 DIM. Insulin area under the curve was higher in PS versus CON at 14 d BEC and 7 DIM. The PS cows produced less milk than CON cows through 60 DIM (40.3 vs. 42.6 kg/d). Exposure to chronic light-dark PS in late gestation decreased mammary development and increased insulin resistance in periparturient cows, which may have caused subsequent lower milk yield.     

Dietary docosahexaenoic acid-rich diacylglycerols ameliorate hepatic steatosis and alter hepatic gene expressions in C57BL/6J-Lep(ob/ob) mice

We examined the effect of docosahexaenoic acid (DHA)-enriched structured lipids-diacylglycerol (SL-DG), which were synthesized using soybean oil (SO) and algae oil (AO), on hepatic lipid metabolism and the mRNA expression of genes involved in hepatic steatosis of C57BL/6J-Lep(ob/ob) compared to the SL-triacylglycerol (TG). The animals were fed a high-fat (10% lard and 10% test oils) and high-cholesterol (0.2% cholesterol) diet for 12 weeks. Mice fed SL-DG showed a lower total white adipose tissue weight and plasma triglyceride concentration than the SO group. Reduction of hepatic triglyceride content in the SL-DG group was related with the suppression of hepatic enzyme activities for fatty acid and triglyceride synthesis along with fecal triglyceride excretion compared to the SL-TG. SL-DG also lowered hepatic cholesterol levels by suppressing cholesterol regulating enzyme activity compared to the SO group. Moreover, SL-DG lowered the mRNA expressions of sterol regulatory element binding protein-1 and its target genes than TG-form oils (SO, AO and SL-TG) in the liver. Thus, the current results suggest that DHA-enriched SL-DG oil used in this study is beneficial for ameliorating hepatic steatosis in obese animal model by improving hepatic fatty acid and cholesterol metabolic enzyme activity and their gene expression.     

Glucose production from cellulose through biological simultaneous enzyme production and saccharification using recombinant bacteria expressing the β-glucosidase gene

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Improved biomass saccharification by Trichoderma reesei through heterologous expression of lacA gene from Trametes sp. AH28-2

The Trametes sp. AH28-2 laccase gene lacA fused to cellobiohydrolase I signal peptide coding sequence was heterologously expressed in T. reesei. The lacA cDNA was under the control of the Aspergillus nidulans glyceraldehyde-3-phosphate dehydrogenase promoter. Native PAGE analysis indicated that two transformants, L8 and L38, were able to secrete recombinant laccase A, and their laccase activities corresponding to ABTS oxidation reached 3.62 IUml(-1) and 1.50 IUml(-1) respectively. Most of the characteristics of the recombinant laccase were similar to those of the native enzyme. Reducing sugar yields of L8 and L38 obtained from saccharification of corn residue by crude enzyme increased by 31.3% and 71.6% respectively compared to the host strain. These results indicated that the engineering strains developed in this work could be potentially used for laccase production and tailoring cellulase properties with laccase proteins through genetic manipulation would be a feasible strategy to improve saccharification efficiency of biomass by cellulase preparation.     

Random and targeted gene integrations through the control of non‐homologous end joining in the yeast Kluyveromyces marxianus

Kluyveromyces marxianus DMKU3‐1042 is a thermotolerant yeast strain suitable for high‐temperature ethanol fermentation and genetic engineering with linear DNA. We have developed a highly efficient random gene integration method with a frequency that exceeds 2.5 × 10⁶ transformants/µg linear DNA, a figure comparable to what is observed with autonomously replicating plasmid transformation in Saccharomyces cerevisiae. To establish the mechanism of random integration in DMKU3‐1042, we identified and deleted the K. marxianus KU70 gene, which is known to be involved in the non‐homologous end‐joining (NHEJ) pathway. In yeast lacking KU70, high‐frequency non‐homologous gene integration was abolished and the Kmku70 mutants showed 82–95% homologous gene targeting efficiencies using homologous sequences of 40–1000 bp. These results indicate that the highly efficient NHEJ pathway can be utilized with random gene disruption techniques such as transposon mutagenesis and plasmid‐free gene manipulations in K. marxianus. Copyright © 2009 John Wiley & Sons, Ltd.     

The Schizosaccharomyces pombe cfr1+ gene participates in mating through a new pathway that is independent of fus1+

Conjugation is a complex event directed to ensure the transfer of genetic material, which is achieved by the union of two cells. In fungi, success of this relevant process requires digestion of the cell wall at the point where both cells have agglutinated and, later, the union of the plasma membranes and nuclei from the mating partners. In order to gain information about cell fusion, we have cloned and disrupted the cfr1+ gene from the fission yeast Schizosaccharomyces pombe. cfr1+ gene is slightly induced at the beginning of mating but Cfr1p protein is degraded soon after the cells are transferred to nitrogen-lacking medium. cfr1Delta mutants present a defect in cell fusion owing to a failure in the digestion of the cell walls between the two parental cells. Finally, cytological and genetic analyses show that cfr1+ acts in a new pathway involved in conjugation that is independent of fus1+, the only gene that has been found to be specifically required for cell fusion during mating in the fission yeast.     

Improvement and induction property of radiation-responsive promoter through DNA shuffling of 5′-flanking regions of the human p21 gene

A promoter that augments gene expression in response to stimulation of ionizing radiation would be a desired tool for radiogenetic therapy, a combination of radiotherapy and gene therapy. Although various promoters occurring naturally or artificially have been used for researches, one showing higher reactivity to ionizing radiation is desirable. In the present study, we attempted to improve a radiation-responsive promoter of the p21 through a technique called DNA shuffling. A library of DNA fragments was constructed by re-ligation of randomly digested promoter fragments and improved promoters were chosen out of the library. We repeated this process twice to obtain a promoter showing 2.6-fold better reactivity to ionizing radiation compared with its parent, p21 promoter after 10 Gy γ-ray irradiation. Nucleotide sequence analyses revealed that the obtained promoter was densely packed with some of the cis-acting elements including binding sites for p53, NF-κB, NRF-2, AP-1 and NF-Y more than p21 promoter. In addition, it was shown that its induction by ionizing radiation was dependent upon p53 status of a cell line, suggesting that the promoter retained properties of the p21 promoter. This technique is simple and efficient to improve a promoter responsive to other stimulus of interest besides IR.