Protective action of dehydroascorbic acid on the Ah receptor-dependent and receptor-independent induction of lipid peroxidation in adipose tissue of male guinea pig caused by TCDD administration

The effect of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on lipid peroxidation, ³H-Me-glucose (³H-Me-glu), and ¹⁴C-dehydroascorbic acid (¹⁴C-DHA) uptakes were studied in adipose tissue of male guinea pig. Under in vitro test conditions, using isolated adipose tissue in a culture medium (explant culture), TCDD reduced the uptake of ³H-Me-glu and ¹⁴C-DHA in a dose- and time-dependent fashion. The IC₅₀ values of TCDD's action were 0.04 and 2 nM on ¹⁴C-DHA and ³H-Me-glu uptakes, respectively. TCDD (10 nM) also suppressed glucose transporting activity within 15 minutes in explant-cultured adipocytes. Cytochalasin B (CB) and nonlabeled D-glucose inhibited ¹⁴C-DHA uptake also in a dose-dependent manner. In addition, TCDD was found to induce lipid peroxidation in ex-plant-cultured adipose tissue. This effect of TCDD was similar to that of a typical lipid peroxidation inducer, CCl⁴, and it was dose and time dependent. TCDD caused a statistically significant rise in lipid peroxidation at a concentration as low as 0.1 nM after 60 minutes of treatment in explant culture. Unexpectedly, the Ah receptor partial antagonists, 4,7-phenanthroline and α-naphthoflavone, did not fully antagonize TCDD-induced lipid peroxidation in explant-cultured adipocytes. In vivo treatment of TCDD also induced lipid peroxidation. Among seven organs of male guinea pig tested, the levels of lipid peroxidation in adipose tissue and in liver increased at 1 and 40 days following a single i.p. dose of TCDD (1 μg/kg). The results of an in vivo time-course study indicated that such an effect of TCDD was most pronounced after 40 days of treatment. Finally, we have tested the protective role of some antioxidants on TCDD-induced lipid peroxidation under explant-culture conditions. The results indicated that DHA, but not ascorbic acid, could completely abolish TCDD-induced lipid peroxidation. The protective effect of DHA on TCDD-induced lipid peroxidation was stronger than that of α-tocopherol and uric acid, and this effect was blocked by CB. We conclude from these studies that TCDD acts in this guinea pig tissue through two different routes: one is the Ah receptor-dependent route causing the reduction of the level of glucose transporters and subsequent decrease of cellular uptake of DHA and the other, the Ah receptor-independent route causing the overall lipid peroxidation. Nevertheless, it appears likely that both events are antagonized by DHA. © 1997 John Wiley & Sons, Inc. J Biochem Toxicol 11: 269–278, 1997.     

猪胰岛素启动子调控EGFP表达载体的优化及体外验证

目的获得猪胰岛素启动子调控外源基因的高效表达载体,为制备转基因猪胰岛特异性表达目的基因奠定基础。方法利用猪胰岛素启动子（PIP,包含5＇调控区、第一外显子、第一内含子及第二外显子ATG前的序列共1.5 kb）构建表达载体,连接PIP和EGFP的酶切位点HindⅢ设计在起始密码子前,命名为PIP-Hind IIIEGFP。鉴于酶切位点的插入位置可能会影响外源基因的表达效率,对载体进行了优化：将Hind III酶切位点删除,实现PIP和EGFP无缝连接,载体命名PIP-EGFP;将第一内含子3＇端的内含子剪接受体位点（splicing acceptor site,SA）突变为Hind III内切酶识别位点,命名为PIP-SA（M）-EGFP。三种载体分别电转染小鼠胰岛素瘤β细胞株MIN-6细胞、猪耳成纤维细胞以及猪肾细胞,48 h后通过荧光强度、流式分析、RT-PCR及Western blot检测,验证载体的表达效率。结果转染细胞后,三种载体都仅在MIN-6胰岛β细胞表达绿色荧光。RT-PCR及其产物测序结果显示三种载体的胰岛素启动子第一内含子的剪切存在差异：载体PIP-Hind III-EGFP和PIP-EGFP的内含子存在剪切和不剪切两种情况,剪切不稳定;载体PIP-SA（M）-EGFP的SA位点突变后内含子不剪切,流式细胞及Western blot检测显示,与另外两种载体相比,该载体目的蛋白的表达量最高。结论通过突变胰岛素启动子第一内含子的3＇端的剪接受体位点,成功获得了胰岛β细胞的高效表达载体,可用于制备胰岛特异表达外源基因的转基因猪。     

赤子爱胜蚓在不同猪粪和农作物秸杆混合物中生长繁殖状况

本文研究了赤子爱胜蚓在长春市城郊不同组合的粮食猪粪、饲料猪粪和玉米、水稻秸杆混合物中生长繁殖状况,结果发现赤子爱胜蚓的质量在0 ～30 d大幅度增加,30 ～ 60 d增加减缓,60～ 90 d呈负增长趋势.赤子爱胜蚓产茧量在试验期间的总趋势是递增的.猪粪种类、秸杆种类和两者的混合比例差异对赤子爱胜蚓的质量变化产生重要影响.粮食猪粪和玉米秸杆混合物的质量比例大致在1∶1时特别适宜赤子爱胜蚓生长和繁殖.赤子爱胜蚓在饲料猪粪和玉米秸杆混合物中的日增重倍数和日产茧量多于饲料猪粪和水稻秸杆混合物.本试验结论可为城郊猪粪和作物秸杆的大规模资源化利用提供参考.     

不同有机肥对土壤镉锌生物有效性的影响

在南方典型稻田设置连续4年施用猪粪、鸡粪、稻草的定位试验,监测施用不同有机肥条件下土壤及水稻植株镉(Cd)、锌(Zn)含量的变化,研究有机肥对土壤Cd、Zn活性及其交互作用的影响.结果表明: 施用有机肥(猪粪、鸡粪、稻草)对土壤全Cd、有效态Cd含量及Cd活性皆无显著影响,但有增加土壤Cd全量的趋势,且显著增加土壤全Zn、有效态Zn含量及Zn活性.施用猪粪、鸡粪、稻草皆可降低稻米Cd含量,降Cd效果为猪粪>鸡粪>稻草,猪粪处理水稻稻米、茎、叶Cd含量分别比对照下降37.5%、44.0%、36.4%;鸡粪处理水稻米、茎、叶Cd含量分别比对照下降22.5%、33.8%、22.7%;而稻草处理水稻米Cd含量比对照下降7.5%,但茎、叶Cd含量比对照分别增加8.2%、22.7%;施用猪粪、鸡粪降低稻米Cd含量主要是降低了水稻植株对土壤Cd的富集,而施用稻草则主要是降低了水稻茎Cd向稻米的转运.施用有机肥还增加了水稻茎Zn含量,施用猪粪、鸡粪、稻草的水稻茎Zn含量比单施化肥分别增加53.4%、41.2%、13.9%,但对水稻稻米、叶Zn含量无显著影响.Zn、Cd在土壤、植株茎中皆表现出显著的拮抗作用,土壤及水稻茎Zn含量的增加显著抑制了水稻米、茎、叶对Cd的吸收积累,且随土壤有效态Zn/Cd含量比值的增加,Zn、Cd竞争土壤吸附不是抑制水稻吸收积累Cd的主控因子,而Zn、Cd竞争吸收才是影响水稻吸收积累Cd的主控因子.     

猪骨骼肌MyHC-Ⅱb基因上调表达的分子机制

哺乳动物骨骼肌由各种不同类型的肌纤维镶嵌而成,不同类型肌球蛋白重链的表达是造成不同类型肌纤维的主要原因.目前已知的肌球蛋白重链家族包含8种亚型,其中长白猪骨骼肌My HC-Ⅱb的表达量显著高于中国地方猪,然而造成这种差异的分子机制未见报道.本研究用荧光定量PCR证明了长白猪背最长肌中My HC-Ⅱb m RNA的表达量显著高于莱芜猪(P=0.013).删除实验结果表明,从转录起始位点上游-1024 bp删除到-187 bp之后,My HC-Ⅱb表达量显著下降,分析发现,在这段启动子区域内存在3个E-box序列;分别突变这3个E-box序列后,My HC-Ⅱb启动子驱动的荧光素酶活性显著下降(P=0.036).另外,在My HC-Ⅱb上游启动子区?1398 bp处发现一个GT的突变,所检测的64头莱芜猪在该位点全部为GG型,65头长白猪中13头为GG型,16头为TT型,36头为GT型.在C2C12细胞系中的转染实验结果显示,G突变为T之后有增加My HC-Ⅱb表达的趋势.Western blot的结果表明,转录因子Myo D在两猪种间表达差异不显著(P=0.136),而Myf-5在长白猪中的表达量极显著高于其在莱芜猪中的表达量(P=0.0036).这些数据表明,Myf-5是造成猪My HC-Ⅱb基因m RNA上调表达的重要因素之一.     

乌金猪FTO基因的克隆和表达分析

目的:克隆乌金猪脂肪和肥胖相关基因(FTO)编码区序列(CDS),分析其序列组成特征及在乌金猪不同组织中的表达情况。方法:采用反转录PCR方法从乌金猪脂肪组织中克隆FTO基因CDS,利用生物信息学方法分析其序列组成特征;采用实时PCR方法分析乌金猪FTO基因mRNA在不同组织中的表达情况。结果:克隆的FTO基因全长1518 bp(已提交至GenBank数据库,登录号为JQ031263),编码由505个氨基酸残基构成的蛋白,推测的相对分子质量为58.16×103,等电点为5.18;乌金猪与牛、羊、人和大鼠的FTO蛋白的氨基酸序列同源性分别为91%、90%、89%和83%;进化树分析显示,推测的乌金猪FTO蛋白的氨基酸组成与牛、羊的亲缘性较近,其次是人、大鼠;推测的氨基酸组成中无跨膜区,无信号肽,为亲水蛋白;分析发现该蛋白有22个磷酸化修饰位点,包括10个丝氨酸蛋白激酶磷酸化位点、7个苏氨酸蛋白激酶磷酸化位点、5个酪氨酸蛋白激酶磷酸化位点,200、246、302位氨基酸残基有糖基化位点;二级结构预测发现该蛋白共有201个螺旋、33个伸展链和271个卷曲结构;实时PCR检测的组织表达谱表明,FTO基因mRNA在乌金猪肝脏组织中的表达量最高,在脂肪、肾、脾中也有大量表达,在心脏、肌肉中的表达量最少。结论:为深入探讨乌金猪FTO基因的生物学功能奠定了重要基础。     

中国家猪高分辨G—带及模式图

采用氨甲喋呤或胸苷阻断法使细胞分裂同步化,并结合胰酶G-带技术,对中国7个家猪品种高分辨G-带进行了研究,发现家猪品种间带型基本一致,从而参照人类细胞遗传学命名法的国际体制,提出了中国家猪高分辨G-带标准化核型及模式图,对显带核型界标进行了少许修改,对每对染色体进行了区带划分和描述。单倍染色体组所显示的G-带数目,包括X和Y染色体,巳达444条,近于中期染色体带纹数目的两倍。     

巴马小型猪肝硬化前后肠道乳杆菌的比较研究

目的比较巴马小型猪诱导型肝硬化造模前(正常猪)和造模成功后(肝硬化猪)肠道乳杆菌的变化情况,探讨小型猪肝硬化模型在肝硬化肠道微生态研究中的适用性。方法收集肝硬化造模前和CCl4诱导肝硬化造模成功后巴马小型猪的新鲜粪便,提取粪便总菌DNA,用乳杆菌特异性引物进行PCR扩增,对扩增产物进行变性梯度凝胶电泳(Denaturing Gradient Gel-Electrophoresis,DGGE),即用PCR-DGGE分析巴马小型猪肝硬化前后肠道乳杆菌的相似性和多样性。结果聚类分析和主成分分析显示巴马小型猪肝硬化前(正常猪)和肝硬化后混杂排列,无明显界限;多样性数据分析显示巴马小型猪肝硬化前后肠道乳杆菌的丰富度(S)、微生物区系Shannon-Wiener指数(H′)和均匀度(E)差异均无统计学意义(P0.05)。结论巴马小型猪肝硬化后肠道乳杆菌与正常猪比较差异无统计学意义。     

Activation of protein kinase C suppresses fragmentation of pig oocytes aged in vitro

When cultured for an extended time, pig oocytes that matured in vitro to the stage of metaphase II undergo the complex process designated as ageing. Under our conditions, some pig oocytes aged 3 days remained at the stage of metaphase II (22%), but others underwent spontaneous parthenogenetic activation (45%), and still others perished through fragmentation (28%) or lysis (5%). Activation of protein kinases C (PKCs) using phorbol-12-myristate-13-acetate (PMA) protects oocytes from fragmentation. None of the oocytes were fragmented after 3 days of aging in 50 nM of PMA. A similar effect (8% of fragmented oocytes) was observed after a 3-day treatment of aging oocytes with 100 μM of 1-stearoyl-2arachidonoyl-sn-glycerol (STEAR). PMA and STEAR activate both calcium-dependent and calcium-independent PKCs. This combined effect on PKCs seems to be essential for the protection of oocytes from fragmentation. Neither the specific activator of calcium-dependent PKCs 1-oleoyl-2-acetyl-sn-glycerol (OLE) nor the specific activator of calcium-independent PKCs dipalmitoyl-l-α-phosphatidylinositol-3,4,5-triphosphate heptaammonium salt (DIPALM) suppressed the fragmentation of aging pig oocytes. Twenty-one percentage of oocytes fragmented when aged for 3 days in 10 μM OLE and 26% of aged oocytes fragmented in 100 nM of DIPALM. However, fragmentation was significantly suppressed to 7% when the oocytes were exposed to the combination of both 10 μM OLE and 100 nM DIPALM. Aging pig oocytes cultured for 1 day with PMA maintained a high capability of being parthenogenetically activated (86% of activated oocytes), using calcium ionophore with 6-dimethylaminopurine. Ageing oocytes treated with PMA also had high capability of cleavage (82%) after their artificial parthenogenetic activation. However, their ability to develop to the stage of blastocyst (12%) was suppressed when compared with oocytes activated immediately after their maturation (29%).