Preliminary survey of estrogenic activity in part of waters in Haihe River, Tianjin

A preliminary survey of estrogenic activity of the contaminant in part of waters （Ziya River and the estuary of Haihe River） in Haihe River, Tianjin has been performed with the plasma vitellogenin （Vtg） in feral fish as a biomarker for estrogenic activity. The concentrations of bisphenol A （BPA）, 4-tert-octylphenol （OP） and 4-nonyl- phenol （NP） in surface water were also determined. The presence of Vtg in male fish plasma as well as that in female can be detected at different sites and different seasons. The results indicate that the water in the sampling sites was contaminated by some estrogenic compounds. Although BPA, OP and NP can be detected in all of the water samples, their concentrations were much lower than the effective concentrations for those chemicals to induce Vtg production in male fish.     

Vitellogenic responses of male Chinese loach (Misgurnus anguillicaudatus) exposed to the individual or binary mixtures of 17β-estradiol and nonylphenol

Male Chinese loaches were exposed to 17β-estradiol (E2) and nonylphenol (NP) both singly and in combination for 42 days using semi-static waterborne exposure system. Plasma vitellogenin (Vtg) was chosen as determining endpoint. The results demonstrated that 0.5 μg/L E2 could induce the enhancement of Vtg contents in male Chinese loaches after exposure for 21 days, which showed a time-related increasing manner; NP was also estrogenic to male Chinese loach, and the vitellogenic responses showed in a time-and dose-related manner, which was less potent than that of E2. The bi- nary mixtures of E2 and NP can significantly elicit the production of Vtg in male Chinese loaches, which was more potent than that of individual compounds, and Vtg induced in the binary mixture groups was higher than the summation of Vtg induced in the corresponding single-compound groups at the same concentration.     

Tissue-specific expression of glutathione S-transferases induced by 2-tridecanone or quercetin in cotton bollworms, Helicoverpa armigera (Hübner)

The tissue-specific expression of glutathione S-transferases (GSTs) in the cotton bollworm and the expression level induced by 2-tridecanone and quercetin were examined using the methods of biochemistry and the quantitative PCR. The relative expression level of GST mRNA was unanimous with the GSTs activity conjugaging with 1-chloro-2, 4-dimitro-benzene (CDNB) in fat bodies, midguts, heads and integuments of cotton bollworms. The GSTs activity in fat bodies was the highest, then midguts, heads and integuments in turn, which was in consistent with the relative expression level of GST mRNA. The specific activity of GSTs and the relative expression level of GST mRNA could be significantly induced by 2-tridecanone and quercetin, and after the induction the order of the GSTs activity and the relative expression level of GST mRNA in the above four tissues in cotton bollworms was not different from the control. The induction of GSTs by 2-tridecanone was stronger than by quercetin in all four tissues, which was in accordance with the relative expression level of GST mRNA. It suggested that the increase of GSTs activity induced by plant allelochemicals was associated with the elevated expression of GST mRNA in cotton bollworms.     

Tissue-specific expression of glutathione S-transferases induced by 2-tridecanone or quercetin in cotton bollworms, Helicoverpa armigera (Hübner)

The tissue-specific expression of glutathione S-transferases (GSTs) in the cotton bollworm and the expression level induced by 2-tridecanone and quercetin were examined using the methods of biochemistry and the quantitative PCR. The relative expression level of GST mRNA was unanimous with the GSTs activity conjugaging with 1-chloro-2, 4-dimitro-benzene (CDNB) in fat bodies, midguts, heads and integuments of cotton bollworms. The GSTs activity in fat bodies was the highest, then midguts, heads and integuments in turn, which was in consistent with the relative expression level of GST mRNA. The specific activity of GSTs and the relative expression level of GST mRNA could be significantly induced by 2-tridecanone and quercetin, and after the induction the order of the GSTs activity and the relative expression level of GST mRNA in the above four tissues in cotton bollworms was not different from the control. The induction of GSTs by 2-tridecanone was stronger than by quercetin in all four tissues, which was in accordance with the relative expression level of GST mRNA. It suggested that the increase of GSTs activity induced by plant allelochemicals was associated with the elevated expression of GST mRNA in cotton bollworms.     

Homoharringtonine induces apoptosis of endothelium and down－regulates VEGF expression of K562 cells

Homoharringtonine (HHT) has currently been used successfully in the treatment of acute and chronic myeloid leukemias and has been shown to induce apoptosis of different types of leukemic cells in vitro. Emerging evidence suggests that angiogenesis may play an important role in hematological malignancies, such as leukemia. However, whether HHT can relieve leukemia by anti-angiogenesis is still unknown. We investigated the anti-angiogenesis potential of HHT with the human umbilical vein endothelial cell line (ECV304) and leukemic cell line (K562) in vitro. Cellular proliferation was determined by MTT assay and apoptosis was analyzed by flow cytometry, The mRNA expression of vascular endothelial growth factor (VEGF) was assessed by RT-PCR and VEGF protein production was detected by Western blot. Inhibition of cell proliferation and induction of apoptosis by HHT were discovered in ECV304 cells, and appeared in a dose- and time-dependent manner, Also, treatment with HHT caused down-regulation of VEGF mRNA expression in K562 cells in similar dose- and time-dependent manner and inhibition of VEGF protein production in K562 cells in response to the enhancing concentration of HHT. The results demonstrated that HHT could also induce apoptosis in endothelium and down-regulate VEGF expression in K562 cells. In conclusion, we believe HHT has anti-angiogenesis potential and speculate that HHT might exert its anti-leukemia effects via reduction of angiogenesis.     

Optimization of cultural conditions for thermostable β-1,3-1,4-glucanase production by Bacillus subtilis ZJF-IA5

The optimization of cultural conditions for β-glucanase production by Bacillus subtilis ZJF-1A5 was investigated in flask trials. Temperature had great effect on β-glucanasc production which maximized atoptimal temperature of 37℃ and decreased significantly when temperature was over 37℃ . Charge quantity affected β-glucanasc production significantly. Adding oxygen vector N-dodecanc or acetic ether benefited β-glucanasc production, but it depended on the concentration and charge quantity. The results of fractional factorialdesign showed that age and size of inoculum and shaking speed were the key factors affecting β-glueanase production and the cultivation time span to reach the highest β-glucanasc activity. The optimal cultural conditionsfor p-glucanase production obtained with CCD were as follows: inoculum age and size (16 h, 3.82% (v/v)),shaking speed 210 r/rain, charge quantity of 30 mL in 250 mL flask and initial pH 7.0, cultured at 37℃ for 50 h. Repeated experimental results accorded with those predicted by a second-order polynomial model. The amount of p-glucanase, a-amylase and neutral protease produced by B subtills ZJF-1A5 was associated partially with cell growth. Those three enzymes‘ activities increased following the cell growth and increased signifi-cantly when cells entered the stationary phase.     

Activation of proteinkinase ERK mediates induction of macrophage MMP-12 by OxLDL

The present study was undertaken to investigate the effect of oxidized low density lipoprotein (oxLDL) on the expression of maerophage matrix metalloproteinase-12 (MMP-12), and the possible mechanisms. Activation of extracellular signal-regulated kinase 1/2 (ERK1/2) was detected by Western blot analysis.Enzymatic activity of MMP-12 was determined by β-casein zymography. RT-PCR analysis was used to measure the mRNA expression level of MMP-12. OxLDL-stimulated macrophages produced increased casein-degrading activities and oxLDL also significantly increased the mRNA level of MMP-12 in a dose-dependent manner.OxLDL stimulated the phosphorylation of ERK1/2 in macrophages. The use of the specific inhibitor indicated that the ERK1/2 signaling pathway was required for the induction of MMP-12.These data demonstrated that oxLDL induced MMP-12 expression in macrophages through an ERK1/2-dependent pathway.     

Effect of vitamin B12 on cleft palate induced by 2,3.7.8-tetrachlorodibenzo-p-dioxin and dexamethasone in mice

The purpose of this study was to investigate the effect of vitamin B12 on palatal development by co-administration of 2,3,7,8-tetrachlorodibenzo-p-dioxin （TCDD） and dexamethasone （DEX）. We examined the morphological and histological features of the palatal shelf and expression levels of key signaling molecules （trans- forming growth factor-β3 （TGF-β3） and TGF-β3 type I receptor （activin receptor-like kinase 5, ALK5）） during pala- togenesis among a control group （Group A）, TCDD＋DEX exposed group （Group B）, and TCDD＋DEX＋vitamin B12 exposed group （Group C）. While we failed to find that vitamin B12 decreased the incidence of cleft palate induced by TCDD＋DEX treatment, the expression levels of key signaling molecules （TGF-~3 and ALK5） during palatogenesis were significantly modulated. In TCDD＋DEX exposed and TCDD＋DEX＋vitamin B12 exposed groups, palatal shelves could not contact in the midline due to their small sizes. Our results suggest that vitamin B12 may inhibit the expression of some cleft palate inducers such as TGF-β3 and ALK5 in DEX＋TCDD exposed mice, which may be beneficial against palatogenesis to some degree, even though we were unable to observe a protective role of vitamin B12 in morphological and histological alterations of palatal shelves induced by DEX and TCDD.     

Characterization of a PDR type ABC transporter gene from wheat （Triticum aestivum L.）

DON, as a virulence factor, plays an important role in the infection of Fusarium graminearum in wheat. The infection ability of F. graminearum depends on its capacity of producing DON. The production of DON by F. graminearum is significantly decreased in the wheat varieties with scab resistance. In this study, GeneChip analysis indicated that an EST encoding an ATP-binding cassette （ABC） transporter was up-regulated by 45 times in a wheat landrace Wangshuibai, which is resistant to DON accumulation. A pair of EST-derived primers were designed based on the EST sequence, and a clone was then isolated from a wheat genomic DNA TAC library. The TAC clone was sequenced using chromosome walking and gene prediction was conducted using Softberry. A cDNA clone of this gene was subsequently isolated from Wangshuibai induced by DON using gene-specific primers designed according to the untranslated sequence of the gene. The genome size of the gene is 7377 bp, consisting of 19 exons with coding sequences of 4308 bp. It encodes a protein with 1435 amino acid residues and the calculated molecular weight is about 161 kD. BLAST analysis indicated that the gene may belong to pleiotropic drug resistance （PDR） sub-family, and hence designated as TaPDR1 （Triticum aestivum pleiotropic drug resistance）. TaPDR1 was located on chromosome 5A of wheat using nullisomic-tetrasomic lines of Chinese Spring. TaPDR1 was up-regulated by induction of both DON and F. graminearum. Expression patterns of TaPDR1 were different in wild-type Wangshuibai and the fast-neutron induced Wangshuibai mutant lacking FHB1, a major QTL of FHB resistance and DON resistance in chromosome arm 3BS. These results suggested that TaPDR1 might be a candidate gene responsible for DON accumulation resistance. The expression profile showed that TaPDR1 expression was neither induced by hormones typically involved in biotic stress, such as JA and SA, nor by abiotic stresses, such as heat, cold, wounding and NaCI. However, TaPDR1 expression was regulated by Al^3＋ and [Ca^2＋], indicating that [Ca^2＋]1 might mediate the signal of TaPDR1 expression.     

苯并〖DK(〗［a］芘(BaP)〖DK)〗对真鲷细胞色素P450和芳香烃受体基因表达的影响

 通过水体暴露方式对海水养殖真鲷进行BaP持续染毒,利用实时定量PCR技术研究了真鲷细胞色素P450基因(CYP1A1)和芳香烃受体基因(AhR2)随BaP暴露剂量、时间的动力学变化。结果发现,01～15 μg/L环境浓度的BaP能够显著性诱导CYP1A1基因和AhR2基因的表达,且AhR2 mRNA早于CYP1A1 mRNA被诱导表达;BaP持续暴露48 h,CYP1A1和AhR2基因的表达水平均随暴露时间的延长而显著升高,染毒72 h后又回复到本底水平,实验表明这两个基因的表达与BaP的暴露剂量和暴露时间之间具有显著性的剂量-效应和时间-效应关系。     

表没食子儿茶素没食子酸酯对IL-4刺激的小鼠腹腔巨噬细胞诱导型一氧化氮合酶和精氨酸酶-1表达的影响

为探讨表没食子儿茶素没食子酸酯(EGCG)对白介素-4(IL-4)刺激的巨噬细胞精氨酸酶1(Arg-1)和诱导型一氧化氮合成酶(iNOS)表达的影响.将6~8周龄Balb/c小鼠经无血清DMEM培养基刺激后,收集腹腔巨噬细胞用于体外培养.体外培养的巨噬细胞经20ng/mL IL-4和不同浓度EGCG处理后,用实时荧光定量PCR(RTqPCR)和酶联免疫吸附试验(ELISA)方法检测Arg-1和iNOS的mRNA表达水平和含量.结果显示IL-4和EGCG单独处理均可显著刺激Arg-1的表达和抑制iNOS的表达,而EGCG(12.5~50μM)增强IL-4刺激的Arg-1表达和IL-4对iNOS表达的抑制作用,且存在剂量依赖效应.上述结果提示EGCG以剂量依赖方式促进IL-4刺激的Arg-1表达、抑制iNOS的表达.     

模拟失重对大鼠肝脏CYP1A2的影响研究

为揭示失重条件下药物代谢酶变化规律,考察了不同模拟失重周期对鼠肝代谢酶CYP1A2的影响.将大鼠尾悬吊3,7,14,21 d模拟失重效应,分别采用Western-blot、Q-PCR及HPLC-UV检测鼠肝微粒体中CYP1A2的蛋白表达、mRNA表达及活性变化.与对照组相比,模拟失重3 d大鼠肝脏CYP1A2的蛋白表达量显著下降（p<0.05）,7,14 d显著上升（p<0.05）,21 d略有下降（p>0.05）.在大鼠肝脏CYP1A2 mRNA量及活性方面,模拟失重3,7,14 d均显著上升（p<0.05）,21 d上升不明显（p>0.05）.实验结果表明模拟失重14 d鼠肝中的CYP1A2蛋白、基因表达及活性显著上升,但21 d各指标有恢复到正常组的趋势.      

检测细胞色素P-450基因族中CYP1A1基因表达的一个简捷方法

NMR1系雄性小鼠被用来研究肺微粒体细胞色素P450家族中一个同功基因CYPlAl在香烟烟雾的诱导下表达．双轮回‘聚合酶链式反应’(PCR)和‘反向转录酶-聚合酶链式反应’(RT-PCR)被用来检测基因CYP1Al的转录水平．代表CYPlAl蛋白活性的7-乙氧基试卤灵-0-去乙基酶(EROD)的活性被用来检测基因CYPlAl的表达．双轮回‘聚合酶链式反应’(PCR)和‘反向转录酶-聚合酶链式反应’(RT-PCR)被用来检测基因CYPlAl的转录水平．测试结果表明在吸入香烟烟雾的情况下。小鼠肺微粒体EROD的活性和基因CYPlAl的转录水平是同步增加的．这个结果不同于以前在同样实验条件的一个自相矛盾的结果，即在CYPlAl蛋白增加时，基因CYPlAl的转录水平却是下降或不变．这说明本文所使用的方法可能要优于以前的实验方法.     

Efficient expression of human factor Ⅸ cDNA in liver mediated by hydrodynamics-based plasmid administration

Hydrodynamics-based administration via tail vein was used to deliver naked plasmid with human factor IX (hFIX) cDNA in 2.2 mL Ringer‘s solution into mice within 7 s. The peak level of expression of hFIX was 2921 ng/mL in mouse plasma. The hFIX cDNA expression increased with increasing the amount of plasmid DNA injected. The peak level of gene expression declined after repeated injection of plasmid (1459 ng/mL). The hFIX cDNA was detected in various organs, but the highest level of gene expression appeared in liver. Transaminase levels and liver histologicalresults showed that rapid intravenous plasmid injection into mice induced transient focal acute liver damage, which was rapidly repaired within 3--10 d. These results suggested that high-level expression of hFIX cDNA can be achieved by hydrodynamics-based plasmid transfer and this method is nowfurther used for gene therapy and gene function study in our lab.     

IGF-1对绵羊皮肤中GHR、IGF-1、IGF-1R、kAP3.2和KAP6-1基因表达的影响

选取36只健康、体况一致的新疆细毛羊随机分成6组。在每只羊的左侧肩胛部皮下局部注射0．5mL（10ng／mL）的IGF-1,右侧肩胛部皮肤为未注射IGF-1的对照组,然后分别于第0、3、6、9、12、50d采集各皮肤样品,用反转录多聚酶链式反应（RT-PCR）方法,定量分析绵羊皮肤中生长激素受体（GHR）、胰岛素样生长因子1（IGF-1）和胰岛素样生长因子1型受体（IGF-1R）、角蛋白关联蛋白KAP3．2和KAP6-1mRNA的相对丰度。试验结果表明,IGF-1对GHR基因的表达有下调的作用,对IGF-1、IGF-1R基因的表达没有显著的影响,而对KAP3．2、KAP6—1基因的表达具有显著的促进作用。说明IGF-1促进绵羊角蛋白关联蛋白基因的表达可能是通过生长轴以外的其他途径实现的。     

Rapid induction of mRNAs forPC3 by partial hepatectomy and epidermal growth factor

The expression of immediate early gene plays a pivotal role in rat hepatocyte proliferation from G₀ to G₁ phases and the progression through G₁ phase of the cell cycle within several hours after 2/3 hepatectomy. We investigated the different gene expressions within 1 h after 2/3 hepatectomy by representational difference analysis. Sequence analysis indicated thatPC3 induced by NGF was a kind of immediate early gene and might be correlated with liver regeneration. Moreover, we found that 2/3 hepatectomy could induce the expressing ofPC3 mRNA by Northern blot with a peak 1–2 h after surgery. In primary cultures of rat hepatocytes, addition of EGF resulted in rapid and transient induction ofPC3 mRNA. It was first reported thatPC3 gene belongs to immediate early gene associated with liver regeneration.     

Preparation of a recombinant adeno-associated viral vector with a mutation of human factor Ⅸ in large scale and its expression in vitro and in vivo

A series of adeno-associated viral vectors containing a mutation of human factor Ⅸ (hFⅨR338A) with different regulation elements were constructed and used to transduce cell lines. The plasmids and the stable transduction cell clones with high expression level of hFⅨR338A were obtained by selecting and optimizing, and then, the recombinant adeno-associated viral vector with hFⅨR338A was prepared via novel rHSV/AAV hybrid virus packaging system on a large scale, which contained the capsid protein genes. A method for producing rAAV-hFⅨR338A viral stocks on a large scale and higher titer was established, which can be used for industrial purpose. The titer of rAAV-hFⅨR338A was more than 1.25×10¹² particle/mL, and then, a mammalian cell line, C2C12 and the factor Ⅸ knock-out mice were transfected with the rAAV-hFⅨR338A in vitro and in vivo. The results show that the high-level expression of rAAV-hFⅨR338A was achieved in cell line and hemophilia B mice. It reached at (2551.32±92.14) ng·(10⁶ cells)^-1·(24 h)^-1 in C2C12 cell in vitro and had a peak concentration of 463.28 ng/mL in mice treated with rAAV-hFⅨR338A, which was as high as the expression of rAAV-hFⅨ-wt (2565.76±64.36) ng·(10⁶ cells)^-1·(24 h)^-1 in C2C12 and 453.92 ng/mL in the mice treated with rAAV-hFⅨ-wt) in vitro and in vivo, there is no any difference between two groups, but the clotting activity of hFⅨR338A is about 2.46 times higher than that of hFⅨ-wt. It was first reported that a mutation of human factor Ⅸ was used into gene therapy research for hemophilia B, meanwhile, a novel packaging system, rAAV/HSV was used for preparation of rAAV-hFⅨR338A on a large scale, which laid the foundation of industrial production for applying rAAV viral stocks to gene therapy clinical trial for hemophilia B mediated with rAAV-hFⅨ.     

Determination of sulfonamides with fluorescent inclusion complex of carbonic anhydrase and dansylamide

A new fluorimetric method for determination of sulfonamides was described based on the formation of a fluorescent inclusion complex of carbonic anhydrase(CA) with dansylamide(DNSA). The binding of DNSA to CA resulted in an enhancement in the fluorescence emission at 460 nm with excitation at 280 nm. Dissociation constants were determined for the carbonic anhydrase sulfonamide complexes. Linear calibration graphs of sulfonamides were obtained within a concentration range of 0- 0.058 μg/mL for DNSA; 0-0.344 μg/mL for sulfanilamide (SAN) and 0-0.286 μg/mL for P toluenesulfonamide (PTSN). The relative standard deviations were within 1.8%-4.2%. Limits of detection for DNSA, SAN and PTSN were 0.84, 19.5 and 6.1 ng/mL, respectively. The method was applied to the determination of sulfonamides in cow milk at ng/mL level.