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Impaired reproduction in transgenic mice overexpressing γ-aminobutyric acid transporter Ⅰ (GAT1)

cell Research 2004年第1期14卷 54-59页

作者： Jia Hua HU~(1,*), Jin Fu zhang~(2,*), Ying Hua MA~1, Jie JIANG~1, Na YANG~1, Xin Bo LI~3. Zhi Guang YU CHI~4, Jian FEI~(1,5,**), Li He GUO~(1,**) ~1Laboratory of Molecular cell biology, {3. of biochemistry and cell biology, shanghai institutes for biological Sciences, chinese academy of Sciences, shanghai {13., china. ~2Department of Urology, Tongji Hospital, Tongji University, 3.9 Xincun Road, shanghai 200065, china. ~3.ew Drug R & D Center, North china Pharmaceutical Group Corporation, 3.8 Heping East Road, Shijia Zhuang 050015, Hebei, china. ~4School of life sciences, Fudan University, 220 Handan Road, shanghai 20043., china. ~5shanghai Research Center for Biomodel Organism, shanghai 201203. china. Laboratory of Molecular cell biology Institute of Biochemistry and Cell Biology Shanghai Institutes for Biological Sciences Chinese Academy of Sciences Shanghai 200031 China.

It is well documented that γ-aminobutyric acid (GABA) system existed in reproductive organs. Recent researches showed that GABA_A and GABA_B receptors were present in testis and sperm, and might mediate the acrosome reaction induced by GABA and progesterone. GABA transporter Ⅰ (GAT1) also existed in testis and sperm, but its physiological function was unknown. In the present study, we used GAT1 overexpressing mice to explore GAT1 function in male reproductive system. We found that the expression level of GAT1 continuously increased in wild-type mouse testis from 1 month to 2 months after birth. GAT1 overexpression in mouse affected testis development, which embodied reduced testis mass and slowed spermatogenesis in transgenic mice. Moreover, transgenic mice showed increase of the percentage of broken sperm. The further study revealed that the reproductive capacity was impaired in GAT1 overexpressing mice. In addition, testosterone level was significantly low in transgenic mice compared with that in wi

关键词： GABA GAT1 testes sperm reproduction transgenic.

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Studies on the Temperature Effect on Bacteriorhodopsin of Purple and Blue Membrane by Fluorescence and Absorption Spectroscopy

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Acta Biochimica et Biophysica Sinica 2006年第10期38卷 691-696页

作者： Lan-Ying CHENG~(1,2) Yue zhang~3.Shi-Gui LIU~1 Kun-Sheng HU~3.Kang-Cheng RUAN~2* 1 College of Life Science,Sichuan University,Chengdu 610064,china 2 State Key Laboratory of Molecular biology,{3. of biochemistry and cell biology,shanghai institutes for biological Sciences,chinese academy of Sciences,shanghai {13.,china 3.{3. of Biophysics,Academia Sinica,Beijing 100101,china State Key Laboratory of Molecular biology Institute of Biochemistry and Cell Biology Shanghai Institutes for Biological Sciences Chinese Academy of Sciences Shanghai 200031 China

Fluorescence and absorption spectra were used to study the temperature effect on theconformation of bacteriorhodopsin (bR) in the blue and purple membranes (termed as bRb and bRprespectively).The maximum emission wavelengths of tryptophan fluorescence in both proteins at roomtemperature are 3.0 nm,and the fluorescence quantum yield of bRb is about 1.4 fold higher than that of *** temperature increases,the tryptophan fluorescence of bRb decreases,while the tryptophan fluorescenceof bRp *** binding study of extrinsic fluorescent probe bis-ANS indicated that the probe can bindonly to bRb,but not to *** results suggest that significant structural difference existed between bRband *** was also found that both kinds of bR are highly thermal *** maximum wavelength of theprotein fluorescence emission only shifted from 3.0 nm to 3.6 nm at 100℃.More interestingly,as tempera-ture increased,the characteristic absorption peak of bRb at 605 nm decreased and a new absorption peak at3.0 nm *** transition occurred at a narrow temperature range (65℃-70℃).These facts indicatedthat an intermediate can be induced by high *** phenomenon has not been reported before.

关键词： bacteriorhodopsin purple membrane blue membrane fluorescence and absorption spectroscopy

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cAMP elevators inhibit LPS-induced IL-12 p40 expression by interfering with phosphorylation of p3. MAPK in Murine Peritoneal Macrophages

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cell Research 2002年第Z2期 331-337页

作者： WEI Guo FENG, YI BING WANG, JIN SONG zhang, XING Yu WANG, CHANG LIN LI, ZONG LIANG CHANGLaboratory of Immune Signaling Transduction, institute of biochemistry and {3. biology, shanghai institutes for biological Sciences, chinese academy of Sciences, shanghai 20003., china

cAMP mediated signaling may play a suppressive role in immune response. We previously found that the cAMP-elevators (CTx and 8-Br-cAMP) inhibited IL-12, IL-la, IL-6 gene expression, but increased the transcriptional levels of IL-10 and IL-IRa in LPS-treated murine peritoneal macrophages. The present study examined a possible molecular mechanism involved in cAMP elevators-induced inhibition of IL-12 p40 expression in response to LPS. Our data demonstrated that cAMP elevators downregulated IL-12 p40 mRNA expression and IL-12 p70 production in murine peritoneal macrophages. Subsequent studies revealed that cAMP-elevators blocked phosphorylation of p3. MAPK, but did not affect the activity of NF-kB binding to IL-12 promoter (-13./-112). This is the first report that cAMP elevators inhibit LPS-induced IL-12 production by a mechanism that is associated, at least in part, with p3.-dependent inhibition by cAMP signaling pathways.

关键词： IL-12 cAMP LPS NF-kB P3. MAPK.

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Identification and Characterization of GH Receptor and Serum GH-binding Protein in chinese Sturgeon (Acipenser sinensis)

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Acta Biochimica et Biophysica Sinica 2004年第12期36卷 811-816页

作者： Zhi-Yong LIAO1,2 and Shang-Quan ZHU2* 1College of Life Sciences, Jiangxi Normal University, Nanchang 3.0027, china 2Key Laboratory of Proteomics biochemistry and cell {3., shanghai institutes for biological Sciences, chinese academy of Sciences, Graduate School of the chinese academy of Sciences, shanghai 20003., china College of Life Sciences Jiangxi Normal UniversityNanchang 330027 China Key Laboratory of Proteomics biochemistry and cell biology Shanghai Institutes for Biological Sciences Chinese Academy of Sciences Graduate School of the Chinese Academy of SciencesShanghai 200031 China

chinese sturgeon, a kind of cartilage ganoid, has a history of over one billion years and it is called the living fossil of aquatic biology since it keeps some evolutionary trace. Here, we characterized the growth hormone receptor (GHR) and serum growth hormone binding protein (GHBP) of chinese sturgeon. It was shown that GHR was expressed in various tissues, mainly in hepatic, kidney and intestine tissues. GHR on the hepatic membrane has high and specific affinity for bream GH (brGH) and Scatchard analysis of the binding data showed a single class of high affinity binding site with an association constant Ka of 3.1×109 M–1. A specific band around 94 kD was detected by SDS-PAGE in cross-linking studies of membrane receptors. After incubation of chinese sturgeon serum with 125I-brGH, a 125I-brGH-GHBP complex was identified by Sephadex G-75, indicating that in the serum exists GHBP specially binding to brGH.

关键词： chinese sturgeon growth hormone receptor growth hormone binding protein ligand binding

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Functional analysis of two Sp1/Sp3.binding sites in murine Nanog gene promoter

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cell Research 2006年第3期16卷 319-322页

作者： Da Yong Wu Zhen Yao Laboratory of Molecular cell biology,Laboratory of Stem cell biology,institute of biochemistry and cell biology,shanghai institutes for biological Sciences,chinese academy of Sciences 3.0 Yue Yang Road,shanghai 20003.,china 1Laboratory of Molecular cell biology Laboratory of Stem Cell Biology Institute of Biochemistry and Cell Biology Shanghai Institutes for Biological Sciences Chinese Academy of Sciences 320 Yue Yang Road Shanghai 200031 China.

Nanog gene plays a key role in maintaining pluripotency of ES cells and early embryonic cells.A 5’ flank sequenceof the Nanog gene has been reported to be regulated differentially,and two regulatory elements within the Nanog pro-moter,namely Oct-4 and Sox-2 binding sites,have been identified to regulate the transcriptional activity ofNanog *** this report,we identified the role of two putative Sp 1 binding sites located in the Nanog gene 5’-flanking region inregulation of murine Nanog gene *** studies showed that the two sites were essential for the Nanogpromoter *** shift and supershift analysis showed that both sites specifically bind Spl and ***,overexpression of dominant-negative Sp 1 or Sp3.mutants significantly inhibits Nanog promoter *** resultssuggest that the transcription factor Spl and Sp3.are important for Murine Nanog gene expression.

关键词： Nanog promoter Sp1 Sp3

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Fusion Protein of Interleukin 4 and Diphtherial Toxin with High Cytotoxicity to Cancer cells

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Acta Biochimica et Biophysica Sinica 2004年第6期36卷 437-442页

作者： Yu-Jian zhang, Xiao-BoHU, Su-Xia LI1, Li-Ping TIAN, Sheng-Li YANG, and Yi GONG* Research Center of Biotechnology, shanghai institutes for {3. Sciences,chinese academy of Sciences, Graduate School of the chinese academy of Sciences, shanghai 20023., china State Key Laboratory of Bioreactor Engineering, East china University of Science and Technology, shanghai 20023., china Research Center of Biotechnology Shanghai Institutes for Biological SciencesChinese Academy of Sciences Graduate School of the Chinese Academy of SciencesShanghai 200233 China State Key Laboratory of Bioreactor Engineering East China University of Science and TechnologyShanghai 200237 China

Receptor of human interleukin 4 (hIL4R) has been found to be present on many types of cancer, so it may be a good target for cancer therapy. Here, fusion toxin gene DT4H has been constructed by fusing DNA sequence encoding the first 3.9 amino acids of diphtherial toxin (DT), which can not bind its own receptor, to human interleukin 4 (hIL4) gene. In order to improve the affinity of fusion toxin for hIL4R, a circularly permuted form of hIL4 (cpIL4) was used. The fusion gene was expressed in Escherichia coli where the fusion toxin DT4H was highly expressed. Purified DT4H was very cytotoxic to cancer cell line U251 cells, and moderate cytotoxic to HepG2 and MCF-7 cells. SGC-7901 cells were insensitive to it. The cytotoxic action of DT4H was specific because it was blocked by excess hIL4. These results suggest that DT4H may be a useful agent in the treatment of certain malignancies.

关键词： fusion toxin diphtherial toxin cancer therapy interleukin 4 IL-4 receptor

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HAL1 mediate salt adaptation in Arabidopsis thaliana

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cell Research 2001年第2期11卷 142-148页

作者： YANG SU XIN, YAN XIU ZHAO, QUAN zhang, YU KE HE, HUI zhang, DA LUO institute of Plant Physiology, {3. institutes for biological Sciences, chinese academy of Science, 3.0 Fenglin Road, {3. 20003., china Key Laboratory of Plant Stress Research, Sh 1institute of Plant Physiology Shanghai Institutes for Biological Sciences Chinese Academyof Science 300 Fenglin Road Shanghai 200032 China 2Key Laboratory of Plant Stress Research Shandong Normal University Jinan 250014 China 2Key Laboratory of Plant Stress Research Shandong Normal University Jinan 250014 China 1institute of Plant Physiology Shanghai Institutes for Biological Sciences Chinese Academyof Science 300 Fenglin Road Shanghai 200032 China 2Key Laboratory of Plant Stress Research Shandong Normal University Jinan 250014 China 1institute of Plant Physiology Shanghai Institutes for Biological Sciences Chinese Academyof Science 300 Fenglin Road Shanghai 200032 China

The yeast HAL1 gene was introduced into Arabidopsis thaliana by Agrobacterium tumefaciens-mediated transformation with vacuum infiltration under the control of CaMV 3.S promoter. Thirty-three individual kanamycin resistant plants were obtained from 75,000 seeds. Southern blotting analysis indicated that HAL1 gene had been integrated into all of the transgenic plants’ genomes. The copy number of HAL1 gene in transgenic plants was mostly 1 to 3.by Southern analysis. Phenotypes of transgenic plants have no differences with wild type plants. several samples of transformants were self-pollinated, and progenies from transformed and non-transformed plants (controls) were evaluated for salt tolerance and gene expression. Measurement of concentrations of intracellular K+ and Na+ showed that transgenic lines were able to retain less Na+ than that of the control under salt stress. Results from different tests indicated the expression of HAL1 gene promotes a higher level of salt tolerance in vivo in the transgenic Arabidopsis plants.

关键词： Salt-tolerant Arabidopsis HAL1 gene engineering.

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一个抗真菌蛋白在绿色木霉中的分泌表达

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生物化学与生物物理学报 2003年第5期35卷 454-458页

作者：徐军龚蓁蓁中国科学院上海生命科学研究院生物化学与细胞生物学研究所分子生物学国家重点实验室中国科学院上海生命科学研究院生物化学与细胞生物学研究所分子生物学国家重点实验室上海20003.

AFP(antifungalprotein)是在丝状真菌巨大曲霉 (AspergillusgiganteusMDH18894 )中分泌的一个抗真菌蛋白。其mRNA含长度为 4 3.bp的开放阅读框 ,编码 94个氨基酸的AFP前体 ,而成熟的AFP为 5 1个氨基酸的多肽。根据推测 ,在巨大曲霉中 ,... 详细信息

AFP(antifungalprotein)是在丝状真菌巨大曲霉 (AspergillusgiganteusMDH18894 )中分泌的一个抗真菌蛋白。其mRNA含长度为 4 3.bp的开放阅读框 ,编码 94个氨基酸的AFP前体 ,而成熟的AFP为 5 1个氨基酸的多肽。根据推测 ,在巨大曲霉中 ,AFP前体可能经两步剪切去除前导序列 (4 3.氨基酸 ) ,并最终形成具有抗真菌活性的成熟AFP ,已有报道证实 ,在另一种丝状真菌绿色木霉 (Trichodermaviride)基因组中存在一个类似AFP基因但不表达的序列 ,该序列与没有内含子的AFPcDNA序列完全一样。为了解巨大曲霉AFP基因可否在绿色木霉中表达 ,将AFP基因开放阅读框插入真菌表达载体trpC基因的启动子和终止子之间 ,并成功的转化了绿色木霉。SDS PAGE和Western印迹分析表明 ,绿色木霉转化子分泌表达了具有抗真菌活性的成熟AFP。为研究在绿色木霉中分泌表达具有重要应用价值的异源真核蛋白质打下了基础。

关键词：绿色木霉巨大曲霉抗真菌蛋白分泌表达

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Effect of two human growth hormone receptor antagonists on glomerulosclerosis in streptozotocin-induced diabetic rats

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Acta Pharmacologica Sinica 2004年第4期25卷 92-97页

作者： Wei LI, Li-hua ZHU, En-bi WANG, Zeng-can YE, Jun LIN, Li-he GUO1, Fei-hong LUO2, Xi-hong LIU2, Xin FANG2, Shui-xian SHEN2 institute of biochemistry and cell {3., shanghai institutes for biological Science, chinese academy of Sciences, 2Children’s Hospital ofshanghaiMedical University, shanghai20003., china institute of biochemistry and cell {3. Shanghai Institutes for Biological Science Chinese Academy of Sciences Children\'s Hospital of Shanghai Medical University Shanghai 200031 China

AIM: To explore the feasibility of human growth hormone (hGH) receptor antagonist in the treatment of end-stage diabetic renal complications. METHODS: Two hGH mutants, hGHA1 (Cys-hGH-del1-4, G120R, K168A, E174A, C182S, del186-191) and hGHA2 (hGH-H21A, G120R, E174A) were expressed in E coli. The IC50 (Mean±SD) values for the mutants for inhibiting 125I-hGH binding to rabbit growth hormone receptor were (65±10) ng for hGHA1, (27±5.6) ng for hGHA2, and (10±0.6) ng for wild type hGH, respectively. RESULTS: After treatment for 12 weeks, the renal histology analysis showed that treatment with hGHA2 at 4 mg/kg body weight daily markedly suppressed glomerulosclerosis in streptozotocin-induced diabetic Sprague-Dawley (SD) rats; hGHA1 at the same dosage slightly increased the renal damage compared with saline; while wild type hGH at 1 U/kg body weight daily severely worsened the glomerulo-sclerosis in diabetic SD rats. CONCLUSION: The data indicated that hGHA2 inhibited the end-stage glomerulosclerosis in diabetic rats, but hGHA1 mildly increased the glomerulosclerosis.

关键词： growth hormone diabetic nephropathies mutation diabetes mellitus

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Embryonic stem cells generated by nuclear transfer of human somatic nuclei into rabbit oocytes

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cell Research 2003年第4期13卷 251-263页

作者： YING CHEN, ZHI Xu HE, AILIAN LIU, KAI WANG, WEN WEI MAO, JIAN XIN CHU, YONG LU, ZHENG Fu FANG, YING TANG SHI, QING zhang YANG, DA YUAN CHEN, MIN RANG WANG, JIN SONG LI, SHAO LIANG HUANG, XIANG YIN KONG, YAO ZHOU SHI, Zm QIANG WANG, JIA HuI XIA, ZHI GAO LONG, ZHI GANG XUE, WEN XIANG DING, HuI ZHEN SHENG Center for Developmental biology, shanghai Second Medical University, shanghai 200092, china Laboratory of Stem cell biology, institute of biochemistry and cell biology, shanghai institutes for {13. Sciences, chinese academy of Sciences, 1665 Kong Jiang Road, shanghai 200092, china Center for Stem cell Research, the Second Affiliated Hospital, Sun Yat-Sen University, Guangzhou 510120,china State Key Laboratory of Reproductive biology, institute of Zoology, chinese academy of Sciences, 19 Zhong-guan Cun Road, Haidian District, Beijing 100080, china Health Science Center, shanghai institutes of {13. Sciences, chinese academy of Sciences and shanghai Second Medical University, shanghai 200025, china National Laboratory of Medical Genetics, Central South University, 88 Xiangya Road, Changsha 410078, china Xinhua Hospital, shanghai Second Medical University, 1665 Kong Jiang Road, shanghai 200092, china 2Laboratory of Stem cell biology Institute of Biochemistry and Cell Biology Shanghai Institutes for Biological Sciences Chinese Academy of Sciences 1665 Kong Jiang Road Shanghai 200092 China. 3.enter for Stem cell Research the Second Affiliated Hospital Sun Yat-Sen University Guangzhou 510120 China. 2Laboratory of Stem cell biology Institute of Biochemistry and Cell Biology Shanghai Institutes for Biological Sciences Chinese Academy of Sciences 1665 Kong Jiang Road Shanghai 200092 China. 2Laboratory of Stem cell biology Institute of Biochemistry and Cell Biology Shanghai Institutes for Biological Sciences Chinese Academy of Sciences 1665 Kong Jiang Road Shanghai 200092 China. 2Laboratory of Stem cell biology Institute of Biochemistry and Cell Biology Shanghai Institutes for Biological Sciences Chinese Academy of Sciences 1665 Kong Jiang Road Shanghai 200092 China. 2Laboratory of Stem cell biology Institute of Biochemistry and Cell Biology Shanghai Institutes for Biological Sciences Chinese Academy of Sciences 1665 Kong Jiang Road Shanghai 200092 China. 2Laboratory of Stem cell biology Institute of Biochemistry and Cell Biology Shanghai Institutes for Biological Sciences Chinese Academy of Sciences 1665 Kong Jiang Road Shanghai 200092 China. 2Laboratory of Stem cell biology Institute of Biochemistry and Cell Biology Shanghai Institutes for Biological Sciences Chinese Academy of Sciences 1665 Kong Jiang Road Shanghai 200092 China. 2Laboratory of Stem cell biology Institute of Biochemistry and Cell Biology Shanghai Institutes for Biological Sciences Chinese Academy of Sciences 1665 Kong Jiang Road Shanghai 200092 China. 2Laboratory of Stem cell biology Institute of Biochemistry and Cell Biology Shanghai Institutes for Biological Sciences Chinese Academy of Sciences 1665 Kong Jiang Road Shanghai 200092 China. 4State Key Laboratory of Reproductive biology Institute of Zoology Chinese Academy of Sciences 19 Zhong-guan Cun Road Haidian District Beijing 10008

To solve the problem of immune incompatibility, nuclear transplantation has been envisaged as a means to produce cells or tissues for human autologous transplantation. Here we have derived embryonic stem cells by the transfer of human somatic nuclei into rabbit oocytes. The number of blastocysts that developed from the fused nuclear transfer was comparable among nuclear donors at ages of 5, 42, 52 and 60 years, and nuclear transfer (NT) embryonic stem cells (ntES cells) were subsequently derived from each of the four age groups. These results suggest that human somatic nuclei can form ntES cells independent of the age of the donor. The derived ntES cells are human based on karyotype, isogenicity, in situ hybridization, PCR and immunocytochemistry with probes that distinguish between the various species. The ntES cells maintain the capability of sustained growth in an undifferentiated state, and form embryoid bodies, which, on further induction, give rise to cell types such as neuron and muscle, as well

关键词： nuclear transfer (NT) somatic cell nuclear transfer (SCNT) embryonic stem cells (ES cell) therapeutic cloning rabbit oocyte.

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