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Adenovirus-mediated expression of pig α(1,3. galactosyltransferase reconstructs Gal α(1,3. Gal epitope on the surface of human tumor cells

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cell Research 2001年第2期11卷 116-124页

作者： XING li, guo HONG XIA, JIAN FEI, FANG HUANG, li he guo institute of biochemistry and cell biology, {3. institutes for biological sciences, chinese academy of sciences, shanghai 20003., china 1institute of biochemistry and cell {3. Shanghai Institutes for Biological Sciences Chinese Academy of Sciences Shanghai 200031 China 1institute of biochemistry and cell {3. Shanghai Institutes for Biological Sciences Chinese Academy of Sciences Shanghai 200031 China 1institute of biochemistry and cell {3. Shanghai Institutes for Biological Sciences Chinese Academy of Sciences Shanghai 200031 China 1institute of biochemistry and cell {3. Shanghai Institutes for Biological Sciences Chinese Academy of Sciences Shanghai 200031 China 1institute of biochemistry and cell {3. Shanghai Institutes for Biological Sciences Chinese Academy of Sciences Shanghai 200031 China

Gal α(1, 3. Gal (gal epitope) is a carbohydrate epitope and synthesized in large amount by α(1, 3. galactosyltransferase [α(1, 3. GT] enzyme on the cells of lower mamma{3.an animals such as pigs and mice. Human has no gal epitope due to the inactivation of α(1, 3. GT gene but produces a large amount of antibodies (anti-Gal) which recognize Gal α(1, 3. Gal structures specifically. In this study, a rep{3.cation deficient recombinant adenoviral vector Ad5sGT containing pig α(1, 3. GT cDNA was constructed and characterized. Adenoviral vector-mediated transfer of pig α(1, 3. GT gene into human tumor cells such as ma{3.gnant melanoma A3.5, stomach cancer SGC-7901, and lung cancer SPC-A-1 was reported for the first time. Results showed that Gal epitope did not increase the sensitivity of human tumor cells to human complement-mediated lysis, although human complement activation and the binding of human IgG and IgM natural antibodies to human tumor cells were enhanced significantly after Ad5sGT transduction. Appearance of gal epitope on the human tumor cells changed the expression of cell surface carbohydrates reacting with Ulex europaeus I (UEA I) lectins, Vicia villosa agglutinin (VVA), Arachis hypogaea agglutinin (PNA), and Glycine max agglutinin (SBA) to different degrees. In addition, no effect of gal epitope on the growth in vitro of human tumor cells was observed in MTT assay.

关键词： Adenoviral vector galactosyltransferase Gal α(1 3. Gal gene expression human tumor cell.

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Transgenic mice overexpressingγ-aminobutyric acid transporter subtypeⅠ develop obesity

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cell Research 2000年第4期10卷 303-310页

作者： MA YING HUA JIA HUA HU XIAO GANG ZHOU RUO WANG ZENG ZheN TONG MEI JIAN FEI li he guo institute of biochemistry and cell biology, shanghai {3. for biological sciences, chinese academy of science, shanghai 20003., china shanghai institute of harmacy.ankara.edu.tr

Transgenic mice ubiquitously overexpressing murine γ aminobutyric acid transporter subtype Ⅰ were created. Unexpectedly, these mice markedly exhibited heritable obesity, which features significantly increased body weight and fat deposition. Behavioral examination revealed that transgeinc mice have slightly reduced spontaneous locomotive capacity and altered feeding pattern. Tills preliminary finding indicates that the inappropriate level of γ-aminobutyric acid transporters may be directly or indirectly involved in the pathogenic mechanism underlying certain types of obesity.

关键词： γ-aminobutyric acid transporter subtype Ⅰ transgenic mouse obesit€

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GABA transporter 1 transcriptional starting site exhibiting tissue specific difference

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cell Research 2001年第2期11卷 161-163页

作者： JIN XIAO PING, FANG HUANG, NA YANG, BU FENG LU, JIAN FEI, li he guo institute of biochemistry and cell biology, shanghai institutes for {3. science, chinese academy of sciences, 3.0 Yueyang Road, shanghai 20003., china institute of biochemistry and cell {3. Chinese Academy of Sciences Shanghai.

GABA transporter 1(GAT1) takes important roles in multiple physiological processes through the uptake and release of GABA, but the regulation of GAT1 gene expression in different tissues is rarely known. To address the question, first, 5’ Rapid amp{3.fication of cDNA end (RACE) was used to determine GAT1 transcriptional starting sites in neonatal mouse cerebral cortex and intestine, adult mouse brain and adult rat testis. The products of 5’RACE were confirmed by DNA sequencing. We found that the transcript of GAT1 in neonatal mouse cerebral cortex and adult mouse brain starts at the same site (inside of exon 1), while in mouse intestine, GAT1 starts transcription in intron 1, and in rat testis, the transcript of GAT1 has an additional untranslation exon to the 5’ direction.

关键词： GABA transporter 1(GAT1) transcriptional starting site 5’ RACE tissue spedcific difference DNA sequencing.

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Overexpression of γ-aminobutyric acid transporter subtype I leads to susceptibility to Kainic acid-induced seizure in transgenic mice

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cell Research 2001年第1期11卷 61-67页

作者： MA YINGHUA, JIA HUA HU, WEN JUAN ZHAO, JIAN FEI, YUN YU, XIAO GANG ZHOU, ZheN TONG MEI, li he {3. (institute of biochemistry and cell biology, shanghai institutes for biological sciences, chinese academy of sciences,{13., china) (shanghai Inst 1institute of biochemistry and cell {3. Shanghai Institutes for Biological Sciences Chinese Academy of Sciences Shanghai 200031 China 1institute of biochemistry and cell {3. Shanghai Institutes for Biological Sciences Chinese Academy of Sciences Shanghai 200031 China 1institute of biochemistry and cell {3. Shanghai Institutes for Biological Sciences Chinese Academy of Sciences Shanghai 200031 China 1institute of biochemistry and cell {3. Shanghai Institutes for Biological Sciences Chinese Academy of Sciences Shanghai 200031 China 1institute of biochemistry and cell {3. Shanghai Institutes for Biological Sciences Chinese Academy of Sciences Shanghai 200031 China 1institute of biochemistry and cell {3. Shanghai Institutes for Biological Sciences Chinese Academy of Sciences Shanghai 200031 China 2shanghai institute of Physiology Chinese Academy of Sciences Shanghai 200031 China 1institute of biochemistry and cell {3. Shanghai Institutes for Biological Sciences Chinese Academy of Sciences Shanghai 200031 China

γ-aminobutyric acid (GABA) is the principal inhibitory neurotransmitter, and the GABAergic synaptic transmission is normally terminated by the rapid uptake through GABA transporters. With transgenic mice ubiquitously overexpressing GABA transporter subtype I (GAT1), the present study explored the pathophysiological role of GAT1 in epileptogenesis. Though displaying no spontaneous seizure activity, these mice exhibit altered electroencephalographic patterns and increased susceptibility to seizure induced by kainic acid. In addition, the GABAA receptor and glutamate transporters are up-regulated in transgenic mice, which perhaps reflects a compensatory or corrective change to the elevated level of GAT1. These preliminary findings support the hypothesis that excitatory and inhibitory neurotransmission, and seizure susceptibility can be altered by neurotransmitter transporters.

关键词： γ-aminobutyric acid transporter seizure susceptibility kainic acid electroencephalography transgenic mice

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Enhancement of germ cell apoptosis induced by ethanol in transgenic mice overexpressing Fas ligand

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cell Research 2003年第5期13卷 361-367页

作者： JIA HUA HU, JIE JIANG, YING HUA MA, NA YANG, MAO HU ZHANG, MIN WU, JIAN FEI, li he guo 1 Laboratory of Molecular {3. biology, institute of biochemistry and {3. biology, shanghai institutes for biological sciences,{13.s, shanghai 20003., china 2 College of life science, Zhejiang University, Hangzhou 3.0027, china 3.shanghai Research Center for Biomodel Organism, shanghai 201203. china 1Laboratory of Molecular cell biology Institute of Biochemistry and Cell Biology Shanghai Institutes for Biological Sciences Chinese Academy of Sciences Shanghai 200031 China. E-mail: mhzhang@*** 2College of life science Zhejiang University Hangzhou 310027 China 1Laboratory of Molecular cell biology Institute of Biochemistry and Cell Biology Shanghai Institutes for Biological Sciences Chinese Academy of Sciences Shanghai 200031 China. E-mail: mhzhang@*** 1Laboratory of Molecular cell biology Institute of Biochemistry and Cell Biology Shanghai Institutes for Biological Sciences Chinese Academy of Sciences Shanghai 200031 China. E-mail: mhzhang@*** 1Laboratory of Molecular cell biology Institute of Biochemistry and Cell Biology Shanghai Institutes for Biological Sciences Chinese Academy of Sciences Shanghai 200031 China. E-mail: mhzhang@*** 2College of life science Zhejiang University Hangzhou 310027 China 3.0} Research Center for Biomodel Organism Shanghai 201203 China 1Laboratory of Molecular cell biology Institute of Biochemistry and Cell Biology Shanghai Institutes for Biological Sciences Chinese Academy of Sciences Shanghai 200031 China. E-mail: mhzhang@***

It was suggested that chronic ethanol exposure could result in testicular germ cell apoptosis, but the mechanism is still unclear. In the present study, we use a model of transgenic mice ubiquitously overexpressing human FasL to investigate whether Fas ligand plays a role in ethanol-induced testicular germ cell apoptosis. Both wild-type (WT) mice and transgenic (TG) mice were treated with acute ethanol (20％ v/v) by introperitoneal injection for five times. After ethanol injection, WT mice displayed up-regulation of Fas ligand in the testes, which was shown by FITC-conjugated flow cytometry and western blotting. Moreover, TG mice exhibited significantly more apoptotic germ cells than WT mice did after ethanol injection, which was demonstrated by DNA fragmentation, PI staining flow cytometry and TUNEL staining. In addition, histopathological examination revealed that degenerative changes of epithelial component of the tubules occurred in FasL overexpressing transgenic mice while testicular morphology was

关键词： Fas ligand ethanol apoptosis testes transgenic mouse.

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Direct transfer of A20 gene into pancreas protected mice from streptozotocin-induced diabetes

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Acta Pharmacologica Sinica 2004年第6期25卷 19-24页

作者： Lu-yang YU, Bo liN, Zhen-lin ZHANG2, li-he guo3. instituteof biochemistryand cell biology, shanghai institutes for biological {13.s, chinese academy of{13.s, shanghai 20003. Center for Preventing and Treating Osteoporosis, Osteoporosis Research Unit, 2 shanghai JiaoTong UniversityAffiliated Sixth People’s Hospital, shanghai 20023., china institute of biochemistry and cell {3. Shanghai Institutes for Biological Sciences Chinese Academy of Sciences Center for Preventing and Treating Osteoporosis Osteoporosis Research Unit Shanghai JiaoTong University Affiliated Sixth People’s Hospi

AIM: To investigate the efficiency of transfer of A20 gene into pancreas against STZ-induced diabetes. METHODS: PVP-plasmid mixture was directly transferred into the pancreatic parenchyma 2 d before STZ injection. The uptake of plasmid pcDNA3.LacZ or pcDNA3.A20 was detected by PCR and the expression of LacZ was confirmed by histological analysis with X-gal. A20 expression in the pancreas of pcDNA3.A20 transgenic mice was measured by RT-PCR and Western blots. Urine amylase, NO generation, and histological examination were examined. RESULTS: Injection of PVP-plasmid mixture directly into the pancreatic parenchyma increased urine amylase concentration 16 h after operation and reversed it to nearly normal 3. h later. On d 3. LacZ expression could be found in spleen, duodenum, and islets. The development of diabetes was prevented by direct A20 gene transferring into the pancreas and A20-mediated protection was correlated with suppression of NO production. The insulitis was ameliorated in A20-treated mice. CONCLUSION: Injection of PVP-plasmid mixture directly into the pancreatic parenchyma led to target gene expression in islets. Direct transfer of A20 gene into the pancreas protected mice from STZ-induced diabetes.

关键词： adenoviridae streptozocin insulin-dependent diabetes mellitus gene therapy nitric oxide glucose amylases reversed transcriptase polymerase chain reaction Western blotting

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Identification of glutamate transporters and receptors in mouse testis

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Acta Pharmacologica Sinica 2004年第03期25卷 112-117页

作者： Jia-hua HU,Na YANG2,Ying-hua MA,Jie JIANG,Jin-fu ZHANG3.Jian FEI4,li-he guo4 Laboratory of Molecular {3. biology,institute of biochemistry and {3. biology,shanghai institutes for biological sciences,{13. academy of sciences,shanghai 20003. 3.epartment of Urology,Tongji Hospital,Tongji University,shanghai 200065,china Laboratory of Molecular cell biology Institute of Biochemistry and Cell BiologyShanghai Institutes for Biological Sciences Chinese Academy of Sciences Department of Urology Tongji Hospital Tongji University

AIM: To investigate the presence of glutamate transporters and receptors in mouse testis. METHODS: Glutamate uptake analysis was performed to study the function of glutamate transporters in mouse testis. Comparative RT-PCR technique and sequencing analysis were used to study the expression of glutamate receptors and transport- ers in mouse testis. RESULTS: Mouse testis possessed glutamate uptake capacity with sodium-dependence. Vmax value of glutamate uptake was (1.60 ± 0.21) pmol/min per mg protein and Km value of glutamate uptake was (11.0±1.6) μmol/L in mouse testis according to saturation analysis. Furthermore, the uptake activity could be inhibited by DHK (GLT1 selective inhibitor) and THA (glutamate uptake inhibitor). In addition, RT-PCR results revealed that glutamate transporters (GLT1 and EAAC1) and ionotropic glutamate receptors (NR1, NR2B, GluR6 and KA2) were expressed in mouse testis. CONCLUSION: Glutamate transporters and receptors do exist in mouse testis.

关键词： glutamates receptors amino acid transport systems testis uptake

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Maternal zinc deficiency impairs brain nestin expression in prenatal and postnatal mice

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cell Research 2001年第2期11卷 135-141页

作者： WANG FU DI, WEI BIAN, liNG WEI KONG, FA JI ZHAO, JUN SheNG guo, NAI he JINGI institute of biochemistry and cell biology, shanghai institutes for biological sciences, chinese academy of sciences, 3.0 Yue Yang Road,{13., china Department of Publ 2Department of Public health Second Military Medical University Shanghai 200433 China 1institute of biochemistry and cell {3. Shanghai Institutes for Biological Sciences Chinese Academy of Sciences 320 Yue Yang Road Shanghai 200031 China 1institute of biochemistry and cell {3. Shanghai Institutes for Biological Sciences Chinese Academy of Sciences 320 Yue Yang Road Shanghai 200031 China 2Department of Public health Second Military Medical University Shanghai 200433 China 2Department of Public health Second Military Medical University Shanghai 200433 China 1institute of biochemistry and cell {3. Shanghai Institutes for Biological Sciences Chinese Academy of Sciences 320 Yue Yang Road Shanghai 200031 China

Effects of maternal dietary zinc deficiency on prenatal and postnatal brain development were investigated in ICR strain mice. From d 1 of pregnancy (EO) until postnatal d 20 (P20), maternal mice were fed experimental diets that contained 1 mg Zn/kg/day (severe zinc deficient, SZD), 5 mg Zn/kg/day (marginal zinc deficient, MZD), 3. mg Zn/kg/day (zinc adequately supplied, ZA) or 100 mg Zn/kg/day (zinc supplemented, ZS and pair-fed, PF). Brains of offspring from these dietary groups were examined at various developmental stages for expression of nestin, an intermediate filament protein found in neural stem cells and young neurons. Immunocytochemistry showed nestin expression in neural tube 10. 5 d post citrus (dpc) as well as in the cerebral cortex and neural tube from 10.5 dpc to postnatal d 10 (P10). Nestin immunoreactivities in both brain and neural tube of those zinc-supplemented control groups (ZA, ZS, PF) were stronger than those in zinc-deficient groups (SZD and MZD). Western blot analysis confirmed that nestin levels in -pooled brain extracts from each of the zinc-supplemented groups (ZA, ZS, PF) were much higher than those from the zinc-deficient groups (SZD and MZD) from 10.5 dpc to P10. Immunostaining and Western blots showed no detectable nestin in any of the experimental and control group brains after P20. These observations of an association between maternal zinc deficiency and decreased nestin protein levels in brains of offspring suggest that zinc deficiency suppresses development of neural stem cells, an effect which may lead to neuroanatomical and behavioral abnormalities in adults.

关键词： Zinc deficiency nestin brain development neural stem cells.

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Six Specific Lysihe Residues are Crucial in Maintaining the Structure and Function of Soluble Manganese Stabilizing Protein

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Acta Biochimica et Biophysica Sinica 2006年第9期 611-619页

作者： Jin-Peng GAO~(1,2#) Feng ZHANG~(1,3#) li ZHANG~4 Yin-Long guo~4 Kang-Cheng RUAN~5 De-An JIANG~3* Chun-he XU~1 1 institute of Plant Physiology and Ecology,shanghai institutes for biological sciences,chinese academy of sciences,shanghai 20003.,china 2 Graduate School of the chinese academy of sciences,shanghai 20003.,china 3 College of life sciences,Zhejiang University,Hangzhou 310029,china 4 shanghai Mass Spectrometry Center,shanghai institute of Organic Chemistry,chinese academy of sciences,shanghai 20003.,china 5 Laboratory of Proteomics,institute of biochemistry and cell biology,shanghai institutes for biological sciences,chinese academy of sciences,shanghai 20003.,china

When manganese stabilizing protein （MSP） was treated with 0.5mM N-succinimidyl propionate（NSP）,the rebinding ability and oxygen-releasing capabilities of the modified MSP were not altered,in spiteof changes of MSP surface Lys ***,far-ultraviolet circular dichroism and intrinsicfluorescence spectra analysis revealed that 0.5 mM NSP-modified MSP retained most of its native secondaryand tertiary *** of the sites of NSP modification by Staphylococcus V8protease digestion ofthe modified protein,as well as analysis by matrix-assisted laser desorption ionization-time of flight massspectrometry,indicated that seven Lys residues were *** results suggested that these residues arenot absolutely essential to the structure and function of ***,when the NSP concentration wasincreased to 4mM,the modified MSP was unable to bind photosystem Ⅱ and completely lost its *** far-ultraviolet circular dichroism and intrinsic fluorescence spectra analysis revealed a clearconformational change in MSP after 4 mM NSP treatment,suggesting that some Lys residues are involved inmaintaining the structure and function of *** by matrix-assisted laser desorption ionization-timeof flight mass spectrometry indicated that another six Lys residues,namely Lys20,Lys101,Lys196,Lys207,Lys13. （or Lys13.） and Lys66 （or Lys76）,were modified by 4 mM ***,these six Lys residuesare crucial in maintaining the structure and function of soluble MSP.

关键词： chemical modification fluorescence spectrum circular dichroism spectrum manganese stabilizing protein Lys residue

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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 biology, shanghai {13. 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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