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Purification and characterization of Moschatin, a novel type Ⅰ ribosomeinactivating protein from the mature seeds of pumpkin (Cucurbita moschata), and preparation of its immunotoxin against human melanoma cells

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Cell Research 2003年第5期13卷 369-374页

作者： HENG CHUAN XIA, FENG LI, ZHEN LI, Zu CHUAN ZHANG~* key laboratory of Proteomics, institute of Biochemistry and Cell biology, Shanghai institutes for Biological sciences, chinese academy of sciences, Shanghai 200031, china. State key laboratory of reproductive biology Institute of Zoology Chinese Academy of SciencesBeijing 100080 China Department of Veterinary Pathobiology University of Missouri-Columbia MO 65211 USA

A novel ribosome-inactivating protein designated Moschatin from the mature seeds of pumpkin （Cucurbita moschata） has been successively purified to homogeneity, using ammonium sulfate precipitation, CM-cellulose 52 column chromatography, Blue Sepharose CL-6B Affinity column chromatography and FPLC size-exclusion column chromatography. Moschatin is a type 1 RIP with a pI of 9.4 and molecular weight of～29 kD. It is a rRNA N-glycosidase and potently blocked the protein synthesis in the rabbit reticulocyte lysate with a IC50 of 0.26 nM. Using the anti-human melanoma McAb Ng76, a novel immunotoxin Moschatin-Ng76 was prepared successfully and it efficiently inhibited the growth of targeted melanoma cells M21 with a IC50 of 0.04 nM, 1500 times lower than that of free Moschatin. The results implied that Moschatin could be used as a new potential anticancer agent.

关键词： ribosome-inactivating protein (RIP) Moschatin immunotoxin.

Generation of male germ cells from mouse induced pluripotent stem cells in vitro

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Generation of male germ cells from mouse induced pluripotent...

2012全国发育生物学大会

作者： Yangfang Li~*,Xiuxia Wang,Xue Feng,Shangying Liao,Chunsheng Han State key laboratory of reproductive biology,institute of zoology,chinese academy of sciences,beijing 100101

Germ cells are the only cell type that passes genetic information to the next *** most metazoan species, primordial germ cells(PGCs) were induced from epiblasts by signals from the neighboring *** vitro derivation of germ cells from the pluripotent stem cells(PSCs) such as embryonic stem cells(ESCs) and induced PSCs(iPSCs) are of great values for the treatment of infertility,for animal breeding,and for studying the mechanism of germ cell development. Although the derivations of male germ cells from PSCs have been previously reported,most of the studies failed to conduct the induction in a well-controlled and highly efficient ***,we report the derivation of induced PGC-like cells (iPGCLCs) from mouse iPSCs via induced epiblast-like cells(iEpiLCs) as being monitored by the expression of enhanced green fluorescent protein gene under the control of the promoter of stimulated by retinoic acid 8(Stra8-EGFP).The identity of iPGCLCs was characterized by examining the expression of multiple marker genes as well as by the recovery of spermatogenesis after they were transplanted to the testis of infertile WW ***,iPGCLCs were either induced to germline stem cell-like cells(iGSCLCs) or reverted back to embryonic germ cell-like cells(iEGLCs). Conclusions:We have established an efficient procedure for inducing iPSCs into iPGCLCs that can be further expanded and induced to more developed germ *** work indicates that the technology of in vitro germ cell induction is becoming more sophisticated and can be further improved.

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用少量样本进行抑制性消减杂交

生物化学与生物物理进展 2003年第6期30卷 889-893页

作者：孙晓阳谈寅飞唐爽王雁玲中国科学院动物研究所计划生育生殖生物学国家重点实验室北京100080

利用根据cap finder方法建立的全长cDNA合成技术 ,扩增获得了恒河猴着床点子宫内膜组织表达mRNA的双链cDNA ,通过抑制性消减杂交 ,成功地构建了恒河猴着床点消减文库 .随机挑选文库中的阳性克隆 ,经点杂交证明 2 7%为着床点差异表达的克... 详细信息

利用根据cap finder方法建立的全长cDNA合成技术 ,扩增获得了恒河猴着床点子宫内膜组织表达mRNA的双链cDNA ,通过抑制性消减杂交 ,成功地构建了恒河猴着床点消减文库 .随机挑选文库中的阳性克隆 ,经点杂交证明 2 7%为着床点差异表达的克隆 .由此表明抑制性消减杂交结合cap finder扩增全长cDNA的方法。

关键词：抑制性消减杂交全长cDNA扩增恒河猴着床点消减文库基因表达

Mining and characterization of ubiquitin E3 ligases from the mouse genome

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Mining and characterization of ubiquitin E3 ligases from the...

“细胞活动生命活力”——中国细胞生物学学会全体会员代表大会暨第十二次学术大会

作者： Xiaojun Hou~1,Wei Zhang~(1,2),Chunsheng Han~1 1.State key laboratory of reproductive biology,institute of zoology,chinese academy of sciences,beijing 100101,china 2.Graduate University of chinese academy of sciences,beijing 100039,china

Ubiquitin-mediated protein modification and degradation are believed to play important roles in mammalian *** our previous studies,we have identified several ubiquitin E3 Iigases that belong to the RING finger protein *** the present study,we first did a genome-wide mining of all potential mouse E3 Iigases,and identified those that are highly or specifically expressed in the *** a result,92 putative E3s belonging to the RING,U-box,and F-box subfamilies were discovered and 21 of them are abundantly expressed in the testis based on microarray expression *** selected 15 such genes for further characterization by examining their expression,cellular localization and E3 ligase activity. RT-PCR results showed that all the selected genes are predominately expressed in *** nuclear localization was observed in CHO and Hela cell *** addition,some novel proteins possess E3 ubiquitin ligase activity indicated by both in vivo and in vitro *** results indicated that large number of E3 Iigases are probably involved in *** will be important to identify their substrate in order to elucidate their molecular functions in spermatogenesis.

关键词： spermatogenesis Ubiquitination E3 ligase

富含脯氨酸小蛋白-2在小鼠子宫中的表达及调节

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生物化学与生物物理进展 2004年第1期31卷 73-76页

作者：谈寅飞孙晓阳唐爽朴允尚王雁玲中国科学院动物研究所

富含脯氨酸小蛋白 (Sprrs)参与构建复层扁平上皮的角化细胞壳 (CE) ,它们在子宫单层上皮中的作用还不清楚 .采用RNA印迹和半定量RT PCR方法 ,研究了Sprr2在小鼠动情周期和妊娠子宫中的表达及其激素调控 .实验结果发现 :Sprr2在动情前期... 详细信息

富含脯氨酸小蛋白 (Sprrs)参与构建复层扁平上皮的角化细胞壳 (CE) ,它们在子宫单层上皮中的作用还不清楚 .采用RNA印迹和半定量RT PCR方法 ,研究了Sprr2在小鼠动情周期和妊娠子宫中的表达及其激素调控 .实验结果发现 :Sprr2在动情前期和动情期表达上调 ,而动情后期和间情期表达下调 .在妊娠初期表达迅速下调 ,直至临产期表达重新受到诱导并在产后达到高峰 .鉴于其在不同生殖阶段子宫中独特的表达模式和在复层上皮中保护性的功能。

关键词：富含脯氨酸小蛋白子宫应激情周期妊娠分娩

Computational analysis of mouse piRNAs reveals multiple features potentially associated with their biogenesis and function

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Computational analysis of mouse piRNAs reveals multiple feat...

“细胞活动生命活力”——中国细胞生物学学会全体会员代表大会暨第十二次学术大会

作者： Haiyun Gan,Xiwen Lin,Zhuqiang Zhang,Wei Zhang,Shangying Liao,Lixian Wang,Chunsheng Han State key laboratory of reproductive biology,institute of zoology,chinese academy of sciences,beijing,china 100101

piRNAs(PlWI-interacting RNAs) are a class of small RNAs abundantly expressed in animal gonads with a length about *** that map to retrotransposons are generated by a "ping-pong" amplification loop to suppress the activity of ***,the biogenesis and function of other categories of piRNAs have yet to be *** the present study,we first profiled the expression of small RNAs in type A spermatogonia,pachytene spermatocytes and round spermatids by deep *** then focused on the computational analysis of the potential piRNAs generated in the present study as well as other published *** found that piRNAs with different characteristics and length were expressed during spermatogenesis in *** piRNAs of 26-27nt relative to repetitive sequences and protein-coding genes were mainly expressed in type A ***,most of the piRNAs of 26-27nt also mainly expressed in pachytene spermatocytes were mapped to intergenic *** piRNAs of 29-30nt mainly expressed in round spermatids were also mapped to intergenic *** were many piRNAs mapping to unique genome location co-localized with exon,rarely with *** there also existed piRNAs of cross-intron,indicating that piRNAs might originate from mature *** generating mRNAs(PRMRs),whose expression positively correlated with their piRNA products,constituted one third of the protein-coding genes and were evolutionarily conserved and enriched with splicing isoforms and antisense *** with piRNAs preferentially mapped to CDSs and 3’UTRs partitioned into three clusters differentially expressed during spermatogenesis and enriched with unique sets of functional annotation terms related to housekeeping activities as well as spermatogenesis specific processes. Intergenic piRNAs were divided into 2992 clusters probably representing novel transcriptional units that have not been *** transcripts of a large number of genes involved in spe

关键词： piRNA miRNA spermatogenesis mitosis meiosis antisense transcript alternative splicing

Ribophorin II is a substrate of spermatid-specific E3 ligase RNF148

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Ribophorin II is a substrate of spermatid-specific E3 ligase...

“细胞活动生命活力”——中国细胞生物学学会全体会员代表大会暨第十二次学术大会

作者： Wei Zhang~(1,2),Zhenyu Xiao~(1,2),Chunsheng Han~1 1.State key laboratory of reproductive biology,institute of zoology,chinese academy of sciences,beijing 100101,china 2.Graduate University of chinese academy of sciences,beijing 100039,china

Ring finger proteins(RNFs) are a subfamily of zinc finger proteins and many of them function as ubiquitin ligases(E3s).Many RNFs are germ cell-specifically expressed and play important roles in spermatogenesis. Previously,we have shown that RNF133 is a testis-specific endoreticulum-associated *** this study,we show that another spermatid-specific gene Rnf148,located 5k-bp upstream of Rnfl33,also encodes an E3 with similar protein domains including a signal peptide,a protease-association domain,a transmembrane domain and a C3HC4 RING finger *** vitro assays showed that RNF148 worked with ubiquitin-conjugating enzymes(E2s) such as UbcH5b and UbcH7,which is involved in endoreticulum associated degradation(ERAD) ***148 co-localized with the reticulum-associated E2 MmUbc6, and co-immunoprecipitated with CD3-.which is a typical substrate in ERAD *** two-hybrid screening revealed that RNF148 physically interacts with ribophorin II(RPNII),which is a component of ***-immunoprecipitation and cycloheximide treatment experiments confirmed that RPNII is not only a partner but also a substrate of *** was highly expressed in spermatogonia and spermatocytes,in which RNF148 was *** negative correlation of the expression of RNF148 and RPNII in mouse testis implied that RPNII is targeted by RNF148 for degradation in *** RPNII is involved in the biosynthesis process of N-glycans on glycoproteins, which has been reported to play key roles in germ cell-Sertoli cell attachment,we propose that RNF148 may inhibit the synthesis of glycoproteins through the degradation of RPNII resulting in the dissociation of germ cell from Sertoli cells and their release into the seminiferous tubules.

关键词： RNF148 RPNII RING E3 ligase ubiquitination spermatogenesis

Genital ridge somatic cells regulated the proliferation but not the directional migration of primordial germ cells (PGCs)

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Genital ridge somatic cells regulated the proliferation but ...

2012全国发育生物学大会

作者： Su-Ren Chen,Qiao-Song Zheng,Jun Zhang,Qing Wen,Min Chen,Fei Gao,Yi-Xun liu State key laboratory of reproductive biology,institute of zoology,chinese academy of sciences,beijing,100101

Previous research suggests that the interaction of germ cells and somatic cells is important in gonad development. However,the exact functions of somatic cells in germ cell development remain largely unknown due to a lack of direct in vivo *** this study,a genital ridge(GR)-deficient mouse model,Wt1,was used to investigate the roles of GR somatic cells in primordial germ cell(PGC) migration and *** demonstrated that GR somatic cells were not required for the directional migration of *** of the PGCs reached the mesenchyme under the coelomic epithelium at E10.5,and no ectopic PGCs were noted in GR-deficient ***,the number of PGCs at E11.5 and E12.5 was dramatically reduced in Wt1 *** study revealed that the reduction of PGC number in GR-deficient embryos was due to the decreased proliferation of PGCs,and not increased ***,we found that the germ cells in Wt1 embryos were still labeled with the proliferative marker Ki67 at E13.5,while germ cells in control embryos underwent mitotic arrest at this *** results indicate that the proliferation of germ cells during gonad development is precisely regulated by somatic cells. Conclusions:In summary,our study provides the first in vivo evidence that GR is not required for the directional migration of PGCs but that proliferation of germ cells is tightly regulated by GR somatic cells.

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用激光共聚焦显微术检测哺乳动物卵母细胞中纺锤体相关蛋白的方法(英文)

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生物化学与生物物理进展 2004年第1期31卷 54-58页

作者：范衡宇孟小倩霍立军佟超陈大元孙青原中国科学院动物研究所

哺乳动物卵母细胞是发育生物学和细胞生物学的重要研究对象 .激光共聚焦显微术是检测卵母细胞中纺锤体相关蛋白的有用技术 ,但是针对哺乳动物卵母细胞的特点需要做许多调整以得到理想结果 .其中 ,卵母细胞的准备、固定、透膜、脂类抽提... 详细信息

哺乳动物卵母细胞是发育生物学和细胞生物学的重要研究对象 .激光共聚焦显微术是检测卵母细胞中纺锤体相关蛋白的有用技术 ,但是针对哺乳动物卵母细胞的特点需要做许多调整以得到理想结果 .其中 ,卵母细胞的准备、固定、透膜、脂类抽提、抗体孵育步骤十分关键 .另外 ,不同物种的卵母细胞也有差异。

关键词：卵母细胞哺乳动物激光共聚焦显微术纺锤体