网翅蝗科三种蝗虫卵子发生时c-kit蛋白表达和比较

为探讨c-kit蛋白在蝗虫卵子发生过程中的表达和调控机制,应用免疫组织化学和统计分析等方法对网翅蝗科(直翅目,蝗总科)3种蝗虫卵子发生过程中8个代表性阶段c-kit蛋白表达进行观测和比较,3种蝗虫分别为:绿牧草蝗 Omocestus viridulus(Linnaeus),素色异爪蝗Euchorthippus unicolor(Ikonn.)和条纹异爪蝗Euchorthippus vittatus Zheng.结果显示蝗虫卵子发生第1～6阶段卵母细胞中有不同程度c-kit蛋白特异性表达,但随着卵黄发生的开始逐渐消失,而且3种蝗虫卵子发生过程中c-kit蛋白表达存在种间差异.以上结果提示c-kit蛋白在卵子发生中的表达暗示它参与和调控卵母细胞增殖与分化,此外c-kit蛋白表达种间差异说明在它的调控下不仅导致蝗虫卵子发生进程的迥异而且可能参与维系种间生殖隔离等机制.     

异翅负蝗配子发生过程中c-kit蛋白表达动态

应用免疫组织化学和生物统计分析相结合的方法,对异翅负蝗Atractomorp haheteroptera Bei Bienko配子发生过程中c-kit特异表达特点和动态进行研究。结果表明,(1)精子发生过程中,精原细胞、初级精母细胞、次级精母细胞和成熟精子中均有不同程度的c-kit蛋白表达,精巢末端还有较粗大的阳性颗粒分布;(2)卵子发生过程中,第1~6阶段卵母细胞中有不同程度的c-kit蛋白特异性表达,但随着卵黄发生的开始逐渐消失;(3)此外,滤泡细胞、输卵管和受精囊的腺细胞中有c-kit蛋白颗粒的存在;(4)从c-kit蛋白季节动态变化看,精子发生和卵子发生中的阳性表达都随着时间的延续出现下降的态势。因此,c-kit蛋白的特异性表达提示该蛋白参与配子发生过程的阶段性调控,具有重要的生理作用。     

类c-kit原癌蛋白在多伊棺头蟋胚后精子发生中的表达和定位

为了了解类c-kit原癌蛋白在多伊棺头蟋Loxoblemmus doenitzi Stein胚后精子发生中的表达、定位及可能的调控作用,采用常规免疫组织化学方法进行了相关研究。结果表明：处于减数分裂中期Ⅰ至末期Ⅱ的初级精母细胞的细胞膜上有类c-kit原癌蛋白阳性颗粒;精巢或受精囊内成熟精子头部也具有类c-kit原癌蛋白阳性颗粒。结果反映了类c-kit蛋白对于维持动物精子发生过程中减数分裂、精子成熟及受精能力具有特殊功能。     

大垫尖翅蝗配子发生过程中c-kit蛋白特异性表达

应用免疫组织化学和生物统计分析相结合的方法,对大垫尖翅蝗Epacromius coerulipes (Ivan.)配子发生过程中c-kit蛋白特异表达特点和动态进行研究.结果表明:(1)精子发生过程中,精原细胞、初级精母细胞、次级精母细胞和成熟精子中均有不同程度的c-kit蛋白表达,精巢末端还有较粗大的阳性颗粒分布;(2)卵子发生过程中,第1～6阶段卵母细胞中有不同程度的c-kit蛋白特异性表达,但随着卵黄发生的开始逐渐消失;(3)滤泡细胞、输卵管和受精囊的腺细胞中有c-kit蛋白颗粒的存在.     

花胫绿纹蝗和疣蝗配子发生时原癌基因c-kit特异性表达比较研究

应用免疫组织化学和生物统计分析相结合的方法,对花胫绿纹蝗Aiolopus tamulus (Fabricius)和疣蝗Trilophidia annulata (Thunberg)配子发生过程中原癌基因c-kit特异表达进行了比较研究。结果表明：（1）精子发生过程中,精原细胞、初级精母细胞、次级精母细胞和成熟精子中均有不同程度的c-kit蛋白表达,精巢末端还有较粗大的阳性颗粒分布;（2）卵子发生过程中,第1～6阶段卵母细胞中有不同程度的c-kit蛋白特异性表达,但随着卵黄发生的开始逐渐消失;（3）2种蝗虫配子发生过程中c-kit表达存在种间差异。     

精子发生过程中相关酶及蛋白因子的功能

精子发生经历了复杂的细胞分化过程,该过程受许多因素的调控,诸多因素中,生精细胞内一些酶和蛋白因子与精子发生密切相关.近年来随着蛋白离子交换柱层析、免疫印迹、免疫定位荧光和双相凝胶电泳等蛋白鉴定技术方法的发展和应用,发现了许多与精子发生相关的酶与蛋白因子.这些酶与蛋白因子,有些是在精子发生的多个阶段有不同程度的表达,有些呈现严格的阶段性特异表达,在精子发生过程中都发挥了重要作用.对这些酶和蛋白因子在精子发生过程中的功能作用进行综述.有助于进一步了解精子发生过程的调控机制.     

MCT在精子发生中的表达规律及调控机制的研究进展

单羧酸转运蛋白(monocarboxylate transporters,MCTs)是哺乳动物细胞膜上一类重要的跨膜转运蛋白,主要负责乳酸盐、丙酮酸盐、酮体等单羧酸类化合物的跨膜转运。MCT基因在睾丸生精上皮细胞发育、分化过程中具有不同程度表达,并通过多种途径调节精子发生过程。开展对MCT基因在精子发生过程的作用研究有助于人们从能量代谢角度进一步阐明生精细胞发育和精子发生的调控机制。本研究着重从MCT在精子发生过程中的表达定位、功能及调节机制进行综述。     

Polycomb基因家族在大鼠精子发生过程中的表达

目的检测大鼠精子发生不同阶段细胞中Polycomb-group（Pc-G）家族在mRNA水平上表达是否有差异。方法提纯大鼠精子发生过程中的精原细胞、精母细胞、圆形精子细胞以及支持细胞,用荧光定量PCR方法检测Pc-G家族基因mRNA表达量。结果Pc-G基因家族中Ezh2、Eed、Bmi-1在精子发生中后期高表达;在各生精细胞中,YY1基因表达量低于支持细胞。结论Pc-G基因家族在精子发生各阶段细胞中特征性表达,与精子发生具有相关性,可能对精子发生分化和维持遗传稳定性都有重要的作用。     

哺乳动物雷帕霉素靶蛋白（mTOR）在不同发育阶段SD大鼠睾丸中的表达及作用

精子发生是男性生殖中的主要过程,精原细胞的不断分裂增殖又保证了精子发生的顺利进行。随着年龄的不断增长,男性精子的数量、质量出现下降趋势。mTOR信号转导通路在细胞增殖分化中发挥着中心调控作用,因此,mTOR信号通路可能在精子发生过程中有着重要的地位。为了探明mTOR信号通路与精子发生的关系,首先,通过SD大鼠睾丸组织切片的免疫组化,发现mTOR是在生精小管的精原细胞胞浆中表达;其次,采用FQ-PCR检测mTOR mRNA在SD大鼠睾丸中的表达。结果显示,80周龄组mTOR的转录与8周龄组相比差异显著。最后利用Western blot检测出mTOR蛋白的表达及其对下游靶蛋白P70S6K的磷酸化效率均随年龄的增长逐渐下降。同时,在用雷帕霉素处理8周龄SD大鼠中,发现精子数量减少,P70S6K磷酸化效率降低并伴随生精小管萎缩和空泡化。通过这些结果,可以看出mTOR信号转导通路可能在精子发生中发挥着重要作用。     

小鼠精子发生中环形RNA生成和功能分析

小鼠精子发生过程中存在大量环状RNA(circRNA,circular RNA),其来源和功能尚不清楚。利用生物信息学手段分析小鼠精子发生过程中5个时期(精原干细胞,原始精原细胞、前细线期精母细胞,粗线期精母细胞及圆形精子细胞)的circRNA,共发现3万余个circRNA。对circRNA两侧内含子中的重复序列分析发现:circRNA两侧内含子中显著富集反向互补重复序列。这些重复序列不仅包括已报道的SINE/Alu序列,还包括SINE/B2、SINE/B4、LINE/L1,LTR/ERVL-MaLR和LTR/ERVK,暗示多种类别的反向互补序列有可能参与了精子发生过程中的circRNA形成。采用sailfish-cir和maSigPro对获得的circRNA进一步定量和差异表达分析,发现5个不同时期的精子细胞中共存在409个差异表达circRNA,它们所在基因能够富集在与精子发生密切相关的功能类群上。对差异表达的circRNA进行miRNA结合预测,共发现有137个circRNA-miRNA结合位点。精子发生相关基因来源的circRNA序列中有93%含有与翻译有关的m~6A基序,暗示精子发生进程中的部分circRNA有形成多肽的潜力。研究发现小鼠精子发生过程中许多circRNA具有RNA结合蛋白(RBP)结合位点,提示circRNA可能具有"RBP海绵"的潜在功能。     

Temporal expression of c-kit in spermatogenesis of two grasshopper species

Two species of grasshoppers, Calliptamus abbreviatus （Ikonn.） and Shirakiacris shirakii （I. Bol.）, were collected randomly in the Siping area of Jilin Province, China. By using immunohistochemical methods and statistical analysis, we observed and compared the temporal expression of c-kit protein in four representative stages of spermatogenesis of the two grasshoppers, namely： spermatogonia; primary spermatocyte; secondary spermatocyte; and mature sperm. Results showed that there was c-kit positive temporal expression at each stage of spermatogenesis, but there were different positive expression levels： （i） weak positive expression of c-kit protein appeared in spermatogonia and the positive granules were thinner; （ii） strong positive expression of c-kit protein existed in primary spermatocyte and positive granules became biggest among all developmental stages; （iii） c-kit positive expression stayed stronger in secondary spermatocyte while positive granules became thinner; （iv） there was a strong positive expression of c-kit and thinner positive granules in mature sperm, which distributed on head and tail; （v） the biggest c-kit positive granules had been found massing at the end of spermary; and （vi） significant differences of c-kit positive expression existed in spermatogenesis between two species of grasshoppers. The results indicated that c-kit protein may play a crucial role in spermatogenesis and even retain the physiological action of sperms and fertilization in grasshoppers.     

c-kit protein, a transmembrane kinase: identification in tissues and characterization.

The proto-oncogene c-kit encodes a transmembrane kinase which is related to the receptors for colony-stimulating factor type 1 and platelet-derived growth factor, as well as to the immunoglobulin superfamily. Antibodies specific for the kinase domain of the P80 gag-kit protein of the Hardy-Zuckerman 4 feline sarcoma virus were prepared. These kit-specific antibodies were used to identify and characterize the c-kit protein in cat brain tissue. The c-kit protein product displays an autophosphorylating activity in immune complex kinase assays, and, in turn, this activity was used to identify the c-kit protein in different tissues. In cat brain, a single 145-kilodalton (kDa) glycoprotein was detected. Its N-linked carbohydrates were found to be sensitive to digestion with the endoglycosidases (neuraminidase, endoglycosidase F, and endoglycosidase H), indicating hybrid and/or complex and high-mannose structures. A partial purification of the c-kit protein was achieved by wheat germ agglutinin affinity chromatography, and the autophosphorylating activity of the partially purified c-kit protein was characterized and found to be specific for tyrosine. The kit antibodies cross-react with the murine c-kit protein product, and variant c-kit proteins in different mouse tissues were identified, with sizes of about 145 kDa (brain), 160 kDa (spleen), and 150 kDa (testis).     

金丝桃属和三腺金丝桃属植物叶的红外光谱分析及其与分类群鉴定的相关性

利用傅里叶红外光谱仪和OMINI采样器直接迅速准确地测定金丝桃属(Hypericum L.)9组43种1亚种1变种和三腺金丝桃属(Triadenum Raf)2种植物的红外光谱,结果表明:各分类群(种)的红外光谱具有高度特异性和重现性,这两属及其金丝桃属组间的红外光谱图存在较大的差异,而组内种间红外光谱图的差异较小,同种不同分布区和不同发育时期的叶的红外光谱几无差别;其红外光谱图的变化可以作为这两属植物的分类依据之.这也暗示利用已知的标准红外光谱图库,可以区分和鉴定出这两属或其他属植物的种类.     

Steel factor-induced tyrosine phosphorylation in murine mast cells. Common elements with IL-3-induced signal transduction pathways.

The c-kit/W gene encodes a transmembrane protein tyrosine kinase, which is the receptor for Steel factor (SLF). SLF shares many general characteristics of hemopoietic growth factors, stimulating the survival, proliferation, and differentiation of stem and progenitor cells. We have investigated the tyrosine phosphorylation events that ensue after SLF binding to the c-kit protein using primary cultures of murine mast cells as a model system and have compared the effects of SLF and IL-3. Proteins that became phosphorylated on tyrosine after treatment of cells with SLF included c-kit itself, and major protein substrates designated p130, p122, p118, p115, p112, p100, p77, p55, p44, and p42. The majority of these proteins were cytosolic and maximally phosphorylated within 2 min of growth factor treatment. Combinations of immunoprecipitation and immunoblotting with antibodies specific for proteins known to be associated with signaling pathways demonstrated that none of the major tyrosine-phosphorylated species correlated with phospholipase C-gamma 1, GTPase activating protein, or phosphatidylinositol 3' kinase. However, stimulation with SLF led to a modest increase in tyrosine phosphorylation of the 85-kDa subunit of the phosphatidylinositol 3' kinase and increased association with a 150-kDa phosphotyrosyl protein, likely to be c-kit. Two species that did correlate with known elements were the 44- and 42-kDa polypeptides, shown to be members of the mitogen-activated protein kinase family. A subset of these proteins (p130, p115/112, p100, p55, p44, p42) were also tyrosine-phosphorylated when cells were stimulated by IL-3. MonoQ ion-exchange chromatography and two dimensional gel analyses were used to demonstrate that at least the p55, p44, and p42 substrates were identical, as well as some more minor species of molecular weights 50, 38, and 36 kDa, thus indicating common pathways of signaling in hemopoietic cells. Whereas in the case of SLF the dose-response characteristics of the proliferative response and the induction of tyrosine phosphorylation were similar, in the case of IL-3, much lower concentrations were required for maximal proliferation than maximal tyrosine phosphorylation. These studies form the basis for further molecular characterization of common components of signal transduction pathways in hemopoietic cells.     

金丝桃属和三腺金丝桃属植物叶的红外光谱分析及其与分类群鉴定的相关性

利用傅里叶红外光谱仪和OMINI采样器直接迅速准确地测定金丝桃属 (Hypericum L.) 9组43种1亚种1变种和三腺金丝桃属(Triadenum Raf.) 2种植物的红外光谱,结果表明:各分类群(种)的红外光谱具有高度特异性和重现性,这两属及其金丝桃属组间的红外光谱图存在较大的差异,而组内种间红外光谱图的差异较小,同种不同分布区和不同发育时期的叶的红外光谱几无差别;其红外光谱图的变化可以作为这两属植物的分类依据之一。这也暗示利用已知的标准红外光谱图库,可以区分和鉴定出这两属或其他属植物的种类。     

Sequencing and G-Quadruplex Folding of the Canine Proto-Oncogene KIT Promoter Region: Might Dog Be Used as a Model for Human Disease?

Downregulation of gene expression by induction of non-canonical DNA structures at promotorial level is a novel attractive anticancer strategy. In human, two guanine-rich sequences (h_kit1 and h_kit2) were identified in the promotorial region of oncogene KIT. Their stabilization into G-quadruplex structures can find applications in the treatment of leukemias, mastocytosis, gastrointestinal stromal tumor, and lung carcinomas which are often associated to c-kit mis-regulation. Also the most common skin cancer in domestic dog, mast cell tumor, is linked to a mutation and/or to an over-expression of c-kit, thus supporting dog as an excellent animal model. In order to assess if the G-quadruplex mediated mechanism of regulation of c-kit expression is conserved among the two species, herein we cloned and sequenced the canine KIT promoter region and we compared it with the human one in terms of sequence and conformational equilibria in physiologically relevant conditions. Our results evidenced a general conserved promotorial sequence between the two species. As experimentally confirmed, this grants that the conformational features of the canine kit1 sequence are substantially shared with the human one. Conversely, two isoforms of the kit2 sequences were identified in the analyzed dog population. In comparison with the human counterpart, both of them showed an altered distribution among several folded conformations.