Somatic Expression and Autosomal Inheritance of Phosphoglycerate Kinase B in Kangaroos

The PGK-B isozyme, currently known as PGK-2 in the mouse nomenclature, is the predominant PGK isozyme in mammalian sperm. In many species it is detectable only in sperm, in spermatogenic testes and in epididymides containing sperm. In this paper, we provide evidence that some kangaroo species express low PGK-B activity in somatic tissues, in addition to high activity in testes. Three kangaroo species, M. rufogriseus, M. robustus and M. giganteus, exhibit polymorphism of PGK-B. Breeding data support the hypothesis of autosomal co-dominant inheritance, as is the case in mice. Population data for the three polymorphisms are discussed. PGK-B is not detectable in somatic tissues or spermatogenic testis extracts of monotreme mammals, birds or lizards; it is probably restricted to therian mammals.     

精子介导GFP基因在小鼠早期胚胎中的表达

利用 DIG 末端标记技术和免疫组化技术分析了小鼠精子体外结合内化外源DNA的效率。试验结果表明,不同小鼠个体的精子结合外源DNA的阳性率有明显差异（P<0.01）,平均为13%。利用考马斯亮蓝染色评价了小鼠精子顶体反应发生的情况,筛选出TYH培养液为较合适的体外受精液。利用小鼠体外受精技术,将体外转染GFP基因并获能的小鼠精子与成熟卵母细胞进行体外受精,受精卵进行体外培养,表达GFP胎的阳性率为4.7%。验证了精子介导制备转基因小鼠胚胎的可行性,并建立了利用精子载体法制备转基因小鼠胚胎的平台。     

精子介导GFP基因在小鼠早期胚胎中的表达

利用DIG末端标记技术和免疫组化技术分析了小鼠精子体外结合内化外源DNA的效率。试验结果表明,不同小鼠个体的精子结合外源DNA的阳性率有明显差异(P<0.01),平均为13%。利用考马斯亮蓝染色评价了小鼠精子顶体反应发生的情况,筛选出TYH培养液为较合适的体外受精液。利用小鼠体外受精技术,将体外转染GFP基因并获能的小鼠精子与成熟卵母细胞进行体外受精,受精卵进行体外培养,表达GFP胚胎的阳性率为4.7%。验证了精子介导制备转基因小鼠胚胎的可行性,并建立了利用精子载体法制备转基因小鼠胚胎的平台。     

高浓度葡萄糖对昆明小鼠早期胚胎发育的影响

建立昆明小鼠受孕模型,分离并体外培养胚胎细胞.检测了各培养浓度下的细胞增殖、分化与凋亡.胚胎细胞在0.2mmol/L和5.56mmol/L葡萄糖浓度的KSOM培养基中能正常发育和孵化;而在浓度为15.56mmol/L和25.56mmol/L葡萄糖培养基中胚胎发育和孵化均受到损害(P<0.005),且总细胞数和内细胞团细胞数也明显减少(P<0.01),但其细胞凋亡率与0.2mmol/L和5.56mmol/L葡萄糖浓度下胚胎细胞凋亡率无显著性差异(P>0.05).随着葡萄糖浓度的增高,胚泡总的表面积无明显变化,但胚胎细胞密度呈增加趋势.高血糖对早期胚胎的发育具有毒性作用,提示高糖可能导致妊娠合并糖尿病患者的流产和胎儿畸形率升高.     

Diverse Routes toward Early Somites in the Mouse Embryo

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Rb基因在小鼠胚胎发育中期肾组织的表达

用原位杂交法检测小鼠肾组织中细胞周期调控基因Rb的表达;用免疫组织化学及缺口末端标记法观察小鼠肾组织发育过程中的细胞增殖与凋亡：结果表明,Rb基因主要在肾上皮细胞表达,随胎龄增长而表达颜色加深。肾组织在第13天有一个较大的增殖增长,到第14天生长趋于稳定。以上结果说明Rb基因在小鼠胚胎肾发育中期有表达,该基因在胚胎肾发育中期起一定的作用;同时胚胎发育中期肾细胞增殖与凋亡相伴存在。     

Differences in Gene Expression between Mouse and Human for Dynamically Regulated Genes in Early Embryo

Infertility is a worldwide concern that can be treated with in vitro fertilization (IVF). Improvements in IVF and infertility treatment depend largely on better understanding of the molecular mechanisms for human preimplantation development. Several large-scale studies have been conducted to identify gene expression patterns for the first five days of human development, and many functional studies utilize mouse as a model system. We have identified genes of possible importance for this time period by analyzing human microarray data and available data from online databases. We selected 70 candidate genes for human preimplantation development and investigated their expression in the early mouse development from oocyte to the 8-cell stage. Maternally loaded genes expectedly decreased in expression during development both in human and mouse. We discovered that 25 significantly upregulated genes after fertilization in human included 13 genes whose orthologs in mouse behaved differently and mimicked the expression profile of maternally expressed genes. Our findings highlight many significant differences in gene expression patterns during mouse and human preimplantation development. We also describe four cancer-testis antigen families that are also highly expressed in human embryos: PRAME, SSX, GAGE and MAGEA.     

IK细胞因子在小鼠早孕期子宫内膜的表达规律及其在胚胎着床中的作用

通过Real-time PCR、Western blot及免疫组织化学方法分析了IK细胞因子(IK cytokine)在早孕小鼠(妊娠D1~D7)子宫内膜中的表达规律及宫角注射IK细胞因子反义寡聚脱氧核苷酸后对胚胎着床的影响。结果显示,IK细胞因子mRNA表达在D1~D4逐渐升高,于D4达到高峰(P<0.05);Western blot和免疫组织化学结果与Real-time PCR结果基本一致,其蛋白表达在D1~D5逐渐升高,于D5达到高峰(P<0.05);IK细胞因子在D5胚胎着床点的表达显著高于着床旁组织;假孕小鼠子宫内膜IK细胞因子蛋白表达明显低于正常妊娠,且整个假孕过程中没有表达高峰;宫角注射IK细胞因子反义寡聚脱氧核苷酸后24 h和48 h(即D4和D5)子宫内膜IK细胞因子表达明显受到抑制,MHCⅡ抗原表达增强,且胚胎着床数量明显减少(P<0.05),提示IK细胞因子在胚胎着床中发挥着重要作用。     

Gm2052基因在小鼠胚胎发育中表达的初步研究

目的:研究预测的编码蛋白基因Gm2052在小鼠胚胎发育阶段的表达模式,为进一步了解该基因的功能奠定基础。方法:通过全胚胎原位杂交技术、组织切片原位杂交技术及半定量RT-PCR方法,对预测的Gm2052基因在小鼠胚胎发育中后期及在新生小鼠中的表达情况进行初步分析。结果:全胚胎原位杂交显示,在E10.5小鼠胚胎中,Gm2052仅在脑中表达;当小鼠胚胎发育至E13.5时,Gm2052在脑、舌、肺、肝脏、胰腺等组织中均有表达。半定量RT-PCR结果显示,在小鼠胚胎中后期(E15.5和E18.5)及新生小鼠(出生后第9 d)中,Gm2052呈动态表达模式。结论:预测基因Gm2052与小鼠脑的发育密切相关,并可能参与小鼠肺、肝脏及胰腺等主要脏器胚期的发育。     

Heterogenous Macromolecular Contributions to Early Mouse Embryo Development

Mouse embryos have been cultured for more than one half of its gestation period by providing sera from definite species of animals as the inducer at the proper stage of development. The processes of normal development at four different but discrete phases have been described.
(1) Fertilized ovum (stage 1) is able to grow up to denuded blastocyst stage and attaches to the culture dish (stage 7) in balanced salt solution with bovine serum albumin as the sole macromolecule. (2) Embryoblast or inner cell mass (ICM) of denuded blastocyst (stage 7) is able to develop in fetal calf serum (FCS), human placenta cord serum (FCS), or sera from mouse (MS), rat (RS) or rabbit (RbS) to the early cylinder stage (stage 11). (3) The early egg cylinder stage (stage 11) of mouse embryo is able to grow in HCS and RS, but not in FCS nor RbS, to the stage of early somite stage (stage 15). (4) Beyond early somite stage (stage 15), mouse embryo is able to develop neural tissues in rat serum. The macromolecular nature of these growth factors in serum has been described (Hsu, 1980).
It indicates that the differential biological activity which induces the early mouse embryogenesis among the sera from different animal species is due to the various degree of sequence homology between the growth factor family among the different species of animals.     

Low Resolution Structure of Yeast Phosphoglycerate Kinase

Yeast phosphoglycerate kinase, like the same enzyme from horse muscle, has two structural lobes, reminiscent of a dimer rather than a monomer.     

HCMV基因在人和小鼠胚胎成纤维细胞中表达的差异性研究

人巨细胞病毒（human cytomegalovirus, HCMV）种属特异性机制尚不清楚。研究通过检测HCMVADl69体外感染人胚胎成纤维细胞（Human embryo fibroblast, HEF）和小鼠胚胎成纤维细胞（mouse embryo fibroblast, MEF）后病毒基因的表达情况,探讨HCMV种属特异性的可能分子机制。首先用HCMV AD169（MOI=5）分别感染HEF和MEF,相差显微镜逐日观察细胞的形态学变化;RT—PCR检测HCMV即刻早期（IE1、IE2）、早期（uL84）和晚期基因（UL83）的表达情况;Western—blot和免疫荧光检测病毒基因编码蛋白表达的情况。形态学观察发现HEF感染HCMV后逐渐变大变圆并相互融合,第4天可见典型的HCMV特征性病变效应,而MEF则未出现上述的变化;RT-PCR和Western—blot表明HEF组表达即刻早期基因IE1和IE2、早期基因uL84和晚期基因UL83 mRNA以及各基因所编码的蛋白,且相对表达量显著高于模拟感染组（P〈0．01）;而MEF组仅IEl和IE2mRNA和蛋白相对表达量显著低于HEF组（P〈0．05）,而高于模拟感染组（P〈0．01）。免疫荧光检测发现HEF感染72h表达IE和UL83蛋白,而MEF则无明显表达。以上结果表明,HC—MV不能在MEF中复制并产生完整子代病毒颗粒,且病毒基因表达阻止在IE2基因表达之后和UL84基因表达之前,其种属特异性可能与即刻早期蛋白低水平的表达量有关。     

小鼠受精卵早期发育过程中PKB/Akt对p21蛋白表达及定位的影响

初步探讨在小鼠受精卵早期发育过程中PKB/Akt对p21 蛋白表达及定位的影响。通过显微操作技术注射野生型、持续激活型及激酶失活型的PKB的mRNA,用免疫荧光方法检测p21蛋白的细胞定位、Western blot 方法检测p21蛋白的表达。结果显示在注射不同形式的PKB mRNA 后p21蛋白的表达无明显差别,但是细胞定位发生改变,PKB被激活后,p21蛋白滞留在胞浆中。因而初步认为在小鼠受精卵中,PKB/Akt通过影响p21 的细胞定位而影响细胞周期的进程。     

小鼠受精卵早期发育过程中PKB/Akt对p21蛋白表达及定位的影响

初步探讨在小鼠受精卵早期发育过程中PKB/Akt对p21蛋白表达及定位的影响。通过显微操作技术注射野生型、持续激活型及激酶失活型的PKB的mRNA,用免疫荧光方法检测p21蛋白的细胞定位、Western blot方法检测p21蛋白的表达。结果显示在注射不同形式的PKB mRNA后p21蛋白的表达无明显差别,但是细胞定位发生改变,PKB被激活后,p21蛋白滞留在胞浆中。因而初步认为在小鼠受精卵中,PKB/Akt通过影响p21的细胞定位而影响细胞周期的进程。     

小鼠的胚胎移植方法

全面而详细地描述了小鼠胚胎移植中所采用的输卵管移植及子宫移植方法,包括作者的改进。介绍了寄母小鼠的选择、配种及孕鼠的编号方法等,具有较强的实用意义。     

Structural Tightening and Interdomain Communication in the Catalytic Cycle of Phosphoglycerate Kinase

Changes in amide-NH chemical shift and hydrogen exchange rates as phosphoglycerate kinase progresses through its catalytic cycle have been measured to assess whether they correlate with changes in hydrogen bonding within the protein. Four representative states were compared: the free enzyme, a product complex containing 3-phosphoglyceric acid (3PG), a substrate complex containing ADP and a transition-state analogue (TSA) complex containing a 3PG-AlF₄⁻-ADP moiety. There are an overall increases in amide protection from hydrogen exchange when the protein binds the substrate and product ligands and an additional increase when the TSA complex is formed. This is consistent with stabilisation of the protein structure by ligand binding. However, there is no correlation between the chemical shift changes and the protection factor changes, indicating that the protection factor changes are not associated with an overall shortening of hydrogen bonds in the protected ground state, but rather can be ascribed to the properties of the high-energy, exchange-competent state. Therefore, an overall structural tightening mechanism is not supported by the data. Instead, we observed that some cooperativity is exhibited in the N-domain, such that within this domain the changes induced upon forming the TSA complex are an intensification of those induced by binding 3PG. Furthermore, chemical shift changes induced by 3PG binding extend through the interdomain region to the C-domain β-sheet, highlighting a network of hydrogen bonds between the domains that suggests interdomain communication. Interdomain communication is also indicated by amide protection in one domain being significantly altered by binding of substrate to the other, even where no associated change in the structure of the substrate-free domain is indicated by chemical shifts. Hence, the communication between domains is also manifested in the accessibility of higher-energy, exchange-competent states. Overall, the data that are consistent with structural tightening relate to defined regions and are close to the 3PG binding site and in the hinge regions of 3-phosphoglycerate kinase.     

Structure of Horse-muscle Phosphoglycerate Kinase at 6 Å Resolution

The single peptide chain of 3-phosphoglycerate kinase is folded into two distinct globular units, only one of which seems to be involved in substrate binding.     

Aurora 激酶A调节小鼠受精卵早期发育

Aurora激酶是参与细胞周期调节的重要激酶,已成为肿瘤研究领域的热点．近年来有研究表明, Aurora激酶A(Aurora kinase A,AURKA)对卵母细胞减数分裂也起到重要的调节作用,但对其在哺乳动物早期胚胎发育中的研究鲜有报道．本研究利用显微注射向受精卵中导入干扰AURKA表达的质粒,观察了AURKA表达敲低对小鼠受精卵早期发育的影响,并检测丝裂原活化蛋白激酶（mitogen activated protein kinase,MAPK）通路抑制后,小鼠受精卵卵裂及AURKA表达与活性变化．实验结果表明,干扰AURKA的表达可导致受精卵发育停滞和异常分裂．MAPK通路的抑制亦可破坏受精卵正常卵裂,并下调AURKA的蛋白表达及活性．实验结果提示,AURKA是小鼠受精卵早期发育所必需的,并与MAPK通路的激活相关．