富含脯氨酸小蛋白-2在小鼠子宫中的表达及调节

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,推测Sprr2与子宫对交配和分娩所产生的应激反应有关.     

G0101C03-3和L0254H10-3 基因在小鼠动情周期子宫中的差异表达

正常动情周期的维持是小鼠子宫多功能的必要条件 ,但对于其分子基础至今尚不十分明了。我们曾通过基因芯片技术分析了小鼠动情期与间情期子宫的基因表达谱 ,发现了许多差异表达的基因或表达序列标签(ESTs)。本实验选取了G0 1 0 1C0 3 3及L0 2 5 4H1 0 3两个差异表达的EST ,通过Northern印迹与原位杂交方法分析了它们在小鼠动情周期子宫中的时空表达模式。结果表明 :这两个基因的表达水平都发生周期性变化 ,在动情期表达量较少 ,而在间情期表达量较高 ;G0 1 0 1C0 3 3在动情期的表达量是间情期的 2 7% ,L0 2 5 4H1 0 3在动情期检测不到有表达 ,提示它们的表达受卵巢类固醇激素的调控 ;G0 1 0 1C0 3 3基因主要在子宫的腔上皮与腺上皮中表达 ,可能与子宫细胞的程序性死亡有关 ;而L0 2 5 4H1 0 3基因则主要位于基质细胞中     

ACOT7在小鼠妊娠早期子宫的表达和调控

目的 分析酰基辅酶A硫酯酶7(ACOT7)在小鼠早期妊娠过程中的表达和调控,探讨其与子宫接受态建立的相关性.方法 分别建立小鼠早期妊娠模型、假孕模型、卵巢摘除后类固醇激素处理模型和延迟着床与激活模型,结合实时定量PCR(qRT-PCR)、原位杂交、免疫荧光检测子宫ACOT7在这些模型中的表达模式.结果 ACOT7的mR...     

Caveolin-1在围植入期小鼠子宫内膜组织中的动态变化

目的检测caveolin-1在胚胎植入过程中小鼠子宫内膜组织中的表达,探讨其在胚胎植入过程中的作用。方法选择成年雌性昆明小白鼠42只,随机均分为7组(处于动情期的未孕组、妊娠3.5天组、妊娠4.5天组、妊娠5.5天组、妊娠6.5天组、妊娠7.5天组、妊娠9天组),采用免疫组织化学和RT-PCR方法检测子宫内膜组织中caveolin-1蛋白及mRNA水平在围植入期的变化。结果 (1)caveolin-1在胚胎植入前期(0d、3.5d)小鼠子宫内膜组织中的表达高于胚胎植入期(4.5d、5.5d、6.5d),差异有显著性(P<0.05)。(2)caveolin-1在胚胎植入后期(7.5d、9d)小鼠子宫内膜组织中的表达高于胚胎植入期(4.5d、5.5d、6.5d),差异有显著性(P<0.05)。(3)caveolin-1在胚胎植入后期小鼠子宫内膜组织中的表达略高于胚胎植入前期,但差异无显著性(P>0.05)。结论 Caveolin-1在胚胎植入前期和后期均高表达,植入期低表达。这种变化提示caveolin-1是影响胚胎植入的重要因素之一。     

蛋白激酶C亚型在人胃癌组织中的表达及其意义

目的研究PKC亚型α、β1、β2及ε在人胃癌组织的表达,并探讨PKC亚型表达与胃癌组织分化程度、临床分期以及与淋巴结转移的关系。方法采用免疫组化方法观察胃癌组织及其癌旁组织中PKC亚型α、β1、β2及ε的表达。结果PKCα在46例胃癌组织中,80.3%呈阳性表达,其中21.74%呈强阳性表达,与癌旁组织相比,二者差异显著(χ2=4.813,P<0.05);PKCβ1、β2则呈阴性;20例癌旁组织中,PKCε70%呈阳性表达,均为( ),46例胃癌组织中84.8%呈阳性表达,其中26.1%呈强阳性,两者阳性率无显著差异(χ2=1.93,P>0.05),但染色强度差异显著(χ2=5.07,P<0.05)。结论1.胃癌组织和癌旁组织均表达PKCα和,εPKCβ1、β2则呈阴性。2.胃癌组织PKC亚型α、ε的表达较癌旁组织显著增强。3.PKCα、ε的表达与胃癌组织的分化程度、临床分期及有无淋巴结转移均无明显相关性。     

一氧化氮对小鼠胚胎围植入期子宫VEGF及其受体表达的调节

为研究NO在胚胎植入中的作用机理 ,本文采用子宫角注射、原位杂交及Westernblot方法研究了一氧化氮 (NO)在小鼠胚胎植入过程中对血管内皮生长因子 (VEGF)及其受体表达的调节。受试小鼠于妊娠第三天 (D3 )在一侧子宫角内注射一氧化氮合酶 (NOS)抑制剂N 硝基 L 精氨酸甲酯 (L NAME)或者L NAME与NO的供体硝普钠 (SNP)合用 ,另一侧子宫角为对照侧 ;收集并分别检测了D5,D6和D7天小鼠子宫中VEGF及其受体mRNA和蛋白的表达情况。结果显示 :与对照侧相比 ,L NAME处理后小鼠胚胎围植入期子宫中VEGF及其受体mRNA的表达有不同程度的下降 ;对VEGF及其受体蛋白表达水平检测表明 ,抑制的NO产生也使VEGF及其受体蛋白在小鼠围植入期子宫中的表达有不同程度的降低。当NOS抑制剂和NO的供体SNP同时注射小鼠时 ,VEGF及其受体mRNA和蛋白表达都恢复到正常水平。以上结果表明 ,在小鼠胚胎植入中NO可通过调节VEGF及其受体的表达参与血管新生 ,从而对胚胎植入起到调节作用     

XBP1在小鼠早期妊娠子宫的表达和调节

转录因子X盒结合蛋白1 (X-box binding protein-1,XBP1)在未折叠蛋白反应中起关键作用.本研究旨在分析XBP1在小鼠早期妊娠过程中的表达和调节,探讨其与胚胎着床的相关性.分别建立小鼠早期妊娠、人工诱导蜕膜化、发情周期和激素调节模型,通过免疫组织化学染色和实时定量RT-PCR技术检测XBP1在这...     

SHBG基因敲除小鼠模型的建立及其表型分析

目的:建立性激素结合球蛋白(SHBG)基因条件敲除小鼠模型,为探讨胎盘组织中SHBG在体内的生理功能及其与妊娠期糖尿病发病关系提供实验手段。方法:首先运用生物信息学手段确定小鼠SHBG基因组序列,构建SHBG打靶载体,以电穿孔方法将其导入小鼠ES细胞,筛选培养阳性ES细胞并行PCR鉴定,并将正确同源重组的ES细胞注射进小鼠囊胚,移入受体小鼠子宫;将获得的嵌合体小鼠与C57BL/6J小鼠交配,筛选后获得Flox小鼠,该小鼠与EIIa-Cre转基因小鼠杂交,子代多次自交获得SHBG全身基因敲除(SHBG~(-/-))的小鼠。结果:运用同源重组及ES细胞技术建立了SHBG基因的Flox小鼠,并利用Cre/Loxp重组酶系统建立了SHBG基因全身敲除小鼠模型,PCR方法从基因水平证明了SHBG基因Flox小鼠及SHBG基因全身敲除小鼠模型建立成功。对基因敲除鼠进行初步表型分析发现:SHBG基因全身敲除小鼠的生长发育与野生型小鼠相比无明显肉眼所见异常,SHBG基因全身敲除雌雄小鼠均具有生殖能力。结论:成功建立SHBG基因全身敲除小鼠模型,通过对基因敲除鼠进行初步表型分析,发现SHBG基因全身敲除小鼠外观上发育正常,为进一步研究SHBG在妊娠期糖尿病中的作用奠定了基础。     

Insights into protein kinase regulation and inhibition by large scale structural comparison

Protein structure determination of soluble globular protein domains has developed into an efficient routine technology which can now be applied to generate and analyze structures of entire human protein families. In the kinase area, several kinase families still lack comprehensive structural analysis. Nevertheless, Structural Genomics (SG) efforts contributed more than 40 kinase catalytic domain structures during the past 4 years providing a rich resource of information for large scale comparisons of kinase active sites. Moreover, many of the released structures are inhibitor complexes that offer chemical starting points for development of selective and potent inhibitors. Here we discuss the currently available structural data and strategies that can be utilized for the development of highly selective inhibitors.     

Differential regulation of p53 function by protein kinase C isoforms revealed by a yeast cell system

The complexity of the mammalian p53 pathway and protein kinase C (PKC) family has hampered the discrimination of the effect of PKC isoforms on p53 activity. Using yeasts co-expressing the human wild-type p53 and a mammalian PKC-α, -δ, -ε or -ζ, we showed a differential regulation of p53 activity and phosphorylation state by PKC isoforms. Whereas PKC-α reduced the p53-induced yeast growth inhibition and cell cycle arrest, PKC-δ and -ε enhanced the p53 activity through p53 phosphorylation, and PKC-ζ had no effect on p53. This work identified positive and negative p53 regulators which represent promising pharmacological targets in anti-cancer therapy.     

Molecular cloning and characterization of three cDNAs encoding putative mitogen-activated protein kinase kinases (MAPKKs) in Arabidopsis thaliana.

We isolated three Arabidopsis thaliana cDNA clones (ATMKK3, ATMKK4 and ATMKK5) encoding protein kinases with extensive homology to the mitogen-activated protein kinase kinases (MAPKKs) of various organisms in the catalytic domain. ATMKK3 shows high homology (85% identity) to NPK2, a tobacco MAPKK homologue. ATMKK4 and 5 are closely related to each other (84% identity). Phylogenetic analysis showed that the plant MAPKKs constitute at least three subgroups. The recombinant ATMKK3 and ATMKK4 were expressed as a fusion protein with glutathione S-transferase (GST) in Escherichia coli. Affinity purified GST-ATMKK3 and GST-ATMKK4 proteins contained phosphorylation activity, which shows that both the ATMKK3 and ATMKK4 genes encode functional protein kinases. Northern blot analysis revealed that the ATMKK3 gene expressed in all the organs. The levels of ATMKK4 and 5 mRNAs were relatively higher in steins and leaves than in flowers and roots. We determined the map positions of the ATMKK3, 4 and 5 genes on Arabidopsis chromosomes by RFLP mapping using P1 genomic clones.     

Involvement of obestatin,cyclin-dependent kinase and protein kinase C in control of feline ovarian cell viability and hormones release

The aim of our in vitro study was to understand the role of obestatin, cyclin-dependent kinase (CDK) and protein kinase C (PKC) in the control of basic feline ovarian cell functions (viability, ovarian hormones release), as well as the role of protein kinases in mediating the effect of obestatin on these processes. For this purpose, we analyzed the effect of obestatin (0, 10 and 100 ng/mL) alone or in combination with CDK blocker olomoucine (100 ng/mL) or PKC blocker calphostin-c (100 ng/mL) on cultured feline ovarian fragments or granulosa cells. The release of progesterone (P4), testosterone (T) and estradiol (E2) by isolated ovarian follicular fragments were evaluated by ELISA. Granulosa cell viability was analysed using the Trypan blue exclusion test. It was observed that the addition of obestatin alone significantly increased the granulosa cell viability (at dose 100 ng/mL), promoted the release of P4 (at all doses added) and IGF-I (at dose 100 ng/mL) but decreased T (at all doses added). E2 output was below the detection limit in all groups. The addition of either olomoucine or calphostin-c reduced cell viability, P4, T and IGF-I release. Both olomoucine and caplhostin-c inverted the stimulatory effect of obestatin on granulosa cell viability and were able to prevent stimulatory action of obestatin on ovarian cell viability and on hormone and growth factor release and change it to an inhibitory action. These observations show that obestatin can directly regulate (mostly promote) basal feline ovarian cell functions (hormone release and viability). The inhibitory action of CDK and PKC blockers on these functions suggests, that both CDK and PKC can be promoters of ovarian cell viability and steroidogenesis in cats. Furthermore, the ability of both CDK and PKC to prevent olomoucine action demonstrates that obestatin action on the feline ovary could be mediated by these kinases.     

Conformational plasticity of the catalytic subunit of protein kinase CK2 and its consequences for regulation and drug design

At the first glance CK2α, the catalytic subunit of protein kinase CK2, is a rigid molecule: in contrast to many eukaryotic protein kinases in CK2α the canonical regulatory key elements like the activation segment occur exclusively in their typical active conformations. This observation fits well to the constitutive activity of the enzyme, meaning, its independence from phosphorylation or other characteristic control factors. Most CK2α structures are based on the enzyme from Zea mays, supplemented by an increasing number of human CK2α structures. In the latter a surprising plasticity of important ATP-binding elements – the interdomain hinge region and the glycine-rich loop – was discovered. In fully active CK2α the hinge region is open and does not anchor the ATP ribose, but alternatively it can adopt a closed conformation, form hydrogen bonds to the ribose moiety and thus retract the γ-phospho group from its functional position. In addition to this partially inactive state human CK2α was recently found in a fully inactive conformation. It is incompatible with ATP-binding due to a combination of a closed hinge and a collapse of the glycine-rich loop into the ATP cavity. These conformational transitions are apparently correlated with the occupation state of a remote docking site located at the interface to the non-catalytic subunit CK2β: if CK2β blocks this site, the fully active conformation of CK2α is stabilized, while the binding of certain small molecule seems to favour the partially and fully inactive states. This observation may be exploited to design effective and selective CK2 inhibitors.     

Mutual regulation of conventional protein kinase C and a ubiquitin ligase complex

Several isoforms of protein kinase C (PKC) are degraded by the ubiquitin-proteasome pathway after phorbol ester-mediated activation. However, little is known about the ubiquitin ligase (E3) that targets activated PKCs. We recently showed that an E3 complex composed of HOIL-1L and HOIP (LUBAC) generates linear polyubiquitin chains and induces the proteasomal degradation of a model substrate. HOIL-1L has also been characterized as a PKC-binding protein. Here we show that LUBAC preferentially binds activated conventional PKCs and their constitutively active mutants. LUBAC efficiently ubiquitinated activated PKC in vitro, and degradation of activated PKCalpha was delayed in HOIL-1L-deficient cells. Conversely, PKC activation induced cleavage of HOIL-1L and led to downregulation of the ligase activity of LUBAC. These results indicate that LUBAC is an E3 for activated conventional PKC, and that PKC and LUBAC regulate each other for proper PKC signaling.     

Combined analysis of microRNome and 3'-UTRome reveals a species-specific regulation of progesterone receptor expression in the endometrium of rhesus monkey

The establishment of endometrial receptivity is a prerequisite for successful pregnancy, which is controlled by a complex mechanism. MicroRNAs (miRNAs) are small non-coding RNAs that have emerged as important regulators of gene expression. However, the contribution of miRNAs in endometrial receptivity is still unknown. Here we used rhesus monkey as an animal model and compared the endometrial miRNA expression profiles during early-secretory (pre-receptive) phase and mid-secretory (receptive) phase by deep sequencing. A set of differentially expressed miRNAs were identified, 8 of which were selected and validated using quantitative RT-PCR. To facilitate the prediction of their target genes, the 3'-UTRome was also determined using tag sequencing of mRNA 3'-termini. Surprisingly, about 50% of the 10,677 genes expressed in the rhesus monkey endometrium exhibited alternative 3'-UTRs. Of special interest, the progesterone receptor (PGR) gene, which is necessary for endometrial receptivity, processes an ultra long 3'-UTR (~10 kb) along with a short variant (~2.5 kb). Evolutionary analysis showed that the 3'-UTR sequences of PGR are poorly conserved between primates and rodents, suggesting a species-biased miRNA binding pattern. We further demonstrated that PGR is a valid target of miR-96 in rhesus monkey and human but not in rodents, whereas the regulation of PGR by miR-375 is rhesus monkey-specific. Additionally, we found that miR-219-5p regulates PGR expression through a primate-specific long non-coding RNA immediately downstream of the PGR locus. Our study provides new insights into the molecular mechanisms underlying endometrial receptivity and presents intriguing species-specific regulatory roles of miRNAs.     

Inhibition of cGMP-dependent protein kinase II by its own splice isoform

cGMP- and cAMP-dependent protein kinases (cGK I, cGK II, and cAK) are important mediators of many signaling pathways that increase cyclic nucleotide concentrations and ultimately phosphorylation of substrates vital to cellular functions. Here we demonstrate a novel mRNA splice isoform of cGK II arising from alternative 5' splicing within exon 11. The novel splice variant encodes a protein (cGK II Delta(441-469)) lacking 29 amino acids of the cGK II Mg-ATP-binding/catalytic domain, including the conserved glycine-rich loop consensus motif Gly-x-Gly-x-x-Gly-x-Val which interacts with ATP in the protein kinase family of enzymes. cGK II Delta(441-469) has no intrinsic enzymatic activity itself, however, it antagonizes cGK II and cGK I, but not cAK. Thus, the activation and cellular functions of cGK II may be determined not only by intracellular cGMP levels but also by alternative splicing which may regulate the balance of expression of cGK II versus its own inhibitor, cGK II Delta(441-469).