Decreased expression and DNA methylation levels of GATAD1 in preeclamptic placentas

Expression of syncytin-1, or the human endogenous retroviral family W member 1 (HERVWE1) in human placental trophoblasts is regulated by DNA methylation. Increased DNA methylation and decreased expression of syncytin-1 have been observed in preeclamptic placentas. The syncytin-1-mediated fusogenic as well as non-fusogenic activities, e.g., cell cycle promotion, anti-apoptosis, and immune suppression, are implicated in the pathogenic changes in preeclamptic placentas. It is noteworthy that in a close vicinity to syncytin-1 there are two genes, peroxisome biogenesis factor 1 (PEX1) and GATA zinc finger domain containing 1 (GATAD1), as well as multiple CpG islands around these genes. In this study we determined if these adjacent genes might, like syncytin-1, subject to epigenetic regulation in preeclamptic placentas. Data from quantitative real-time PCR and Western blotting indicated that while PEX1 expression remained stable, GATAD1 expression was significantly decreased in the third-trimester placentas associated with preeclampsia than those associated with normal pregnancy. Immunohistochemistry detected high GATAD1 expression in trophoblast linage, and confirmed its reduced levels in preeclamptic placentas. However, COBRA and bisulfate sequencing detected decreased DNA methylation in levels in the 3 [prime] region of GATAD1 gene in preeclamptic placentas. The positive correlation between 3 [prime] methylation and GATAD1 expression was confirmed by treatment of choriocarcinoma JAR cells with DNMT inhibitor. These data pointed to a potential role of GATAD1 for the syncytium deficiency often associated with preeclamptic placentas. The sharp contrast of the methylation alterations for the closely positioned GATAD1 and HERVWE1 may provide a useful model for studying the accurate control of DNA methylation as well as their positive and negative impact on gene expression in placental trophoblasts.     

Identification of differential gene expression profiles in placentas from preeclamptic pregnancies versus normal pregnancies by DNA microarrays

The purpose of this study was to perform a comprehensive analysis of gene expression profiles in placentas from preeclamptic pregnancies versus normal placentas. Placental tissues were obtained immediately after delivery from women with normal pregnancies (n=6) and patients with preeclampsia (n=6). The gene expression profile was assessed by oligonucleotide-based DNA microarrays and validated by quantitative real-time RT-PCR. Functional relationships and canonical pathways/networks of differentially-expressed genes were evaluated by GeneSpring? GX 11.0 software, and ingenuity pathways analysis (IPA). A total of 939 genes were identified that differed significantly in expression: 483 genes were upregulated and 456 genes were downregulated in preeclamptic placentas compared with normal placentas (fold change ≥ 2 and p<0.05 by unpaired t-test corrected with Bonferroni multiple testing). The IPA revealed that the primary molecular functions of these genes are involved in cellular function and maintenance, cellular development, cell signaling, and lipid metabolism. Pathway analysis provided evidence that a number of biological pathways, including Notch, Wnt, NF-κB, and transforming growth factor-β (TGF-β) signaling pathways, were aberrantly regulated in preeclampsia. In conclusion, our microarray analysis represents a comprehensive list of placental gene expression profiles and various dysregulated signaling pathways that are altered in preeclampsia. These observations may provide the basis for developing novel predictive, diagnostic, and prognostic biomarkers of preeclampsia to improve reproductive outcomes and reduce the risk for subsequent cardiovascular disease.     

Analysis of nitroso-proteomes in normotensive and severe preeclamptic human placentas

Nitric oxide (NO) plays a key role in placental biology, and placental dysfunction is the main pathogenesis pathway for preeclampsia, yet the direct placental targets of NO actions have not been determined. Covalent adduction of an NO moiety to cysteines, termed S-nitrosylation (SNO), is emerging as a key route by which NO can directly modulate protein functions. This study was conducted to analyze global S-nitroso (SNO)-proteins in human placentas and to determine if their levels differ in normotensive versus severe preeclamptic placentas. Although total nitrite/nitrate increased, total levels of SNO-proteins and nitrosylated forms of endothelial NO synthase and heat shock protein 90 were decreased by preeclampsia. We further compared normotensive and preeclamptic placental nitroso-proteomes (total SNO-protein profiles) by using a biotin and CyDye switch test combined with two-dimensional fluorescence difference gel electrophoresis (2D-DIGE) and identified SNO-proteins by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Numerous SNO-proteins were displayed as spots on 2D-DIGE gels. One hundred spots of interest were excised; 46 spots were identified, of which 8 spots were novel SNO-proteins; levels of 15 spots were increased, and 6 spots were decreased, and the rest were unchanged by preeclampsia. Pathway analysis suggested that placental SNO-proteins are involved in regulating various cellular functions including protein synthesis, cell movement and metabolism, cell signaling, and other functions. These data therefore show for the first time that SNO is a crucial mechanism by which NO directly regulates placental proteins linked to various biological pathways. The significantly altered placental nitroso-proteome in preeclampsia suggests that SNO plays a role in the placental pathophysiology in preeclampsia.     

胰岛素样生长因子结合蛋白-7基因的克隆和表达

IGFBP-7是胰岛素样生长因子结合蛋白(IGFBP)家族中的一员,具有一些抑癌基因的特征。利用重组PCR技术获得了IGFBP-7基因的全长编码区序列,并将其克隆到pcDNA3．1／His—Myc真核表达载体中,得到重组质粒pcDNA3．1／His—Myc—IGFBP-7。将该重组质粒瞬时转染人胚胎肾293T细胞,Westem-blot分析表明,IGFBP-7在293T细胞中获得了表达,为进一步研究IGFBP-7的功能奠定了基础。     

Effects of aging and caloric restriction on IGF-I, IGF-I receptor, IGFBP-3 and IGFBP-4 gene expression in the rat stomach and colon

The purpose of this study was to determine the effects of aging and caloric restriction (CR) on insulin-like growth factor-I (IGF-I), IGF-I receptor (IGF-IR), IGF-binding protein-3 (IGFBP-3) and IGFBP-4 expression in the stomach and colon of male Fischer 344 rats. Stomach and colonic RNA were prepared from ad libitum (AL) fed or long-term CR rats. Stomach IGF-I, IGFBP-3 and IGFBP-4 mRNA levels increased significantly (P相似文献   

The expression of cell cycle related proteins PCNA,Ki67, p27 and p57 in normal and preeclamptic human placentas

Placenta is a transitional area making many physiological activities between mother and fetus and therefore, it is a critical organ influencing the outcome of pregnancy. Fetal growth is directly related to placental development. Accurate placental development depends on coordinated action of trophoblasts’ proliferation, differentiation and invasion. Information on cell cycle related proteins that control these events is limited and how they are affected in preeclampsia is not fully understood yet. Therefore, in this study, in order to understand the role of cell cycle regulators in preeclamptic placentas we aimed to determine the spatio-temporal immunolocalizations of cell cycle regulators in preeclamptic and normal human term placentas. Term placentas were obtained from women diagnosed with preeclampsia and from normal pregnancies with informed consent following cesarean deliveries. Placental samples were stained via immunohistochemistry with PCNA, Ki67, p27, p57, vimentin and cytokeratin 7 antibodies and were examined by light microscopy. PCNA and Ki67 staining intensities significantly increased in villous parts, significantly decreased in basal plates of PE group and did not change in chorionic plates. Staining intensities of cell cycle inhibitors p27 and p57 significantly increased in all parts of preeclamptic placentas compared to control. Placental abnormalities of preeclamptic placentas might be associated with proliferation and cell cycle arrest mechanisms’ alterations occurred in preeclampsia.     

Differential regulation of IGFBP-2 and IGFBP-5 gene expression by vitamin A status in Japanese quail

To investigate the involvement of the insulin-like growth factor (IGF) system in vitamin A (VA)-supported growth, we examined the effects of VA status on IGF binding protein (IGFBP)-2 and -5 gene expression in Japanese quail. VA deficiency caused a reduction in IGFBP-2 mRNA only in lung, without effect in other tissues. However, the expression of IGFBP-5 mRNA was more sensitive to the change of VA status. IGFBP-5 mRNA levels were significantly reduced by VA depletion in a tissue-specific manner, which preceded the decrease in body weight. A single injection of retinoic acid or retinol to VA-deficient quail did not affect the levels of IGFBP-2 mRNA, but it rapidly induced the expression of IGFBP-5 mRNAs in some tissues. These results are the first to show that gene expression of some IGFBPs in vivo are under the control of VA status and suggest a possible involvement of the IGF system in mediating the physiological actions of VA in the growth of Japanese quail.     

pGhrelin对离体猪脂肪细胞Caspase-3活性及基因表达的影响

研究新近发现的猪Ghrelin (porcine ghrelin,pGhrelin)对猪前体脂肪细胞Caspase-3活性及其基因表达的影响．采用细胞培养技术,以仔猪背部皮下前体脂肪细胞为靶细胞,经0、1、10和100 nmol/L pGhrelin处理细胞48 h后,于倒置生物显微镜下进行脂肪细胞形态学观察．利用MTT法测定pGhrelin对细胞增殖的影响．采用分光光度法检测Caspase-3活性．以实时荧光定量RT-PCR方法测定Caspase-3的基因表达．结果显示,10 nmol/L pGhrelin可以显著降低脂肪细胞Caspase-3的活性与mRNA的表达水平(P < 0.05),100 nmol/L pGhrelin对猪脂肪前体细胞增殖有极显著促进作用(P < 0.01)．上述结果表明,pGhrelin可以下调Caspase-3的活性与基因表达,促进脂肪细胞增殖,抑制脂肪细胞凋亡,其机制可能与Caspase-3依赖性凋亡调节信号通路有关．     

Impairment of mitochondrial respiration in platelets and placentas: a pilot study in preeclamptic pregnancies

Molecular and Cellular Biochemistry - Preeclampsia (PE) is a major complication of pregnancy with partially elucidated pathophysiology. Placental mitochondrial dysfunction has been increasingly...     

Death effector domain-only polypeptides of caspase-8 and -10 specifically inhibit death receptor-induced cell death

Caspase-8 and -10 are thought to be involved in a signaling pathway leading to death receptor-mediated apoptosis. The prodomains of these caspases are known to form fibrous structures in the perinuclear region when overexpressed, though the meaning of the structures remains unclear. In a previous study we showed that the overexpressed caspase-8 or -10 prodomain (PDCasp8 or PDCasp10) did not induce cell death, and we hypothesized that these prodomains interfere with the receptor-mediated cell death signaling pathway. Indeed, in 293, HeLa and Jurkat cells, cell death mediated by agonistic anti-Fas antibody, TRAIL or overexpression of full-length caspase-8 was significantly inhibited by overexpression of PDCasp8 or PDCasp10 which colocalized with the Golgi complex and with overexpressed FADD. However, when about 20 amino acid residues were deleted from either terminus of the caspase-10 prodomain (amino acid residue 1 to 219), the ability to inhibit Fas-mediated cell death was lost. Interestingly, these deletion mutants also lost the ability to make fibrous structures and to bind FADD, suggesting that FADD binding is important for their function, and that PDCasp8 and PDCasp10 act as dominant-negative inhibitors.     

Macrophage-mediated clearance of cells undergoing caspase-3-independent death

Little is known of the functions of caspases in mediating the surface changes required for phagocytosis of dying cells. Here we investigate the role played by the effector caspase, caspase-3 in this process using the caspase-3-defective MCF-7 breast carcinoma line and derived caspase-3-expressing transfectants. Our results indicate that, while certain typical features of apoptosis induced by etoposide--namely classical morphological changes and the ability to degrade DNA into oligonucleosomal fragments - are caspase-3-dependent, loss of cell adhesion to plastic and the capacity to interact with, and to be phagocytosed by, human monocyte-derived macrophages - both by CD14-dependent and CD14-independent mechanisms--do not require caspase-3. Furthermore, both etoposide-induced caspase-3-positive and -negative MCF-7 cells suppressed proinflammatory cytokine release by macrophages. These results demonstrate directly that cell surface changes that are sufficient for anti-inflammatory clearance by human macrophages can be regulated independently of stereotypical features of the apoptosis programme that require caspase-3.     

Islet-specific expression of IL-10 promotes diabetes in nonobese diabetic mice independent of Fas, perforin, TNF receptor-1, and TNF receptor-2 molecules

Several death-signaling or death-inducing molecules have been implicated in beta cell destruction, including Fas, perforin, and TNFR-1. In this study, we examined the role of each death-signaling molecule in the IL-10-accelerated diabetes of nonobese diabetic (NOD) mice. Groups of IL-10-NOD mice, each deficient in either Fas, perforin, or TNFR-1 molecules, readily developed insulitis, and subsequently succumbed to diabetes with an accelerated kinetics and incidence similar to that observed in their wild-type or heterozygous IL-10-NOD littermates. Similarly, a TNFR-2 deficiency did not block accelerated diabetes in IL-10-NOD mice and spontaneous diabetes in NOD mice. These results demonstrate that pancreatic IL-10 promotes diabetes independent of Fas, perforin, TNFR-1, and TNFR-2 molecules. Subsequently, when cyclophosphamide, a diabetes-inducing agent, was injected into insulitis-free NOD. lpr/lpr mice, none of these mice developed insulitis or diabetes. Our data suggest that cyclophosphamide- but not IL-10-induced diabetes is Fas dependent. Overall, these findings provide evidence that pancreatic expression of IL-10 promotes diabetes independent of the major death pathways and provide impetus for identification of novel death pathways precipitating autoimmune destruction of insulin-producing beta cells.     

Molecular cloning, expression, and functional analysis of caspase-10 from Japanese flounder Paralichthys olivaceus

We isolated and sequenced caspase-10 cDNA and gene from Japanese flounder, Paralichthys olivaceus. The Japanese flounder (JF)-caspase-10 cDNA consisted of 2282 bp and encoded 495 amino acid residues. The characteristic death effector domains (DEDs) of caspases were observed in JF-caspase-10 as well as the three aspartic acid residues (D-186, -382 and -392), which are potential cleavage sites for the large and small subunit structures. The amino acid residue (His-325) and pentapeptide (QACQG), which are involved in catalytic activity, were absolutely conserved in Japanese flounder-caspase-10. JF-caspase-10 gene has a length of 6.6 kb and consists of 11 exons and 10 introns similar to that of human. The strong expression of JF-caspase-10 mRNA was detected in the gills, peripheral blood leukocytes, spleen and posterior kidney, while the weak expression was observed in the head kidney, heart, intestine, skin and stomach. The over-expression analysis of JF-caspase-10 in Japanese flounder cell line HINAE was shown to induce apoptosis 24h post-transfection using TUNEL assay.     

草地贪夜蛾半胱氨酸蛋白酶Sf-caspase-3基因克隆与表达分析

半胱氨酸蛋白酶caspases是细胞凋亡过程中重要的酶类,参与调控昆虫变态发育及响应外界压力等多种生理过程。本研究通过转录组数据及逆转录PCR鉴定并验证得到Sf-caspase-3基因编码区全长。该基因编码区长840 bp,编码279个氨基酸,预测蛋白分子量为31.43 kDa。氨基酸序列分析表明Sf-caspase-3具有保守的QACRG五肽序列,且与Lep-caspase-3高度保守。进化树分析表明Sf-caspase-3与甜菜夜蛾Spodoptera exigua Se-caspase-3亲缘关系最近。RT-qPCR检测结果表明Sf-caspase-3在草地贪夜蛾Spodoptera frugiperda各个龄期均有表达,其中3~6龄幼虫期表达量最高,蛹期和卵期表达量最低。此外Sf-caspase-3在6龄幼虫中肠组织中表达量最高,而在表皮、脂肪体及马氏管中表达量极低。本研究成功构建Sf-caspase-3原核表达重组质粒,在28℃下0.4 mmol/L异丙基硫代半乳糖苷（IPTG）诱导重组菌株4 h得到约50 kDa大小条带,与预期条带大小相符。蛋白可溶性分析表明Sf-caspase-3主要以不可溶的形式存在包涵体中。本研究为草地贪夜蛾Sf-caspase-3功能及鳞翅目昆虫细胞凋亡机制研究提供一定的理论依据。     

Altered protein expression in gestational diabetes mellitus placentas provides insight into insulin resistance and coagulation/fibrinolysis pathways

Objective

To investigate the placental proteome differences between pregnant women complicated with gestational diabetes mellitus (GDM) and those with normal glucose tolerance (NGT).

Methods

We used two-dimensional electrophoresis (2DE) to separate and compare placental protein levels from GDM and NGT groups. Differentially expressed proteins between the two groups were identified by MALDI-TOF/TOF mass spectrometry and further confirmed by Western blotting. The mRNA levels of related proteins were measured by realtime RT-PCR. Immunohistochemistry (IHC) was performed to examine the cellular location of the proteins expressed in placenta villi.

Results

Twenty-one protein spots were differentially expressed between GDM and NGT placenta villi in the tested samples, fifteen of which were successfully identified by mass spectrometry. The molecular functions of these differentially expressed proteins include blood coagulation, signal transduction, anti-apoptosis, ATP binding, phospholipid binding, calcium ion binding, platelet activation, and tryptophan-tRNA ligase activity. Both protein and mRNA levels of Annexin A2, Annexin A5 and 14-3-3 protein ζ/δ were up-regulated, while the expression of the Ras-related protein Rap1A was down-regulated in the GDM placenta group.

Conclusion

Placenta villi derived from GDM pregnant women exhibit significant proteome differences compared to those of NGT mothers. The identified differentially expressed proteins are mainly associated with the development of insulin resistance, transplacental transportation of glucose, hyperglucose-mediated coagulation and fibrinolysis disorders in the GDM placenta villi.