The null mutation of cardiac Na+-Ca2+ exchanger (NCX1) gene in mice caused death of embryo in utero at embryonic day (ED) 9.0-9.5 and this embryonic lethality appears resulted from abnormal heart development. In the present study, we investigated whether transgenic re-expression of NCX1 in mutant cardiac myocytes could rescue these lethal defects. Transgenic mice expressing the canine NCX1 in a cardiac specific manner were bred into the NCX1 knock-out background but did not prevent the fetal lethality associated with the NCX1 null allele. However, the NCX1 knock-out embryos with an NCX1 transgene survived with heart beatings until ED 10.5 which was one day longer than the survival of the NCX1 knock-out embryos (ED 9.5). At ED 10.5, however, the partially rescued NCX1 embryos might have succumbed to the lack of an organized vasculature in the yolk sacs. The placental labyrinth layer was reduced in size and largely avascular. The transgenic re-expression of NCX1 rescued heart beatings and survived longer, but was still insufficient for the mice to be completely rescued. Importantly, NCX1 was observed to express in the yolk sac and the placenta of wild type mice. The results suggest that defects in extra-embryonic compartments are causal to the lethality, and that NCX1 may play an important role in establishing vascularization in extra-embryonic tissues.
Animals ; Embryo/*metabolism/pathology ; Embryo Loss ; Female ; Gene Deletion ; *Gene Expression ; Genetic Complementation Test ; Mice ; Mice, Knockout ; Mice, Transgenic ; Myocytes, Cardiac/metabolism ; Phenotype ; Placenta/metabolism/pathology ; Sodium-Calcium Exchanger/*genetics/*metabolism ; Survival Rate ; Yolk Sac/embryology/metabolism/pathology

Animals ; Cell Nucleus Size ; genetics ; Cell Proliferation ; genetics ; Embryo Loss ; genetics ; pathology ; Embryo, Mammalian ; metabolism ; pathology ; Embryonic Stem Cells ; cytology ; Endoplasmic Reticulum ; genetics ; Homologous Recombination ; Mice ; rab GTP-Binding Proteins ; deficiency ; genetics ; metabolism

OBJECTIVETo investigate the role of nitric oxide (NO) in the pathogenesis of early endometriosis-associated infertility.

METHODSThe volume of peritoneal fluids was recorded and the concentration of NO in peritoneal fluid and serum was measured with a fluorescence method using 4, 5-diaminofluorescein (DAF-2) as an indicator, in 60 patients with early endometriosis-associated infertility ( endometriosis group), 60 patients with tubal infertility (tubal infertility group) and 20 patients without infertility (control group). The IVF-ET outcomes between patients with endometriosis and tubal infertility were compared.

RESULTThe volume of peritoneal fluids from endometriosis group patients increased significantly compared with that in tubal infertility group patients and control groups. The concentration of NO in peritoneal fluid of the control group,the tubal infertility group and the endometriosis group was 9.98, 13.76 and 20.72, respectively (P<0.017). Furthermore the concentration of NO in serum of the patients of control group,tubal infertility group and endometriosis group was 12.25, 13.00, 13.60, respectively; there were no significant differences among these three groups. There were no significant differences in implantation rate, pregnancy rate and abortion rate of IVF-ET between endometriosis group patients and tubal infertility group patients. However, the fertilization rate was significantly lower in endometriosis group patients than that in tubal infertility group patients.

CONCLUSIONChanges of nitric oxide in peritoneal fluids may play an important role in the pathogenesis of early endometriosis-associated infertility and IVF-ET may serve as an alternative method for this type of infertility.

Adult ; Ascitic Fluid ; metabolism ; Biomarkers ; blood ; Embryo Transfer ; Endometriosis ; blood ; complications ; metabolism ; pathology ; Female ; Fertilization in Vitro ; Humans ; Infertility, Female ; blood ; etiology ; metabolism ; Nitric Oxide ; blood ; metabolism

OBJECTIVE: To examine the association among malondialdehyde (MDA), superoxide dismutase (SOD) and incidence of apoptosis of granulosa cells in follicular fluid with the outcome of in vitro fertilization-embryo transfer (IVF-ET). METHODS: We recruited 51 women undergoing an IVF-ET programme for tubal factor infertility. The women with endometriosis or endocrine diseases and those with male factor infertility were excluded. All the 51 patients underwent a long gonadotropin-releasing hormone (GnRH) agonist protocol for pituitary downregulation followed by controlled ovarian hyperstimulation with rFSH. Granulosa cells were isolated from all aspirated follicular fluid using gradient centrifugation at oocyte retrieval. The level of MDA and the activity of the SOD were measured by the thiobarbituric acid(TBA)and the chemiluminescence method, respectively. The apoptosis was studied by flow cytometry using propidium iodide. RESULTS: Twenty-six out of the 51 patients (51.0%) were pregnant after IVF-ET. Non-pregnant patients showed significantly higher MDA level [(1.7±0.72) nmol/(g × prot) vs. (1.1±0.56) nmol/(g × prot), P<0.05)], higher incidence of apoptosis (24.8%±6.57% vs.19.0%±5.59%, P<0.05) and lower SOD level [(3.5±1.08)×10(3)NU/(g × prot) vs. (4.4±0.99)×10(3)NU/(g × prot), P<0.05)] in the granulose cells and lower good-embryo rate (54.9±20.22% vs. 65.9±16.16%, P<0.05) compared with the pregnant patients. No correlation was detected among SOD and the number of retrieved oocytes, oocyte maturity, embryo quality, fertilization, or cleavage. A significant negative correlation was detected between MDA and fertilization rate (r=-0.425, P=0.002). No significant correlation was detected between MDA and age, the number of retrieved oocytes, oocyte maturity, cleavage, or good-embryo rate. A significant negative correlation was detected between the incidence of apoptosis and the number of retrieved oocytes (r=-0.286, P=0.042), mature oocytes (r=-0.330, P=0.020) and good-embryo rate (r=-0.311, P=0.026). There was significant negative correlation between MDA and SOD levels (r=-0.471, P<0.001); and significant positive correlation between MDA level and incidence of apoptosis (r=0.475, P<0.001). CONCLUSION Oxidative stress may induce apoptosis in granulose cells and subsequently lower oocyte quality and lead to poor outcome of IVF-ET.
Adult ; Apoptosis ; physiology ; Embryo Transfer ; Female ; Fertilization in Vitro ; Granulosa Cells ; metabolism ; pathology ; Humans ; Malondialdehyde ; metabolism ; Ovary ; cytology ; metabolism ; Oxidative Stress ; Pregnancy ; Pregnancy Rate ; Superoxide Dismutase ; metabolism ; Treatment Outcome

OBJECTIVETo study the relationship between late mRNA and the cytopathic effect(CPE) and ultrastructural features after human cytomegalovirus (HCMV) infection in vitro.

METHODSHuman embryo fibroblast cells(HEL) were infected with HCMV AD169 strain. The expression of the HCMV late mRNA was measured by semiquantitative RT-PCR, the cytopathic effect (CPE) and the cell ultrastructure were observed by means of light microscopy and electron microscopy, respectively.

RESULTSThe HCMV late mRNA could be detected 12 hours postinfection and increased gradually, but the CPE appeared 48 hours postinfection in HEL cells. The HCMV infected cells exhibited significant mitochondrial enlargement and the number of mitochondrial ridge deletion, the cisternae lumen of endoplasmic reticulum (ER) dilation and vacuolization (at the end age). The mature nucleocapsid could be observed 96 hours postinfection.

CONCLUSIONThe ultrastructural changes have an intimate correlation with the expression of HCMV late mRNA and play an important role in the life circle of the virus. HCMV late mRNA may serve as a indicator of the clinical effect of treatment in active HCMV infection.

Cytomegalovirus ; genetics ; physiology ; Cytopathogenic Effect, Viral ; Embryo, Mammalian ; Endoplasmic Reticulum ; pathology ; virology ; Fibroblasts ; metabolism ; ultrastructure ; virology ; Humans ; Inclusion Bodies ; pathology ; virology ; Mitochondrial Swelling ; RNA, Messenger ; metabolism

OBJECTIVETo explore the role of telomerase in asbestos dust induced malignant transformation of human embryonic lung fibroblasts in vitro.

METHODSHuman telomerase catalytic subunit (hTERT) was transferred into human embryonic lung fibroblasts (HELF). Chrysotile dust at concentration of 2.5 microg/cm(2) was added to HELF transduced with and without hTERT (HELF-T+), respectively, and their transduced foci were separated. Biological characteristics of the cells, telomerase activity, length of telomere and cell growth curve were observed. Colony forming test was performed on soft agar to evaluate the nature of transformation.

RESULTSThe hTERT gene was transferred into HELF steadily, and HELF-T+ was established. Malignant transformation occurred in both HELF and HELF-T+ by asbestos stimulation. Asbestos dusts could induce higher rate of transformations in HELF-T+ [(2.08 +/- 1.08)/utensil] than in HELF [(1.08 +/- 0.10)/utensil], P < 0.05. Telomerase activity in both transformed malignant cells and HELF-T+ was higher, as well as the longer length of telomere in them.

CONCLUSIONRate of malignant transformation in cells with more activity of telomerase and longer length of the telomere was higher after stimulation with asbestos, indicating telomerase could play an important role in asbestos induced human cells malignant transformation.

Asbestos, Serpentine ; toxicity ; Cell Transformation, Neoplastic ; drug effects ; Cells, Cultured ; DNA-Binding Proteins ; Embryo, Mammalian ; Fibroblasts ; pathology ; Gene Transfer Techniques ; Humans ; Lung ; pathology ; Telomerase ; genetics ; metabolism

OBJECTIVETo investigate the protective effect of reactive oxygen species (ROS) scavenger, N-acetyl-L-cysteine (NAC), against H9c2 cardiomyocytes from injuries induced by chemical hypoxia.

METHODSH9c2 cells were treated with cobalt chloride (CoCl2), a chemical hypoxia-mimetic agent, to establish the chemical hypoxia-induced cardiomyocyte injury model. NAC was added into the cell medium 60 min prior to CoCl2 exposure. The cell viability was evaluated using cell counter kit (CCK-8), and the intercellular ROS level was measured by 2', 7'- dichlorfluorescein-diacetate (DCFH-DA) staining and photofluorography. Mitochondrial membrane potential (MMP) of the cells was observed by Rhodamine123 (Rh123) staining and photofluorography, and the ratio of GSSG/ (GSSG+GSH) was calculated according to detection results of the GSSG kit.

RESULTSExposure of H9c2 cardiomyocytes to 600 micromol/L CoCl2 for 36 h resulted in significantly reduced cell viability. Pretreatment with NAC at the concentrations ranging from 500 to 2000 micromol/L 60 min before CoCl2 exposure dose-dependently inhibited CoCl2-induced H9c2 cell injuries, and obviously increased the cell viability. NAC at 2000 micromol/L obviously inhibited the oxidative stress induced by CoCl2, decreased the ratio of GSSG/(GSSG+GSH), increased ROS level, and antagonized CoCl2-induced inhibition on MMP.

CONCLUSIONNAC offers obvious protective effect on H9c2 cardiomyocytes against injuries induced by chemical hypoxia by decreasing in the ratio of GSSG/(GSSG+GSH) and ROS level and ameliorating MMP.

Animals ; Cell Hypoxia ; drug effects ; Cells, Cultured ; Embryo, Mammalian ; Free Radical Scavengers ; pharmacology ; Myocytes, Cardiac ; drug effects ; metabolism ; pathology ; Oxidative Stress ; drug effects ; Rats ; Reactive Oxygen Species ; metabolism

Apoptosis has an important role in maintaining tissue homeostasis in cellular stress responses such as inflammation, endoplasmic reticulum stress, and oxidative stress. T-cell death-associated gene 51 (TDAG51) is a member of the pleckstrin homology-like domain family and was first identified as a pro-apoptotic gene in T-cell receptor-mediated cell death. However, its pro-apoptotic function remains controversial. In this study, we investigated the role of TDAG51 in oxidative stress-induced apoptotic cell death in mouse embryonic fibroblasts (MEFs). TDAG51 expression was highly increased by oxidative stress responses. In response to oxidative stress, the production of intracellular reactive oxygen species was significantly enhanced in TDAG51-deficient MEFs, resulting in the activation of caspase-3. Thus, TDAG51 deficiency promotes apoptotic cell death in MEFs, and these results indicate that TDAG51 has a protective role in oxidative stress-induced cell death in MEFs.
Animals ; *Apoptosis ; Embryo, Mammalian/*cytology ; Fibroblasts/enzymology/*metabolism/pathology ; Gene Expression Regulation ; Intracellular Space/metabolism ; Mice ; Mitogen-Activated Protein Kinases/metabolism ; NF-kappa B/metabolism ; *Oxidative Stress/genetics ; Reactive Oxygen Species/*metabolism ; Signal Transduction ; Transcription Factors/*deficiency/genetics/metabolism

The establishment of a polarized cellular morphology is essential for a variety of processes including neural tube morphogenesis and the development of the brain. Cdc42 is a Ras-related GTPase that plays an essential role in controlling cell polarity through the regulation of the actin and microtubule cytoskeleton architecture. Previous studies have shown that Cdc42 plays an indispensable role in telencephalon development in earlier embryo developmental stage (before E12.5). However, the functions of Cdc42 in other parts of brain in later embryo developmental stage or in adult brain remain unclear. Thus, in order to address the role of Cdc42 in the whole brain in later embryo developmental stage or in adulthood, we used Cre/loxP technology to generate two lines of tissue-specific Cdc42-knock-out mice. Inactivation of Cdc42 was achieved in neuroepithelial cells by crossing Cdc42/ flox mice with Nestin-Cre mice and resulted in hydrocephalus, causing death to occur within the postnatal stage. Histological analyses of the brains from these mice showed that ependymal cell differentiation was disrupted, resulting in aqueductal stenosis. Deletion of Cdc42 in the cerebral cortex also induced obvious defects in interkinetic nuclear migration and hypoplasia. To further explore the role of Cdc42 in adult mice brain, we examined the effects of knocking-out Cdc42 in radial glial cells by crossing Cdc42/flox mice with human glial fibrillary acidic protein (GFAP)-Cre mice. Inactivation of Cdc42 in radial glial cells resulted in hydrocephalus and ependymal cell denudation. Taken together, these results highlight the importance of Cdc42 for ependymal cell differentiation and maintaining, and suggest that these functions likely contribute to the essential roles played by Cdc42 in the development of the brain.
Animals ; Brain ; metabolism ; pathology ; Cell Differentiation ; Cell Polarity ; Cerebral Cortex ; cytology ; metabolism ; Constriction, Pathologic ; Embryo, Mammalian ; metabolism ; Embryonic Development ; Ependyma ; cytology ; metabolism ; Glial Fibrillary Acidic Protein ; genetics ; metabolism ; Humans ; Hydrocephalus ; metabolism ; pathology ; Integrases ; genetics ; metabolism ; Mice ; Mice, Knockout ; cdc42 GTP-Binding Protein ; genetics ; metabolism

The tyrosine kinase system angiopoietin (Ang)/Tie interacts with vascular endothelial growth factor pathway and regulates vessel quiescence in adults as well as later steps of the angiogenic cascade related to vessel maturation. Since all Angs are able to bind to Tie-2 but none binds to Tie-1, the function of Tie-2 and its ligands have captured attention. However, emerging evidence indicates unique roles of the orphan receptor Tie-1 in angiogenesis under physiological and pathological conditions. It is required for maintaining vascular endothelial cell integrity and survival during murine embryo development and in adult and may be involved in modulating differentiation of hematopoietic cells in adult. Tie-1 exhibits poor tyrosine kinase activity and signals via forming heterodimers with Tie-2, inhibiting Tie-2 signaling mediated by Angs. This inhibition can be relieved by Tie-1 ectodomain cleavage mediated by tumor- and inflammatory-related factors, which causes destabilization of vessels and initiates vessel remodeling. Up-regulated Tie-1 expression has been found not only in some leukemia cells and tumor related endothelial cells but also in cytoplasm of carcinoma cells of a variety of human solid tumors, which is associated with tumor progression. In addition, it has pro-inflammatory functions in endothelial cells and is involved in some inflammatory diseases associated with angiogenesis. Recent research indicated that Tie-1 gene ablation exhibited significant effects on tumor blood- and lymph-angiogenesis and improved anti-Ang therapy, suggesting Tie-1 may be a potential target for tumor anti-angiogenesis treatment.
Angiogenesis Inhibitors ; therapeutic use ; Angiopoietins ; genetics ; metabolism ; Animals ; Embryo, Mammalian ; Embryonic Development ; genetics ; Endothelial Cells ; drug effects ; metabolism ; pathology ; Gene Expression Regulation, Developmental ; Gene Expression Regulation, Neoplastic ; Humans ; Mice ; Neoplasms ; drug therapy ; genetics ; metabolism ; pathology ; Neovascularization, Pathologic ; drug therapy ; genetics ; metabolism ; pathology ; Protein Binding ; Receptor, TIE-1 ; antagonists & inhibitors ; genetics ; metabolism ; Receptor, TIE-2 ; genetics ; metabolism ; Signal Transduction