Comparative Transcriptome Analysis of Cell-Free Fetal RNA from Amniotic Fluid and RNA from Amniocytes in Uncomplicated Pregnancies.

OBJECTIVES: We aimed to compare tissue-specific expression profiles and biological pathways of RNA from amniocytes and amniotic fluid supernatant (AFS) from second-trimester pregnancies by using transcriptome analysis. Additionally, we wanted to explore whether cell-free RNA from AFS exhibits a unique gene expression signature that more adequately reflects the fetal developmental process than amniocyte RNA. METHODS: Amniotic fluid samples were prospectively collected in the second trimester of pregnancy from euploid fetuses. Total RNA was extracted from amniocytes and AFS and hybridized to Affymetrix GeneChip Human Arrays. Significantly differentially expressed transcripts between amniocytes and AFS were obtained by using Welch's t-test. Unsupervised hierarchical clustering was used to visualize overall expression characteristics and differences in transcripts between AFS and amniocytes. The biological functions of selected genes were analyzed using various online Gene Ontology databases. RESULTS: A total of 3,072 and 15,633 transcripts were detected in the second-trimester AFS and amniocytes, respectively. Hierarchical clustering revealed differential transcript expression between AFS and amniocytes. We found 353 genes that were specifically enriched in the AFS only, and tissue expression analysis showed enrichment of brain-specific genes in the AFS. Biological pathway analysis revealed that AFS-specific transcripts were mainly involved in embryonic development, cardiovascular development, and cellular morphology pathways. CONCLUSION: This study demonstrated differential tissue-specific gene expression profiles and biological pathways between AFS and amniocytes. The results suggested that AFS is the preferred RNA source to investigate potential biomarkers of fetal neurodevelopment.

Transcriptional profiling with a pathway-oriented analysis in the placental villi of unexplained miscarriage.

INTRODUCTION: Miscarriage is the most common placental-related complication of pregnancy. It has been extensively investigated to discover the underlying mechanism(s) by which miscarriage occurs, but in many cases the etiology still remains unclear. The aim of this study was to analyze genome-wide expression profiles of placental villi (PV) from unexplained miscarriage with a pathway-oriented method for identifying underlying mechanism(s) of unexplained miscarriage. METHODS: We investigated PV of 18 women with unexplained miscarriage and 11 women underwent normal pregnancy. Each PV was obtained through dilatation & evacuation and chorionic villous sampling, respectively. Genome-wide expression profiles of PV were analyzed by Gene Set Enrichment Analysis (GSEA) to find dysregulated signaling pathways in PV of unexplained miscarriage. RESULTS: Unsupervised hierarchical clustering showed heterogeneity of expression profiles between PV of normal developing pregnancy and unexplained miscarriage. GSEA, a supervised analysis, with KEGG pathways revealed that several gene sets associated with mitochondrial function including glutathione metabolism and oxidative phosphorylation are dysregulated in PV from unexplained miscarriage. RT-PCR, real-time RT-PCR and/or immunohistochemistry reinforced that expression of genes constituting these gene sets enriched in normal pregnancy and Cu/Zn-superoxide dismutase was down-regulated in PV of unexplained miscarriage. DISCUSSION: Structural vulnerability of placental villi for reactive oxygen species (ROS), which is caused by systemic down-regulation of mitochondrial pathways involved in mitochondrial redox balance and functions, aggravates oxidative stress with increased ROS production in PV of unexplained miscarriage. CONCLUSION: Systemic vulnerability for ROS in PV could be a major cause of unexplained miscarriage.

Chlorpromazine attenuates pancreatic beta-cell function and mass through IRS2 degradation, while exercise partially reverses the attenuation.

We investigated the effect and mechanism of exercise and chlorpromazine (CPZ), a conventional anti-psychotic drug, on beta-cell function and mass in 90% pancreatectomized (Px) male rats. The diabetic Px rats were divided into two groups, one of which was provided with exercise whereas the other was not. Both groups were subdivided into the three groups and administered with 0, 5 or 50 mg CPZ per kg body weight (control, low dosage of chlorpromazine (LCPZ), high dosage chlorpromazine (HCPZ)) for 8 weeks. LCPZ did not modulate glucose homeostasis. HCPZ impaired acute phase and second phase insulin secretion during hyperglycemic clamp. Apoptosis of pancreatic beta-cells increased in the HCPZ group, and proliferation decreased, contributing to reduced beta-cell mass. Exercise partially improved glucose-stimulated insulin secretion and beta-cell mass in HCPZ-treated rats. Interestingly, insulin receptor substrate-2 (IRS2) protein levels in islets decreased by increased degradation in the HCPZ group, whereas exercise partially reversed this trend by induction of IRS2 expression. In isolated islets, 50 microM CPZ decreased IRS2 expression by promoting ubiquitin-proteasome degradation, which had been prevented by proteasome inhibitors. Furthermore, similar to the effect of HCPZ treatment, a high dosage of rottlerin, a protein kinase C-delta inhibitor, reduced IRS2 levels in the islets. In conclusion, exercise partially recovered the diabetic symptoms exacerbated by HCPZ through enhancement of beta-cell function and mass in diabetic rats. This modulation by HCPZ and exercise was associated with increasing intracellular IRS2 protein levels in independent pathways.

Long-term consumption of fermented soybean-derived Chungkookjang attenuates hepatic insulin resistance in 90% pancreatectomized diabetic rats.

Soy protein and isoflavonoids in soybeans exhibit the improvement of insulin resistance. Our previous IN VITRO study showed that Chungkookjang (CKJ), fermented unsalted soybeans, had better antidiabetic actions than cooked unfermented soybeans (CSB) by increasing isoflavones aglycones and small peptides. We investigated whether 40% fat diets with different protein sources such as CSB, CKJ, and casein modulated peripheral insulin resistance in 90% pancreatectomized (Px) diabetic rats. The Px rats weighing 209+/-14 g were freely provided casein, CSB, or CKJ diets for 8 weeks. Both CKJ and CSB increased whole body glucose disposal rates and glucose uptake into skeletal muscles of Px rats as much as rosiglitazone plus casein treated rats during euglycemic hyperinsulinemic clamp. In addition, CKJ and CSB decreased hepatic glucose output at hyperinsulinemic clamped states, compared to the Casein group. The reduction of hepatic glucose output was greater in CKJ than CSB. This reduction was associated with enhanced tyrosine phosphorylation of IRS2 and serine (473) phosphporylation of Akt, indicating improved hepatic insulin signaling. This improved signaling led to decreased phosphoenolpyruvate carboxykinase expression to reduce hepatic glucose output. In conclusion, fermented soybeans mainly with BACILLUS SUBTILIS improved hepatic insulin sensitivity better than unfermented soybeans by enhancing hepatic insulin signaling cascade in diabetic rats.

Survival of conditionally immortalized hepatocytes in the spleen of syngeneic rats.

BACKGROUND: Hepatocyte transplantation has been shown to be effective in the treatment of liver failure; however, the shortage of donor organs limits its clinical application. Several reports have suggested that conditionally immortalized hepatocytes (CIH) could be an alternative to primary hepatocytes. However, CIH are known to undergo apoptosis in vitro at a non-permissive temperature, which is similar to body temperature. METHODS: To investigate the duration of survival and in vivo apoptosis of CIH in the syngeneic host, the L2A2 cells (a kind of CIH) that were established from hepatocytes of a Lewis rat with a gene for a temperature-sensitive Simian Virus 40 (SV40) large T antigen were transplanted into the spleen. Cells were isolated from the spleen that was removed periodically up to 6 months, and used to detect the presence of the L2A2 cells among them with the selective culture for CIH and T-antigen PCR. In situ apoptosis of L2A2 cells was also examined. In order to improve the survival of transplanted L2A2 cells in the host, a group of rats were partially hepatectomized 1 day before transplantation was performed. RESULTS: The L2A2 cells secreted albumin at a rate of 1.17 +/- 0.18 microg/24 h per 10(6) cells in vitro. After transplantation, L2A2 cell colonies and PCR amplification bands appeared up to 14 and 7 days, respectively, but this duration was not prolonged by a partial hepatectomy. The spleen showed a large number of hepatocytes that were in the process of dying on the 5th day, and only a number of ghost hepatocytes were present on the 7th day of transplantation. No tumors were found during the 6-month observation period. CONCLUSIONS: Conditionally immortalized hepatocytes can survive in the spleen for a limited period, in spite of the growth stimulation, most likely because they undergo apoptosis in vivo as well as in vitro at a non-permissive temperature. These data suggest that the use of these cells in hepatocyte transplantation be limited to temporary hepatic support.

Dedifferentiation of conditionally immortalized hepatocytes with long-term in vitro passage.

The rat hepatocytes were immortalized using a temperature-sensitive mutant of SV40 large T antigen (tsT) to develop as a possible substitute for primary hepatocytes. Four rat hepatocyte lines that have been developed and maintained more than passage 50, were characterized for their cellular morphology, T antigen and p53 expression, chromosomes, liver-specific differentiation, telomerase activity and anchorage independent growth. All of four cell lines showed a typical epithelial cell morphology, but the population-doubling time became short with passage: 18 to 60%. T antigen expression was increased with passage about 3 to 65 times at permissive temperature but decreased significantly at non-permissive temperature. The expression level of p53 unchanged during passages was also decreased at non-permissive temperature. The distribution of chromosome number changed somewhat with passage. The production levels of albumin and urea in four cell lines were 2.4 to 13.0% and 7.5 to 19.9% of those produced in primary hepatocytes, respectively and were decreased to an undetectable level with passage. Telomerase activity was increased 10 fold following immortalization of cells, but anchorage independent growth of cells did not develop. These results indicate that conditionally immortalized hepatocytes become dedifferentiated with in vitro passage, which may be caused by marked chromosomal damages that occur with compulsive and continuous replications by the increment of T antigen content with passage and its sequential inhibition of p53 function.