Characterization of a putative cis-regulatory element that controls transcriptional activity of the pig uroplakin II gene promoter

Vaspin, an adipocytokine recently identified in a rat model of type 2 diabetes, has been suggested to have an insulin-sensitizing effect. However, the exact mechanism underlying this action has not been fully elucidated. Furthermore, the specific function of vaspin is largely unknown, especially in vascular cells. We examined whether vaspin affects the insulin-signaling pathway in cultured endothelial cells and is capable of preventing free fatty acid (FFA)-induced apoptosis in endothelial cells through its insulin sensitizing effect, specifically, through its stimulatory effect on PI3-kinase/Akt signaling pathways. Vaspin significantly increased Akt phosphorylation and prevented the impairment of Akt phosphorylation by linoleic acid (LA) in insulin-stimulated endothelial cells, which effects were abolished by pretreatment with the PI3-kinase inhibitor, Wortmannin. Moreover, pretreatment with vaspin prevented LA-induced apoptosis in insulin-stimulated endothelial cells; this anti-apoptotic effect of vaspin was also eliminated by pretreatment with Wortmannin. The present study indicates that vaspin protects vascular endothelial cells from FFA-induced apoptosis through upregulation of the PI3-kinase/Akt signaling pathway. Our study is the first to demonstrate that vascular cells can be targets of vaspin. Our results further suggest that vaspin could have beneficial effects on the atherosclerosis.     

Comparative proteomic analysis associated with term placental insufficiency in cloned pig

Somatic cell-derived nuclear transfer (scNT) is a method of animal cloning in which the oocyte reprograms a somatic cell nucleus to divide and execute developmental programs. Despite many successes in this field, cloning by scNT remains very inefficient. Unlike other cloned animals, pigs derived by scNT have placentas with severe villous hypoplasia. To obtain a better understanding of the protein networks involved in this phenomenon, we assessed global protein expression profiles in term placentas from scNT-derived and control animals. Proteomic analysis of term placentas from scNT-derived animals identified 43 proteins that were differentially expressed compared to control animals. Among them, 14-3-3 proteins and Annexin V, which are closely involved in the apoptotic signaling pathway, were significantly down- and up-regulated, respectively. Western blot analysis and immunohistochemistry indicated that down-regulation of 14-3-3 proteins in scNT-derived placentas induced apoptosis of cytotrophoblast cells via mitochondria-mediated apoptosis. Taken together, our results suggest that placental insufficiency in scNT-derived placentas may be due to apoptosis, induced in part by the down-regulation of 14-3-3 proteins and up-regulation of Annexin V. They also indicate that proteomic maps represent an important tool for future studies of placental insufficiency and pathology.     

A simple,rapid, efficient and inexpensive strategy for sequencing clones from cDNA libraries

In this report, we describe a simple, rapid, efficient and inexpensive strategy for sequencing inserted DNAs from clones of cDNA or gDNA libraries. This strategy uses PCR products directly amplified from transformed bacterial colonies, with universal primers within the vector. The method can be applied for sequencing cDNA or gDNA libraries with up to 4 ∼ 5 kb insert sizes, without overnight liquid culture or plasmid DNA preparation steps. We successfully used this method to analyze clones from full-length, enriched cDNA libraries. Although simple, following this strategy will significantly help researchers to avoid unnecessary steps in the analysis of a cDNA library.     

Murine male germ cell apoptosis induced by busulfan treatment correlates with loss of c-kit-expression in a Fas/FasL- and p53-independent manner

Male germ cell apoptosis has been extensively explored in rodents. In contrast, very little is known about the susceptibility of developing germ cells to apoptosis in response to busulfan treatment. Spontaneous apoptosis of germ cells is rarely observed in the adult mouse testis, but under the experimental conditions described here, busulfan-treated mice exhibited a marked increase in apoptosis and a decrease in testis weight. TdT-mediated dUTP-X nicked end labeling analysis indicates that at one week following busulfan treatment, apoptosis was confined mainly to spermatogonia, with lesser effects on spermatocytes. The percentage of apoptosis-positive tubules and the apoptotic cell index increased in a time-dependent manner. An immediate effect was observed in spermatogonia within one week of treatment, and in the following week, secondary effects were observed in spermatocytes. RT-PCR analysis showed that expression of the spermatogonia-specific markers c-kit and Stra 8 was reduced but that Gli I gene expression remained constant, which is indicative of primary apoptosis of differentiating type A spermatogonia. Three and four weeks after busulfan treatment, RAD51 and FasL expression decreased to nearly undetectable levels, indicating that meiotic spermatocytes and post-meiotic cells, respectively, were lost. The period of germ cell depletion did not coincide with increased p53 or Fas/FasL expression in the busulfan-treated testis, although p110Rb phosphorylation and PCNA expression were inhibited. These data suggest that increased depletion of male germ cells in the busulfan-treated mouse is mediated by loss of c-kit/SCF signaling but not by p53- or Fas/FasL-dependent mechanisms. Spermatogonial stem cells may be protected from cell death by modulating cell cycle signaling such that E2F-dependent protein expression, which is critical for G1 phase progression, is inhibited.     

A rare and often unrecognized cerebromeningitis and hemodynamic disorder: a major cause of sudden death in somatic cell cloned piglets

In this study, we generated 40 somatic cell cloned (scNT) piglets. Of these, five piglets were stillborn, 22 scNT piglets died suddenly within the first week of life, and 1 piglet died after 40 days. Twelve scNT piglets are still healthy. The birth weights of compromised scNT piglets in comparison with those of normal scNT piglets are significantly reduced (0.80 +/- 0.29 vs 1.27 +/- 0.30 kg, p < 0.05), in spite of longer gestation (114 versus 120 day). Significant findings from histological examinations showed that approximately 25% (7/28) of scNT piglets showed severe congestion of lung and liver or neutrophilic inflammation in brain indicating that unexpected phenotypes can appear as a result of somatic cell cloning. Two-dimensional gel electrophoresis experiments revealed changes in the responses of several detoxification-related proteins related to stress and inflammation and found significant alterations in myocardium-specific proteins, indicating hemodynamic disorder. scNT piglets that survived to adulthood did not show any abnormality except skin and hair color depigmentation. The present study suggests that cerebromeningitis and hemodynamic disorder are a major risk factor for sudden early death of scNT piglets. Although we cannot completely exclude the possibility that scNT piglets are susceptible to specific respiratory infections, our data suggests that the early death of scNT clones is due to cardiopulmonary functional abnormalities and cerebromeningitis.     

Genetic Diversity and mRNA Expression of Porcine MHC Class I Chain-Related 2 (SLA-MIC2) Gene and Development of a High-Resolution Typing Method

The genetic structure and function of MHC class I chain-related (MIC) genes in the pig genome have not been well characterized, and show discordance in available data. Therefore, we have experimentally characterized the exon-intron structure and functional copy expression pattern of the pig MIC gene, SLA-MIC2. We have also studied the genetic diversity of SLA-MIC2 from seven different breeds using a high-resolution genomic sequence-based typing (GSBT) method. Our results showed that the SLA-MIC2 gene has a similar molecular organization as the human and cattle orthologs, and is expressed in only a few tissues including the small intestine, lung, and heart. A total of fifteen SLA-MIC2 alleles were identified from typing 145 animals, ten of which were previously unreported. Our analysis showed that the previously reported and tentatively named SLA-MIC2*05, 07, and 01 alleles occurred most frequently. The observed heterozygosity varied from 0.26 to 0.73 among breeds. The number of alleles of the SLA-MIC2 gene in pigs is somewhat lower compared to the number of alleles of the porcine MHC class I and II genes; however, the level of heterozygosity was similar. Our results indicate the comprehensiveness of using genomic DNA-based typing for the systemic study of the SLA-MIC2 gene. The method developed for this study, as well as the detailed information that was obtained, could serve as fundamental tools for understanding the influence of the SLA-MIC2 gene on porcine immune responses.     

Activation of PPARδ counteracts angiotensin II-induced ROS generation by inhibiting rac1 translocation in vascular smooth muscle cells

Angiotensin II (Ang II)-mediated modification of the redox milieu of vascular smooth muscle cells (VSMCs) has been implicated in several pathophysiological processes, including cell proliferation, migration and differentiation. In this study, we demonstrate that the peroxisome proliferator-activated receptor (PPAR) δ counteracts Ang II-induced production of reactive oxygen species (ROS) in VSMCs. Activation of PPARδ by GW501516, a specific ligand for PPARδ, significantly reduced Ang II-induced ROS generation in VSMCs. This effect was, however, reversed in the presence of small interfering (si)RNA against PPARδ. The marked increase in ROS levels induced by Ang II was also eliminated by the inhibition of phosphatidylinositol 3-kinase (PI3K) but not of protein kinase C, suggesting the involvement of the PI3K/Akt signalling pathway in this process. Accordingly, ablation of Akt with siRNA further enhanced the inhibitory effects of GW501516 in Ang II-induced superoxide production. Ligand-activated PPARδ also blocked Ang II-induced translocation of Rac1 to the cell membrane, inhibiting the activation of NADPH oxidases and consequently ROS generation. These results indicate that ligand-activated PPARδ plays an important role in the cellular response to oxidative stress by decreasing ROS generated by Ang II in vascular cells.     

Methylation status of putative differentially methylated regions of porcine IGF2 and H19

This study was designed to identify the putative differentially methylated regions (DMRs) of the porcine imprinted genes insulin-like growth factor 2 and H19 (IGF2-H19), and to assess the genomic imprinting status of IGF2-H19 by identifying the methylation patterns of these regions in germ cells, and in tissues from porcine fetuses, an adult pig, as well as cloned offspring produced by somatic cell nuclear transfer (SCNT). Porcine IGF2-H19 DMRs exhibit a normal monoallelic methylation pattern (i.e., either the paternally- or the maternally derived allele is methylated) similar to the pattern observed for the same genes in the human and mice genomes. Examination of the methylation patterns of the IGF2-H19 DMRs revealed that the zinc finger protein binding sites CTCF1 and 2 did not exhibit differential methylation in both control and cloned offspring. In contrast, the CTCF3 and DMR2 loci of the IGF2 gene showed abnormal methylation in cloned offspring, but a normal differential or moderate methylation pattern in tissues from control offspring and an adult pig. Our data thus suggest that regulation of genomic imprinting at the porcine IGF2-H19 loci is conserved among species, and that the abnormal methylation pattern in the regulatory elements of imprinted genes may lead to an alteration in the coordinated expression of genes required for successful reprogramming, which, in consequence, may contribute to the low efficiency of porcine genome reprogramming induced by nuclear transfer.