Ran suppresses paclitaxel-induced apoptosis in human glioblastoma cells

Yeast-based functional screening of a human glioblastoma cDNA library identified ras-related nuclear protein (Ran) as a novel suppressor of Bcl-2-associated X protein (Bax), a pro-apoptotic member of the Bcl-2 family of proteins. Yeast cells that expressed human Ran were resistant to Bax-induced cell death. In U373MG glioblastoma cells, stable overexpression of Ran significantly attenuated apoptotic cell death induced by the chemotherapeutic agent paclitaxel. FACS analysis demonstrated that Ran is involved in paclitaxel-induced cell cycle arrest. Stable overexpression of Ran also markedly inhibited the phosphorylation of Bcl-2 by paclitaxel, and inhibited the translocation of Bax, the release of cytochrome c and activation of caspase-3. Paclitaxel-induced phosphorylation of c-JUN N-terminal kinase (JNK), but not p38, extracellular signal-regulated kinase and Akt, was markedly suppressed in U373MG cells that stably expressed Ran. These results suggest that Ran suppresses paclitaxel-induced cell death through the downregulation of JNK-mediated signal pathways. Im Sun Woo and Han-Su Jang contributed equally to this work.     

Detection of rare Leydig cell hypoplasia in somatic cell cloned male piglets

In this investigation, 22 cloned male piglets were obtained by male fetal fibroblast-cell-derived nuclear transfer. Eighteen of the cloned animals died. The two cell lines did not differ significantly with regard to efficiency of live piglet production. The gross anatomy of the testes of male piglets that died was normal. However, one piglet displayed Leydig cell hypoplasia (LCH). No anatomical defects were detected in the testes of other cloned male piglets. TUNEL analysis of the testis with LCH revealed significant apoptosis in the Leydig cells, while apoptosis was rarely detected in Sertoli cells and spermatogonia. In contrast, testes from the remaining 17 piglets that died appeared normal in size, and their Sertoli and Leydig cell numbers were comparable to those in control piglet testes. Although cloned piglets were derived from fibroblasts obtained from the same fetus, phenotypic instability between cells used for the production of somatic cell cloned piglets suggests that abnormalities in male cloned piglets are caused not by technical problems and/or reprogramming effects, but rather by epigenetically and/or genetically damaged cell-specific effects.     

Lack of Cytosolic Carboxypeptidase 1 Leads to Subfertility due to the Reduced Number of Antral Follicles in pcd3J-/- Females

Females homozygous for the Purkinje cell degeneration mutation (pcd) are fertile, although the success rate is much lower than in the wild type. We performed detailed analysis of reproductive abnormalities of pcd females. The number of oocytes produced following exogenous gonadotropin treatment was much lower in pcd ^3J-/- females than in pcd ^3J+/+ females. Furthermore, the estrous cyclicity of pcd ^3J-/- females according to the appearance of the vagina was almost undetectable comparing to that of the wild type. Histological analyses and follicle counting of 4- and 8-week-old pcd ^3J-/- ovaries showed an increase in the number of secondary follicles and a decrease in the number of antral follicles, indicating that AGTPBP1/ CCP1 plays an important role in the development of secondary follicles into antral follicles. Consistent with a previous analysis of the pcd cerebellum, pcd ^3J-/- ovaries also showed a clear increase in the level of polyglutamylation. Gene expression analysis showed that both oocytes and cumulus cells express CCP1. However, Ccp4 and CCP6, which can compensate the function of CCP1, were not expressed in mouse ovaries. Failure of microtubule deglutamylation did not affect the structure and function of the meiotic spindle in properly aligning chromosomes in the center of the nucleus during meiosis in pcd ^3J-/- females. We also showed that the pituitary-derived growth and reproduction-related endocrine system functions normally in pcd ^3J-/- mice. The results of this study provide insight into additional functions of CCP1, which cannot be fully explained by the side chain deglutamylation of microtubules alone.     

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.     

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.     

The effects of artificial activation timing on the development of SCNT-derived embryos and newborn piglets

This study investigated the effects of two different activation regimens on the developmental potential of somatic cell nuclear transfer (SCNT) embryos and postnatal survivability of the cloned piglets. In vitro matured oocytes were enucleated and reconstructed with porcine fetal fibroblasts. On the basis of the activation regimen used, the reconstructed porcine embryos were allocated into two groups: Group 1—simultaneous electrical pulses and activation group (SFA group); and Group 2—electrical fusion without calcium followed by electrical pulses with calcium after colcemid and cytochalasin B treatment for 5 h (DA group). Embryonic development in both SFA and DA groups was determined at day 6 of culture in NSCU-23 medium. To investigate the post-implantation development after the two activation methods, embryos were cultured for 1 day and then transferred into the oviducts of estrus-synchronized recipients. DA group had significantly (p < 0.05) higher cleavage rates than SFA group. However, the developmental rate to the blastocyst stage and the mean cell number of blastocysts did not differ (p > 0.05) between SFA and DA groups. Moreover, the pregnancy rate of SFA group was not significantly different compared to DA group. A total of 20 cloned piglets (SFA group-8 live piglets, DA group-11 live piglets and one stillborn) were obtained in the present study. The birth weight of the cloned piglets (live births) did not differ (p > 0.05) between the two groups. Furthermore, no difference was observed in the postnatal survival rates of the cloned piglets obtained using two different activation regimens. These results suggest that the timing of artificial activation and additional chemical treatments do not affect the developmental rate of porcine SCNT embryos. Remarkably, the pregnancy rate and postnatal survivability of the cloned piglets did not vary between SFA and DA groups.     

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.     

Phorbol ester potentiates the growth inhibitory effects of troglitazone via up-regulation of PPARgamma in A549 cells

The activation of peroxisome proliferator-activated receptor gamma (PPARgamma) has been shown to induce growth arrest and differentiation of various cancer cells. In the current study, we investigated the effect of 12-O-tetradecanoylphorbol-13-acetate (TPA) on the expression of PPARgamma and proliferation of A549 cells. TPA elicited a dose- and time-dependent increase in PPARgamma mRNA and protein levels. PPARgamma expression in response to TPA was attenuated by pretreatment with bisindolylmaleimide I, N-acetyl-L-cysteine (NAC) and PD98059. TPA-induced protein kinase C (PKC) activation was linked to the generation of reactive oxygen species (ROS), both of which were indispensable for PPARgamma expression in A549 cells. Pretreatment with bisindolylmaleimide I or NAC blocked TPA-induced phosphorylation of extracellular signal-regulated kinase (ERK), suggesting that ERK-mediated signaling is also involved in the induction of PPARgamma. Furthermore, the growth inhibitory effect of troglitazone was significantly potentiated by prolonged incubation with TPA and was attenuated in the presence of GW9662, a specific inhibitor of PPARgamma. These effects were associated with an induction of cell cycle arrest at G0/G1 phase, which was accompanied by the induction of p21Waf1/Cip1 expression and decreased cyclin D1 expression. Taken together, these observations indicate that TPA synergizes with PPARgamma ligand to inhibit cell growth through up-regulation of PPARgamma expression.