Isoquinolinequinone Derivatives from a Marine Sponge (Haliclona sp.) Regulate Inflammation in In Vitro System of Intestine

Using bio-guided fractionation and based on the inhibitory activities of nitric oxide (NO) and prostaglandin E2 (PGE2), eight isoquinolinequinone derivatives (1–8) were isolated from the marine sponge Haliclona sp. Among these, methyl O-demethylrenierate (1) is a noble ester, whereas compounds 2 and 3 are new O-demethyl derivatives of known isoquinolinequinones. Compound 8 was assigned as a new 21-dehydroxyrenieramycin F. Anti-inflammatory activities of the isolated compounds were tested in a co-culture system of human epithelial Caco-2 and THP-1 macrophages. The isolated derivatives showed variable activities. O-demethyl renierone (5) showed the highest activity, while 3 and 7 showed moderate activities. These bioactive isoquinolinequinones inhibited lipopolysaccharide and interferon gamma-induced production of NO and PGE2. Expression of inducible nitric oxide synthase, cyclooxygenase-2, and the phosphorylation of MAPKs were down-regulated in response to the inhibition of NF-κB nuclear translocation. In addition, nuclear translocation was markedly promoted with a subsequent increase in the expression of HO-1. Structure-activity relationship studies showed that the hydroxyl group in 3 and 5, and the N-formyl group in 7 may be key functional groups responsible for their anti-inflammatory activities. These findings suggest the potential use of Haliclona sp. and its metabolites as pharmaceuticals treating inflammation-related diseases including inflammatory bowel disease.     

New Constituents from the Korean Sponge Plakortis simplex

Six new cyclic peroxides (1–6) were isolated from the Korean sponge Plakortis simplex, along with two new alkylpyridinium alkaloids (7 and 8). The structures of these compounds were completely determined by a combination of NMR analysis and chemical reactions. Compounds 1–6 exhibited cytotoxic/antifungal activities against RAW264.7 cells and Candida albicans.     

Efficient Production of Cloned Bovine Embryos Using cdc2 kinase Inhibitor

This study was carried out to compare the effects of the combination of ionomycin with a H1‐histone kinase inhibitor (dimethylaminopurine, DMAP) or cdc2 kinase inhibitor (sodium pyrophosphate, SPP) on the development of reconstituted bovine eggs. For this study, the enucleated bovine oocytes were injected with a presumptive primordial germ cell pre‐treated with 1% sodium citrate, and randomly allocated into three activation groups: Group 1 (ionomycin 5 μm , 5 min), Group 2 (ionomycin + DMAP 1.9 mm , 3 h), and Group 3 (ionomycin + SPP 2 mm , 3 h). The reconstituted eggs were compared on the rates of cleavage and development with the blastocyst stage and the ploidy of embryos at 96 h post‐activation. Cleavage rates and blastocyst development in Groups 1, 2 and 3 were 7 and 0%, 63 and 17%, and 53 and 14%, respectively. The chromosomal composition differed significantly (p < 0.05) among treatments. Although the embryos in Group 1 had significantly lower developments, 60% of embryos evaluated had diploid chromosomal sets. In contrast, ～60% of embryos in Group 2 had abnormal ploidy (21% polyploid and 38% mixoploid). In Group 3, the appearance of abnormal chromosome sets was reduced with the proportion of diploid embryos being increased to 86% (19 of 22), significantly higher (p < 0.05) than in Group 2. It can be concluded that the use of SPP with ionomycin reduces greatly the incidence of chromosomal abnormalities, and may be applicable for the activation of nuclear transplant bovine embryos.     

Antibacterial Bicyclic Fatty Acids from a Korean Colonial Tunicate Didemnum sp.

Five new bicyclic carboxylic acids were obtained by antibacterial activity-guided isolation from a Korean colonial tunicate Didemnum sp. Their structures were elucidated by the interpretation of NMR, MS and CD spectroscopic data. They all belong to the class of aplidic acids. Three of them were amide derivatives (1–3), and the other two were dicarboxylic derivatives (4 and 5). The absolute configurations were determined by a bisignate pattern of CD spectroscopy, which revealed that the absolute configurations of amides were opposite to those of dicarboxylates at every stereogenic centers. Compound 2 exhibited the most potent antibacterial activity (MIC, 2 μg/mL).     

Characterization of Transgenic Pigs That Express Human Decay Accelerating Factor and Cell Membrane‐tethered Human Tissue Factor Pathway Inhibitor

The expression of human complement regulatory proteins (hCRP; hDAF, hCD59, and hMCP) in pig tissues has been suggested as one of strategies to overcome the hyperacute rejection (HAR) in pig‐to‐human transplantation. Expression of human tissue factor pathway inhibitor (hTFPI) in porcine endothelial cells has been suggested as a remedy to overcome microvascular thrombosis. To investigate the effects of these combined transgenes, we established transformed pig cells expressing human decay accelerating factor (hDAF) under the control of enhancer promoter (5′LTR‐PCMVIE), and the fusion protein (hTFPI/hCD4) consisting of the functional domains (K1 and K2) of hTFPI and membrane‐tethering domains (D3 and D4) of hCD4 under the control of PCMVIE. Transgenic pigs were generated with the transformed porcine cells through somatic cell nuclear transfer (SCNT) technology. Analysis of quantitative PCR and real‐time quantitative RT‐PCR showed that four copies of hDAF were integrated and 391 copies of hDAF mRNA expressed in the cells of the transgenic pig. The enhancing activity of 5′LTR was approximately 2 fold compared to CMVIE promoter only. The cell viability test showed that more than 80% of ear cells were viable in the presence of 50% human serum. The chromogenic substrate assay and immunocytochemical staining with tail cells showed that the TFPI activity of fusion protein was observed on the cell membrane. The membrane localization of hDAF and hTFPI proteins was observed by immunocytochemical staining, and the expression of transgenes in heart and liver tissues was also confirmed by immunohistochemistry.     

Different Culture Media Requirements of IVF and Nuclear Transfer Bovine Embryos

Important differences exist between in vitro fertilized (IVF) and nuclear transfer (NT) bovine embryos. Studies have shown that although in vitro development is comparable, post-implantation survival is greatly reduced in NT embryos. In this study, we compare serum and bovine serum albumin (BSA) supplementation during oocyte maturation and embryo culture of IVF and NT embryos. In experiment 1, oocytes and embryos were randomly distributed into different treatment groups consisting of synthetic oviductal fluid (SOF) medium supplemented with either serum, fatty acid-free BSA (FAF) or fraction V BSA during maturation and/or culture to assess IVF embryo development. In experiment 2, oocytes were matured in SOF + serum or SOF + FAF and reconstructed embryos were cultured in SOF + FAF to assess NT embryo development. Among the IVF treatment groups, a greater number of blastocysts were observed in the steer serum (SER) group (IVM and IVC in SOF + serum) on day 6; however, no significant differences were seen in blastocyst development from day 8 onwards. Hatching frequencies on days 8 and 9 were significantly greater in groups with serum, with the exception of FAF (IVM and IVC in SOF + FAF) on day 9. For the NT treatment groups, the presence of serum during IVM resulted in a higher proportion of MII oocytes and increased blastocyst development and hatching rates were compared with supplementation of FAF. These results indicate that both serum and FAF provide comparable embryo development for IVF but not for NT bovine embryos.     

Supplement of autologous ooplasm into porcine somatic cell nuclear transfer embryos does not alter embryo development

Somatic cell nuclear transfer (SCNT) is considered as the technique in which a somatic cell is introduced into an enucleated oocyte to make a cloned animal. However, it is unavoidable to lose a small amount of the ooplasm during enucleation step during SCNT procedure. The present study was aimed to uncover whether the supplement of autologous ooplasm could ameliorate the oocyte competence so as to improve low efficiency of embryo development in porcine SCNT. Autologous ooplasm‐transferred (AOT) embryos were generated by the supplementation with autologous ooplasm into SCNT embryos. They were comparatively evaluated with respect to embryo developmental potential, the number of apoptotic body formation and gene expression including embryonic lineage differentiation, apoptosis, epigenetics and mitochondrial activity in comparison with parthenogenetic, in vitro‐fertilized (IVF) and SCNT embryos. Although AOT embryos showed perfect fusion of autologous donor ooplasm with recipient SCNT embryos, the supplement of autologous ooplasm could not ameliorate embryo developmental potential in regard to the rate of blastocyst formation, total cell number and the number of apoptotic body. Furthermore, overall gene expression of AOT embryos was presented with no significant alterations in comparison with that of SCNT embryos. Taken together, the results of AOT demonstrated inability to make relevant values improved from the level of SCNT embryos to their IVF counterparts.     

In vitro developmental potential of nuclear transfer embryos cloned with enucleation methods using pre-denuded bovine oocytes

Enucleation of a recipient oocyte is an important essential process in the procedure of somatic cell nuclear transfer (SCNT). The present study investigated a method for the improvement of enucleation efficiency. Oocytes were denuded of cumulus cells before the completion of nuclear maturation (pre-denuded) after 12 h of culture at MI stage and subsequently cultured for additional 6 h until the completion of nuclear maturation and extrusion of the first polar body (PB1). The extrusion rate of PB1 was not significantly different in the pre-denuded oocyte group, compared with control oocyte group matured for 18 h. However, the number of oocytes showing the metaphase II (MII) located just underneath the PB1 was significantly higher (p<0.05) in the pre-denuded oocyte group than those in control oocyte group. To test the effect of pre-denuding on the enucleation rate and developmental potential of embryos to blastocyst stage, subsequent somatic cell nuclear transfer comparisons were made with three different methods of enucleation at MII stage using vital dyes (demicoline and Hoescht) or the PB1 (blind enucleation) to localize the chromosome plate. Enucleation rate of the oocytes with demicoline, Hoechst and pre-denuding enucleation groups were significantly higher (p<0.05) than those of blind enucleation groups. However, cleavage rate to two-cell stage and, developmental rate to blastocyst and hatched blastocyst stage, the mean numbers of total and ICM cells in the SCNT embryos with Hoechst enucleation groups were significantly decreased (p<0.05), compared to those of blind, demicoline and pre-denuding enucleation groups. Moreover, the level of telomerase activity was also significantly (p<0.05) decreased in SCNT blastocysts of Hoechst enucleation group, compared to those of blind, demicoline and pre-denuding enucleation groups. Taken together, pre-denuding enucleation group using pre-denuded oocytes was a useful and simple enucleation method for bovine SCNT embryos.     

Effect of histone acetylation modification with sodium butyrate, a histone deacetylase inhibitor, on cell cycle, apoptosis, ploidy and gene expression in porcine fetal fibroblasts

The present study evaluated the effective dose of sodium butyrate (NaB), a histone deacetylase (HDAC) inhibitor, for determination of the level of enhancement of histone acetylation in porcine fetal fibroblasts (PFFs) based on their morphology, growth, apoptosis and cell cycle status. Cells were analyzed for their histone acetylation levels at H3, H4 and H2A and expression of genes related to histone deacetylation (HDAC1, HDAC2 and HDAC3), pro-apoptosis (Bax and Bak) and anti-apoptosis (Bcl-2). PFFs at passage 3-4 were cultured with 0, 0.5, 1.0, 2.0 and 3.0 mM NaB for 96 h. NaB inhibited cell proliferation at all tested concentrations in a dose-dependent manner. However, there was slow cell growth for PFFs treated with 2.0 and 3.0 mM NaB compared with those of untreated PFFs and those treated with other lower concentrations (0.5 and 1.0 mM). More than 85% of the cells that were untreated or treated with 0.5 or 1.0 mM NaB had intact membranes, whereas, approximately 30% of the cells treated with 2.0 or 3.0 mM NaB had increased cell sizes and a more flattened and elongated appearance. NaB induced apoptosis in a dose-dependent manner; the rates of apoptosis were 2.5 +/- 0.4% for 1.0 mM NaB, 7.6 +/- 1.1% for 2.0 mM NaB and 11.2 +/- 1.4% for 3.0 mM NaB. The chromosomal sets of PFFs treated with 0.5 and 1.0 mM NaB were normal, whereas a lower proportion of PFFs treated with 2.0 and 3.0 mM were classified as normal. NaB at 0.5 and 1.0 mM showed little effect on cell cycle. However, 2.0 and 3.0 mM resulted in an increased cell population at the G(0)/G(1) phase. Increased NaB concentrations led to elevated acetylation of H3, H4 and H2A. NaB altered the expression of histone deacetylation and apoptosis-related genes. In conclusion, 1.0 mM NaB induced histone hyperacetylation in the PFFs and produced less deleterious effects than other concentrations; these PFFs might serve as suitable donors for porcine somatic cell nuclear transfer (SCNT).