Sphingosine‐1‐phosphate (S1P) analog phytosphingosine‐1‐phosphate (P1P) improves the in vitro maturation efficiency of porcine oocytes via regulation of oxidative stress and apoptosis

Phytosphingosine‐1‐phosphate (P1P) is a signaling sphingolipid that regulates various physiological activities. However, little is known about the effect of P1P in the context of reproduction. Thus, we aimed to investigate the influence of P1P on oocyte maturation during porcine in vitro maturation (IVM). Here, we report the expression of S1PR1–3 among P1P receptors (S1PR1–4) in cumulus cells and oocytes. When P1P was administered at concentrations of 10, 50, 100, and 1,000 nM during IVM, the metaphase II rate was significantly increased in the 1,000 nM (1 μM) P1P treatment group. Maturation rate improvement by P1P supplementation was observed only in the presence of epidermal growth factor (EGF). Oocytes under the influence of P1P showed decreased intracellular reactive oxygen species levels but no significant differences in glutathione levels. In our molecular studies, P1P treatment upregulated gene expression involved in cumulus expansion (Has2 and EGF), antioxidant enzymes (SOD3 and Cat), and developmental competence (Oct4) while activating extracellular signal‐regulated kinase1/2 and Akt signaling. P1P treatment also influenced oocyte survival by shifting the ratio of Bcl‐2 to Bax while inactivating JNK signaling. We further demonstrated that oocytes matured with P1P displayed significantly higher developmental competence (cleavage and blastocyst [BL] formation rate) and greater BL quality (total cell number and the ratio of apoptotic cells) when activated via parthenogenetic activation (PA) and in vitro fertilization. Despite the low levels of endogenous P1P found in animals, exogenous P1P influenced animal reproduction, as shown by increased porcine oocyte maturation as well as preimplantation embryo development. This study and its findings are potentially relevant for both human and animal‐assisted reproduction.     

SUMO‐modified insulin‐like growth factor 1 receptor (IGF‐1R) increases cell cycle progression and cell proliferation

Increasing number of studies have shown nuclear localization of the insulin‐like growth factor 1 receptor (nIGF‐1R) in tumor cells and its links to adverse clinical outcome in various cancers. Any obvious cell physiological roles of nIGF‐1R have, however, still not been disclosed. Previously, we reported that IGF‐1R translocates to cell nucleus and modulates gene expression by binding to enhancers, provided that the receptor is SUMOylated. In this study, we constructed stable transfectants of wild type IGF1R (WT) and triple‐SUMO‐site‐mutated IGF1R (TSM) using igf1r knockout mouse fibroblasts (R‐). Cell clones (R‐WT and R‐TSM) expressing equal amounts of IGF‐1R were selected for experiments. Phosphorylation of IGF‐1R, Akt, and Erk upon IGF‐1 stimulation was equal in R‐WT and R‐TSM. WT was confirmed to enter nuclei. TSM did also undergo nuclear translocation, although to a lesser extent. This may be explained by that TSM heterodimerizes with insulin receptor, which is known to translocate to cell nuclei. R‐WT proliferated substantially faster than R‐TSM, which did not differ significantly from the empty vector control. Upon IGF‐1 stimulation G1‐S‐phase progression of R‐WT increased from 12 to 38%, compared to 13 to 20% of R‐TSM. The G1‐S progression of R‐WT correlated with increased expression of cyclin D1, A, and CDK2, as well as downregulation of p27. This suggests that SUMO‐IGF‐1R affects upstream mechanisms that control and coordinate expression of cell cycle regulators. Further studies to identify such SUMO‐IGF‐1R dependent mechanisms seem important.     

Fluorescence enhancement of europium (III) perchlorate by 1,10‐phenanthroline on the 1‐(naphthalen‐2‐yl)‐2‐(phenylsulthio)ethanone complex and luminescence mechanism

A novel ligand, 1‐(naphthalen‐2‐yl)‐2‐(phenylsulthio)ethanone was synthesized using a new method and its two europium (Eu) (III) complexes were synthesized. The compounds were characterized by elemental analysis, coordination titration analysis, molar conductivity, infrared, thermo gravimetric analyzer‐differential scanning calorimetry (TGA‐DSC), ¹H NMR and UV spectra. The composition was suggested as EuL₅ · (ClO₄)₃ · 2H₂O and EuL₄ · phen(ClO₄)₃ · 2H₂O (L = C₁₀H₇COCH₂SOC₆H₅). The fluorescence spectra showed that the Eu(III) displayed strong characteristic metal‐centered fluorescence in the solid state. The ternary rare earth complex showed stronger fluorescence intensity than the binary rare earth complex in such material. The strongest characteristic fluorescence emission intensity of the ternary system was 1.49 times as strong as that of the binary system. The phosphorescence spectra were also discussed. Copyright © 2014 John Wiley & Sons, Ltd.     

SREBP‐1c,Pdx‐1, and GLP‐1R involved in palmitate–EPA regulated glucose‐stimulated insulin secretion in INS‐1 cells

Impairment of glucose‐stimulated insulin secretion (GSIS) caused by glucolipotoxicity is an essential feature in type 2 diabetes mellitus (T2DM). Palmitate and eicosapentaenoate (EPA), because of their lipotoxicity and protection effect, were found to impair or restore the GSIS in beta cells. Furthermore, palmitate was found to up‐regulate the expression level of sterol regulatory element‐binding protein (SREBP)‐1c and down‐regulate the levels of pancreatic and duodenal homeobox (Pdx)‐1 and glucagon‐like peptide (GLP)‐1 receptor (GLP‐1R) in INS‐1 cells. To investigate the underlying mechanism, the lentiviral system was used to knock‐down or over‐express SREBP‐1c and Pdx‐1, respectively. It was found that palmitate failed to suppress the expression of Pdx‐1 and GLP‐1R in SREBP‐1c‐deficient INS‐1 cells. Moreover, down‐regulation of Pdx‐1 could cause the low expression of GLP‐1R with/without palmitate treatment. Additionally, either SREBP‐1c down‐regulation or Pdx‐1 over‐expression could partially alleviate palmitate‐induced GSIS impairment. These results suggested that sequent SREBP‐1c‐Pdx‐1‐GLP‐1R signal pathway was involved in the palmitate‐caused GSIS impairment in beta cells. J. Cell. Biochem. 111: 634–642, 2010. © 2010 Wiley‐Liss, Inc.     

Anti‐adhesive property of P‐selectin glycoprotein ligand‐1 (PSGL‐1) due to steric hindrance effect

P‐selectin glycoprotein ligand‐1 (PSGL‐1) is an adhesive molecule that is known to be a ligand for P‐selectin. An anti‐adhesive property of PSGL‐1 has not been previously reported. In this study, we show that PSGL‐1 expression is anti‐adhesive for adherent cells and we have elucidated the underlying mechanism. Overexpression of PSGL‐1 induced cell rounding and floating in HEK293T cells. Similar phenomena were demonstrated in other adherent cell lines with overexpression of PSGL‐1. PSGL‐1 overexpression inhibits access of antibodies to cell surface molecules such as integrins, HLA and CD25. Cells transfected with PSGL‐1 deletion mutants that lack a large part of the extracellular domain and chimeric construct expressing extracellular CD86 and intracellular PSGL‐1 only showed rounded morphology, but there are no floating cells. These results indicated that PSGL‐1 causes steric hindrance due to the extended structure of its extracellular domain that is highly O‐glycosylated, but intracellular domain also has some effect on cell rounding. This study implies that PSGL‐1 has Janus‐faced functions, being both adhesive and anti‐adhesive. J. Cell. Biochem. 114: 1271–1285, 2013. © 2013 Wiley Periodicals, Inc.     

Grp1‐associated scaffold protein (GRASP) is a regulator of the ADP ribosylation factor 6 (Arf6)‐dependent membrane trafficking pathway

GRASP interacts with Grp1 (g eneral r eceptor for p hosphoinositides 1; cytohesin 3), which catalyses nucleotide exchange on and activation of Arf6 (ADP‐ribosylation factor‐6). Arf6 is a low‐molecular‐mass GTPase that regulates key aspects of endocytic recycling pathways. Overexpressed GRASP accumulated in the juxtanuclear ERC (endocytic recycling compartment). GRASP co‐localized with a constitutively inactive mutant of Arf6 in the ERC such that it was reversed by expression of wild‐type Grp1. Co‐expression of GRASP and Grp1 promoted membrane ruffling, a cellular hallmark of Arf6 activation. GRASP accumulation in ERC was found to block recycling of the MHC‐I (major histocompatibility complex‐I), which is trafficked by the Arf6‐dependent pathway. In contrast, overexpression of GRASP had no effect on the recycling of transferrin receptors, which are trafficked by a clathrin‐dependent pathway. The findings suggest that GRASP regulates the non‐clathrin/Arf6‐dependent, plasma membrane recycling and signalling pathways.     

CHIP interacts with heat shock factor 1 during heat stress

Heat shock factor 1 (HSF1) is a major transactivator of heat shock genes in response to stress and mediates cell protection against various harmful conditions. In this study, we identified the interaction of CHIP (carboxyl terminus of the heat shock cognate protein 70-interacting protein) with the N-terminus of HSF1. Using GST full-down assay, we found that CHIP directly interacts with C-terminal deleted HSF1 (a.a. 1-290) but not with full-length HSF1 under non-stressed conditions. Interestingly, interaction of CHIP with full-length HSF1 was induced by heat shock treatment. The structural change of HSF1 was observed under heat stressed conditions by CD spectra. These observations demonstrate the direct interaction between HSF1 and CHIP and this interaction requires conformational change of HSF1 by heat stress.     

Synthesis and Biological Evaluation of Novel 1‐(4‐(Hydroxy(1‐oxo‐1,3‐dihydro‐2H‐inden‐2‐ylidene)methyl)phenyl)‐3‐phenylurea Derivatives

A series of novel phenylurea containing 2‐benzoylindan‐1‐one derivatives 3a  –  3j were synthesized from the reaction of phenylurea‐substituted acetophenones 1a  –  1j with phthalaldehyde 2 under mild reaction conditions in good yields. All synthesized compounds were characterized by spectroscopic methods. The obtained compounds ( 3a  –  3j ) were evaluated for anticancer activity against HeLa and C6 cell lines. Antiproliferative activity was determined by the BrdU proliferation ELISA assay, 3f and 3g were found to be most active compounds. The compounds were also screened for antimicrobial activity and all compounds showed remarkable activity against used microorganisms.     

A cell‐survival factor (N‐acetyl‐L‐cysteine) alters the in vivo fate of constitutively proliferating subependymal cells in the adult forebrain

The adult mouse brain contains a population of constitutively proliferating subependymal cells that surround the lateral ventricle and are the direct progeny of the neural stem cell. Constitutively proliferating cells divide rapidly; 6 days after labeling, 60% of their progeny undergo cell death, 25% migrate to the olfactory bulbs, and 15% continue to proliferate within the subependyma. We have intraventricularly infused a cell survival factor N‐acetyl‐L ‐cysteine (NAC), which is known to have survival effects without concomitant proliferative effects on cells in vitro, and examined the resulting fate of cells spared from the normally occurring cell death. NAC infusion for 5 days results in a five‐fold increase in the number of retrovirally labeled subependymal cells compared to saline‐infused controls. The increase in the number of subependymal cells is directly proportional to the amount of time during which NAC is present and is not due to increased proliferation. While NAC is able to keep all the normally dying progeny alive, the cells spared from death remain confined to the subependyma lining the lateral ventricles and do not migrate to the olfactory bulbs (one normal fate of constitutively proliferating progeny) or into the surrounding brain parenchyma. When animals survive for an additional 6 days following NAC infusion, the number of retrovirally labeled subependymal cells returns to control values, indicating that the continued presence of NAC is necessary for cell survival. These data suggest that preventing cell death is not sufficient to keep all of the progeny of these cells in a proliferative mode. © 2000 John Wiley & Sons, Inc. J Neurobiol 42: 338–346, 2000     

Wnt inhibitory factor (WIF)‐1 promotes melanogenesis in normal human melanocytes

Wnt signaling plays a role in the differentiation as well as the development of melanocytes. Using a microarray analysis, hyperpigmentary skin of melasma expressed high levels of Wnt inhibitory factor‐1 (WIF‐1) compared with perilesional normal skin. In this study, the expression and functional roles of WIF‐1 on melanocytes were investigated. WIF‐1 was expressed both in the melanocytes of normal human skin and in cultured melanocytes. The upregulation of WIF‐1 on cultured normal human melanocytes significantly induced expressions of MITF and tyrosinase, which were associated with increased melanin content and tyrosinase activity. Consistent with the stimulatory effect of WIF‐1, WIF‐1 siRNA reduced melanogenesis in the cells. Moreover, WIF‐1 increases pigmentation in melanocytes co‐cultured with WIF‐1‐overexpressed fibroblasts and of organ‐cultured human skin. These findings suggest that melanocytes express WIF‐1 constitutively in vivo and in vitro and that WIF‐1 promotes melanogenesis in normal human melanocytes.     

Posttranslational oxidative modification of (R)‐2‐(2,4‐dichlorophenoxy)propionate/α‐ketoglutarate‐dependent dioxygenases (RdpA) leads to improved degradation of 2,4‐dichlorophenoxyacetate (2,4‐D)

Microbial activities and the versatility gained through adaptation to xenobiotic compounds are the main biological forces to counteract environmental pollution. The current results present a new adaptive mechanism that is mediated through posttranslational modifications. Strains of Delftia acidovorans incapable of growing autochthonously on 2,4‐dichlorophenoxyacetate (2,4‐D) were cultivated in a chemostat on 2,4‐D in the presence of (R)‐2‐(2,4‐dichlorophenoxy)propionate. Long‐term cultivation led to enhanced 2,4‐D degradation, as demonstrated by improved values of the Michaelis–Menten constant K_m for 2,4‐D and the catalytic efficiency k_cat/K_m of the initial degradative key enzyme (R)‐2‐(2,4‐dichlorophenoxy)propionate/α‐ketoglutarate‐dependent dioxygenases (RdpA). Analyses of the rdpA gene did not reveal any mutations, indicating a nongenetic mechanism of adaptation. 2‐DE of enzyme preparations, however, showed a series of RdpA forms varying in their pI. During adaptation increased numbers of RdpA variants were observed. Subsequent immunoassays of the RdpA variants showed a specific reaction with 2,4‐dinitrophenylhydrazine (DNPH), characteristic of carbonylation modifications. Together these results indicate that posttranslational carbonylation modified the substrate specificity of RdpA. A model was implemented explaining the segregation of clones with improved degradative activity within the chemostat. The process described is capable of quickly responding to environmental conditions by reversibly adapting the degradative potential to various phenoxyalkanoate herbicides.     

Green synthesis of enantiopure (S)‐1‐(benzofuran‐2‐yl)ethanol by whole‐cell biocatalyst

Optically active aromatic alcohols are valuable chiral building blocks of many natural products and chiral drugs. Lactobacillus paracasei BD87E6, which was isolated from a cereal‐based fermented beverage, was shown as a biocatalyst for the bioreduction of 1‐(benzofuran‐2‐yl) ethanone to (S)‐1‐(benzofuran‐2‐yl) ethanol with highly stereoselectivity. The bioreduction conditions were optimized using L. paracasei BD87E6 to obtain high enantiomeric excess (ee) and conversion. After optimization of the bioreduction conditions, it was shown that the bioreduction of 1‐(benzofuran‐2‐yl)ethanone was performed in mild reaction conditions. The asymmetric bioreduction of the 1‐(benzofuran‐2‐yl)ethanone had reached 92% yield with ee of higher than 99.9% at 6.73 g of substrate. Our study gave the first example for enantiopure production of (S)‐1‐(benzofuran‐2‐yl)ethanol by a biological green method. This process is also scalable and has potential in application. In this study, a basic and novel whole‐cell mediated biocatalytic method was performed for the enantiopure production of (S)‐1‐(benzofuran‐2‐yl)ethanol in the aqueous medium, which empowered the synthesis of a precious chiral intermediary process to be converted into a sophisticated molecule for drug production.