JAK2 inhibitor CEP-33779 prevents mouse oocyte maturation in vitro.

The inhibitor CEP-33779 is a specific selective inhibitor of Janus kinase 2 (JAK2). In most somatic cells, JAK2 plays essential roles in cellular signal transduction and in the regulation of cell cycle. Little is known regarding the effects of JAK2 on mammalian oocyte maturation. In the present study, we investigated the effects of CEP-33779 on mouse oocytes' meiosis and the possible mechanisms of JAK2 during mouse oocyte maturation. We detected the distribution of JAK2 during the mouse oocyte maturation. The results showed that JAK2 was mainly distributed in the cytoplasm during maturation. We cultured mouse oocytes with CEP-33779, examined the maturation rate, spindle morphology, and organization of microfilaments during the mouse oocyte maturation. While the rate of germinal vesicle breakdown (GVBD) did not differ between the treated and control groups, the rate of oocyte maturation decreased significantly when treated with CEP-33779. The rate of maturation was 21.14% in treated group and was 81.44% in control group. The results show that CEP-33779 inhibits the maturation of mouse oocytes. There was no obvious difference in the meiotic spindle morphology between the treated and control groups. The results show that CEP-33779 treatment did not disrupt the reorganization of microtubules. The microfilament observation shows that the microfilament did not form actin cap and the spindle stayed at the center of the oocyte in the treated group. CEP-33779 treatment inhibited the maturation of mouse oocytes which might be because of the disruption of formation of the actin cap. These results suggest that JAK2 regulated the microfilaments aggregation during the mouse oocyte maturation.

Volume-activated chloride currents in fetal human nasopharyngeal epithelial cells.

Volume-activated chloride channels have been studied by us extensively in human nasopharyngeal carcinoma cells. However, the chloride channels in the counterpart of the carcinoma cells have not been investigated. In this study, volume-activated chloride currents (I(cl,vol)) were characterized in normal fetal human nasopharyngeal epithelial cells using the whole-cell patch-clamp technique. Under isotonic conditions, nasopharyngeal epithelial cells displayed only a weak background current. Exposure to 47% hypotonic solution activated a volume-sensitive current. The reversal potential of the current was close to the calculated equilibrium potential for Cl(-). The peak values of the hypotonicity-activated current at +80 mV ranged from 0.82 to 2.71 nA in 23 cells. Further analysis indicated that the density of the hypotonicity-activated current in most cells (18/23) was smaller than 60 pA/pF. Only five cells presented a current larger than 60 pA/pF. The hypotonicity-activated current was independent of the exogenous ATP. Chloride channel inhibitors ATP, tamoxifen and 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB), inhibited the current dramatically. The anion permeability of the hypotonicity-activated chloride channels was I(-) > Br(-) > Cl(-) > gluconate. Unexpectedly, in isotonic conditions, ATP (10 mM) activated an inward-rectified current, which had not been observed in the nasopharyngeal carcinoma cells. These results suggest that, under hypotonic challenges, fetal human nasopharyngeal epithelial cells can produce I(cl,vol), which might be involved in cell volume regulation.

Binding analyses between Human PPARgamma-LBD and ligands.

The binding characteristics of a series of PPARgamma ligands (GW9662, GI 262570, cis-parinaric acid, 15-deoxy-Delta(12,14)-prostaglandin J(2), LY171883, indomethacin, linoleic acid, palmitic acid and troglitazone) to human PPARgamma ligand binding domain have been investigated for the first time by using surface plasmon resonance biosensor technology, CD spectroscopy and molecular docking simulation. The surface plasmon resonance biosensor determined equilibrium dissociation constants (KD values) are in agreement with the results reported in the literature measured by other methods, indicating that the surface plasmon resonance biosensor can assume a direct assay method in screening new PPARgamma agonists or antagonists. Conformational changes of PPARgamma caused by the ligand binding were detected by CD determination. It is interesting that the thermal stability of the receptor, reflected by the increase of the transition temperature (T(m)), was enhanced by the binding of the ligands. The increment of the transition temperature (DeltaT(m)) of PPARgamma owing to ligand binding correlated well with the binding affinity. This finding implies that CD could possibly be a complementary technology with which to determine the binding affinities of ligands to PPARgamma. Molecular docking simulation provided reasonable and reliable binding models of the ligands to PPARgamma at the atomic level, which gave a good explanation of the structure-binding affinity relationship for the ligands interacting with PPARgamma. Moreover, the predicted binding free energies for the ligands correlated well with the binding constants measured by the surface plasmon resonance biosensor, indicating that the docking paradigm used in this study could possibly be employed in virtual screening to discover new PPARgamma ligands, although the docking program cannot accurately predict the absolute ligand-PPARgamma binding affinity.

Cell cycle-dependent expression of volume-activated chloride currents in nasopharyngeal carcinoma cells.

Patch-clamping and cell image analysis techniques were used to study the expression of the volume-activated Cl(-) current, I(Cl(vol)), and regulatory volume decrease (RVD) capacity in the cell cycle in nasopharyngeal carcinoma cells (CNE-2Z). Hypotonic challenge caused CNE-2Z cells to swell and activated a Cl(-) current with a linear conductance, negligible time-dependent inactivation, and a reversal potential close to the Cl(-) equilibrium potential. The sequence of anion permeability was I(-) > Br(-) > Cl(-) > gluconate. The Cl(-) channel blockers tamoxifen, 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB), and ATP inhibited I(Cl(vol)). Synchronous cultures of cells were obtained by the mitotic shake-off technique and by a double chemical-block (thymidine and hydroxyurea) technique. The expression of I(Cl(vol)) was cell cycle dependent, being high in G(1) phase, downregulated in S phase, but increasing again in M phase. Hypotonic solution activated RVD, which was cell cycle dependent and inhibited by the Cl(-) channel blockers NPPB, tamoxifen, and ATP. The expression of I(Cl(vol)) was closely correlated with the RVD capacity in the cell cycle, suggesting a functional relationship. Inhibition of I(Cl(vol)) by NPPB (100 microM) arrested cells in G(0)/G(1). The data also suggest that expression of I(Cl(vol)) and RVD capacity are actively modulated during the cell cycle. The volume-activated Cl(-) current associated with RVD may therefore play an important role during the cell cycle progress.