MicroRNA-205 affects mouse granulosa cell apoptosis and estradiol synthesis by targeting CREB1

MicroRNA-205 (miR-205) is involved in various physiological and pathological processes, but its biological function in follicular atresia remains unclear. In this study, we investigated miR-205 expression in mouse granulosa cells (mGCs) and analyzed its functions in primary mGCs by performing a series of in vitro experiments. Quantitative real-time polymerase chain reaction showed that miR-205 expression was significantly higher in early atretic follicles and progressively atretic follicles than in healthy follicles. miR-205 overexpression in mGCs significantly promoted apoptosis and caspase-3/9 activities, as well as inhibited estrogen (E2) release and cytochrome P450 family 19 subfamily A polypeptide 1 (CYP19A1, a key gene in E2 production) expression. Bioinformatics and luciferase reporter assays revealed that the gene encoding cyclic AMP response element (CRE)-binding protein 1 (CREB1) was a direct target of miR-205 in mGCs. CREB1 upregulation partially rescued the effects of miR-205 on apoptosis, caspase-3/9 activities, E2 production, and CYP19A1 expression on mGCs. These results indicate that miR-205 might play an important role in ovarian follicular development and provide new insights into follicular atresia     

The effect of the diameter of cyclic peptide nanotube on its chirality discrimination

In this work, the transport behaviors of the enantiomers of lactic acid (LA) in two cyclic peptide nanotubes (CPNTs) with different diameters were studied using steered molecular dynamic (SMD) simulation to investigate the effect of the diameter of CPNT on the discrimination of the enantiomers of LA. For this purpose, two cyclic peptides with two different sizes ([Ala-_D-Ala-_L]₅ and [Ala-_D-Ala-_L]₄) were used for constructing two CPNTs so that each CPNT was composed of eight cyclic peptide units. The docking calculations were performed to obtain the appropriate position of each enantiomer at the lumen of each CPNT. The variation of the pulling force versus time, exerted on the enantiomers moving in the CPNTs was calculated using the SMD simulations with two different strategies (positional and directional).The obtained results showed that the diameter of CPNT has considerable effect on the discrimination of the LA enantiomers so that the increase of the diameter of CPNT, increased the velocity difference between two enantiomers and improved the performance of CPNT for the chirality discrimination. The SMD simulations indicated that the velocity of S-enantiomer became more than R-enantiomer and its motion became more comfortable than R-enantiomer when the diameter of CNPT increased. The RDFs of the H and O atoms of the LA enantiomers relative to the O atoms of CPNT were calculated and it was found that the increase of the diameter of CPNT creates the significant changes in the RDFs of H1, H2 and H3 atoms of the enantiomers.     

Energy storage and optical cross-section of PS I in the cyanobacterium Synechococcus PCC 7002 and a psaE- mutant

In vivo electron flow in the unicellular cyanobacterium Synechococcus sp. PCC 7002 was studied by pulsed, time- resolved photoacoustics (PTRPA). Using 1-s, 2 J or 10¹³ hcm-2 pulses at 695 nm, we observed large (42 ± 2%) Photosystem I (PS I) cyclic energy storage (ES) in the period of 2 to 12 ms after excitation with wild type (WT) intact cells. This cyclic ES was insensitive to flash interval from 0.3 to 10 s and to the presence of 1 m DCMU (3-(3,4-dichlorophenyl)-1,1-dimethylurea). At this low flash energy and in the absence of continuous background light (in the dark), antimycin A, carbonylcyanide-m- chlorophenylhydrazone (CCCP), 2,5-dibromo-3-methyl-6-isopropyl- p-benzoquinone (DBMIB), DCMU, 2- n-heptyl-4- hydroxyquinoline-N-oxide (HQNO), myxothiazol and N- ethylmaleimide (NEM) caused little or no inhibition of PS I cyclic electron flow. When continuous background far-red light ( > 715 nm) was added during the measurement, strong inhibition by DBMIB and NEM and less by HQNO was observed, the amplitude of which was related to both concentration and the intensity of the background light. Analysis of the data with DBMIB yields its binding constant, 1 m, and the turnover time of the system (> 20 ms). A turnover time of the uninhibited system of 2–3 ms was obtained by a pump-probe method. A dramatic lifting of the partial inhibition in the presence of far-red light was caused by antimycin A and a smaller effect by myxothiazol. The rescuing effect was assigned to a short circuiting of the electron flow about the cytochrome (cyt) b6/f system. Progressively increasing the laser pulse energy allowed us to calculate the PS I optical cross-section (54 ± 2 Å). Analysis by the sensitive method of convolutions revealed a possible energy loss on the few ms time scale by antimycin A in the dark. The analysis also revealed a similar effect or artifact in uninhibited samples using the same sample illuminated with saturating continuous light, the standard procedure in photoacoustics (PA). A psaE- mutant showed more inhibition in the dark by DBMIB and with far-red light by HQNO, but less inhibition in the far-red light by myxothiazol than the WT. Under normal growth conditions, maximum ES for the psaE- mutant (38 ± 2%) was similar to that of the WT (42 ± 2%). However, under mild heat stress, maximum ES for the psaE- mutant dropped to 26% while the WT maximum ES stayed unchanged at 41%, within batch-to-batch variation. These results would indicate that the PsaE protein is not essential for PS I cyclic electron flow under our experimental conditions but plays a stabilizing role in the PS I complex under a mild thermal stress.     

An Analysis of the Mechanism of the Low-wave Phenomenon of Chlorophyll Fluorescence

The low-wave phenomenon, i.e., the transient drop of yield of modulated chlorophyll fluorescence shortly after application of a pulse of saturating light, was investigated in intact leaves of tobacco and Camellia by measuring fluorescence, CO(2) assimilation and absorption at 830 nm simultaneously. Limitations on linear electron flow, due to low electron acceptor levels that were induced by low CO(2), induced the low waves of chlorophyll fluorescence. Low-wave amplitudes obtained under different CO(2) concentrations and photon-flux densities yielded single-peak curves when plotted as functions of fluorescence parameters such as PhiPS II (quantum yield of Photosystem II) and qN (coefficient of non-photochemical quenching), suggesting that low-wave formation depends on the redox state of the electron transport chain. Low waves paralleled redox changes of P700, the reaction center of Photosystem I (PS I), and an additional electron flow through PS I was detected during the application of saturating pulses that induced low-waves. It is suggested that low waves of chlorophyll fluorescence are induced by increased non-photochemical quenching, as a result of the formation of a trans-thylakoid proton gradient due to cyclic electron flow around PS I.     

Bovine sperm capacitation: assessment of phosphodiesterase activity and intracellular alkalinization on capacitation-associated protein tyrosine phosphorylation

Mammalian sperm capacitation is the obligatory maturational process leading to the development of the fertilization-competent state. Heparin is known to be a unique species-specific inducer of bovine sperm capacitation in vitro and glucose a unique inhibitor of this induction. Heparin-induced capacitation of bovine sperm has been shown to correlate with protein kinase A (PKA)-dependent protein tyrosine phosphorylation driven by an increase in intracellular cAMP. This study examines the possible roles of cyclic nucleotide phosphodiesterase (PDE) activity and intracellular alkalinization on bovine sperm capacitation and the protein tyrosine phosphorylation associated with it. Measurement of whole cell PDE kinetics during capacitation reveals neither a substantial change with heparin nor one with glucose: PDE activity is effectively constitutive in maintaining intracellular cAMP levels during capacitation. In contrast to a transient increase in intracellular pH, a sustained increase in medium pH by switching from 5% CO(2)/95% air incubation to 1% CO(2)/99% air incubation over 4 hr in the absence of heparin resulted in an increase in protein tyrosine phosphorylation and in the extent of induced acrosome reaction comparable to that observed following heparin-induced capacitation in 5% CO(2). These results suggest that increased bicarbonate-dependent adenylyl cyclase activity, driven by alkalinization, increases intracellular cAMP and so increases PKA activity mediating protein tyrosine phosphorylation. Quantitative analysis of the lactic acid production rate by bovine sperm glycolysis accounts fully for intracellular acidification sufficient to offset heparin-induced alkalinization, thus inhibiting capacitation. The mechanism by which heparin uniquely induces intracellular alkalinization in bovine sperm leading to capacitation remains obscure, inviting future investigation.     

Depolarization-induced ERK phosphorylation depends on the cytosolic Ca2+ level rather than on the Ca2+ channel subtype of chromaffin cells

The contribution of Ca2+ entry through different voltage-activated Ca2+ channel (VACC) subtypes to the phosphorylation of extracellular signal regulated kinase (ERK) was examined in bovine adrenal-medullary chromaffin cells. High K+ depolarization (40 mM, 3 min) induced ERK phosphorylation, an effect that was inhibited by specific mitogen-activated protein kinase kinase inhibitors. By using selective inhibitors, we observed that depolarization-induced ERK phosphorylation completely depended on protein kinase C-alpha (PKC-alpha), but not on Ca2+/calmodulin-dependent protein kinase nor cyclic AMP-dependent protein kinase. Blockade of L-type Ca2+ channels by 3 microm furnidipine, or blockade of N channels by 1 micromomega-conotoxin GVIA reduced ERK phosphorylation by 70%, while the inhibition of P/Q channels by 1 micromomega-agatoxin IVA only caused a 40% reduction. The simultaneous blockade of L and N, or P/Q and N channels completely abolished this response, yet 23% ERK phosphorylation remained when L and P/Q channels were simultaneously blocked. Confocal imaging of cytosolic Ca2+ elevations elicited by 40 mm K+, showed that Ca2+ levels increased throughout the entire cytosol, both in the presence and the absence of Ca2+ channel blockers. Fifty-eight percent of the fluorescence rise depended on Ca2+ entering through N channels. Thus, ERK phosphorylation seems to depend on a critical level of Ca2+ in the cytosol rather than on activation of a given Ca2+ channel subtype.     

Contribution of bradykinin and nitric oxide to AT2 receptor-mediated differentiation in PC12 W cells

We investigated the effect of angiotensin II on intracellular cyclic GMP content and neurite outgrowth as an indicator of cell differentiation in PC12 W cells. Neurite outgrowth was examined by phase-contrast microscopy. Outgrown neurites were classified as small, medium and large, and were expressed as neurites per 100 cells. Angiotensin II (10-7 m) increased the outgrowth of medium and large neurites by mean +/- SEM 20.2 +/- 2.3 and 6.6 +/- 1.4 compared with 1.66 +/- 0.5 and 0.1 +/- 0.06 neurites per 100 cells in control. Cellular cyclic GMP content increased by 50-250% with angiotensin II at concentrations of 10-6-10-4 m. Both blockade of AT2 receptors and of nitric oxide synthase markedly reduced angiotensin II-induced neurite outgrowth and cyclic GMP production. In contrast, B2 receptor blockade had no effect or even increased these angiotensin II effects. Sodium nitroprusside and 8-bromo-cyclic GMP both mimicked the effects of angiotensin II on cell differentiation. The protein kinase G inhibitor KT-5823 inhibited the neurite outgrowth induced by both angiotensin II and 8-bromo-cyclic GMP. Our results demonstrate that angiotensin II can stimulate cell differentiation in PC12 W cells by nitric oxide-related and cyclic GMP-dependent mechanisms. The effects of angiotensin II on cell differentiation and cyclic GMP production were mediated via the AT2 receptor and further enhanced by bradykinin B2 receptor blockade.     

Spatio-temporal propagation of Ca2+ signals by cyclic ADP-ribose in 3T3 cells stimulated via purinergic P2Y receptors

The role of cyclic ADP-ribose in the amplification of subcellular and global Ca2+ signaling upon stimulation of P2Y purinergic receptors was studied in 3T3 fibroblasts. Either (1) 3T3 fibroblasts (CD38- cells), (2) 3T3 fibroblasts preloaded by incubation with extracellular cyclic ADP-ribose (cADPR), (3) 3T3 fibroblasts microinjected with ryanodine, or (4) 3T3 fibroblasts transfected to express the ADP-ribosyl cyclase CD38 (CD38+ cells) were used. Both preincubation with cADPR and CD38 expression resulted in comparable intracellular amounts of cyclic ADP-ribose (42.3 +/- 5.2 and 50.5 +/- 8.0 pmol/mg protein). P2Y receptor stimulation of CD38- cells yielded a small increase of intracellular Ca2+ concentration and a much higher Ca2+ signal in CD38-transfected cells, in cADPR-preloaded cells, or in cells microinjected with ryanodine. Confocal Ca2+ imaging revealed that stimulation of ryanodine receptors by cADPR or ryanodine amplified localized pacemaker Ca2+ signals with properties resembling Ca2+ quarks and triggered the propagation of such localized signals from the plasma membrane toward the internal environment, thereby initiating a global Ca2+ wave.