Alteration of stimulus-specific guard cell calcium oscillations and stomatal closing in Arabidopsis det3 mutant

Cytosolic calcium oscillations control signaling in animal cells, whereas in plants their importance remains largely unknown. In wild-type Arabidopsis guard cells abscisic acid, oxidative stress, cold, and external calcium elicited cytosolic calcium oscillations of differing amplitudes and frequencies and induced stomatal closure. In guard cells of the V-ATPase mutant det3, external calcium and oxidative stress elicited prolonged calcium increases, which did not oscillate, and stomatal closure was abolished. Conversely, cold and abscisic acid elicited calcium oscillations in det3, and stomatal closure occurred normally. Moreover, in det3 guard cells, experimentally imposing external calcium-induced oscillations rescued stomatal closure. These data provide genetic evidence that stimulus-specific calcium oscillations are necessary for stomatal closure.     

Multicolor and electron microscopic imaging of connexin trafficking

Recombinant proteins containing tetracysteine tags can be successively labeled in living cells with different colors of biarsenical fluorophores so that older and younger protein molecules can be sharply distinguished by both fluorescence and electron microscopy. Here we used this approach to show that newly synthesized connexin43 was transported predominantly in 100- to 150-nanometer vesicles to the plasma membrane and incorporated at the periphery of existing gap junctions, whereas older connexins were removed from the center of the plaques into pleiomorphic vesicles of widely varying sizes. Selective imaging by correlated optical and electron microscopy of protein molecules of known ages will clarify fundamental processes of protein trafficking in situ.     

Calcium rises abruptly and briefly throughout the cell at the onset of anaphase

Continuous measurement and imaging of the intracellular free calcium ion concentration ([Ca2+]i) of mitotic and interphase PtK1 cells was accomplished with the new fluorescent Ca2+ indicator fura-2. No statistically significant difference between basal [Ca2+]i of interphase and mitotic cells was detected. However, mitotic cells showed a rapid elevation of [Ca2+]i from basal levels of 130 nM to 500 to 800 nM at the metaphase-anaphase transition. The [Ca2+]i transient was brief, lasting approximately 20 seconds and the elevated [Ca2+]i appeared uniformly distributed over the entire spindle and central region of the cell. The close temporal association of the [Ca2+]i transient with the onset of anaphase suggests that calcium may have a signaling role in this event.     

Use of Porcine Luteinizing Hormone at Oestrous Onset in a Protocol for Fixed‐Time Artificial Insemination in Gilts

The aim of this study was to evaluate the effect of porcine luteinizing hormone (pLH) given at oestrous onset in gilts, by different routes and doses, on the interval between onset of oestrus and ovulation (IOEO) and reproductive performance using a single fixed‐time artificial insemination (FTAI). A total of 153 gilts were submitted to oestrous detection at 8‐h intervals and assigned to three groups: control – without hormone application and inseminated at 0, 24 and 48 h after oestrous onset; VS2.5FTAI – 2.5 mg pLH by the vulvar submucosal route at oestrous onset and a single FTAI 16 h later; IM5FTAI – 5 mg pLH by the intramuscular route at oestrous onset and a single FTAI 16 h later. More VS2.5FTAI gilts (47.1%; p < 0.05) ovulated within 24 h after oestrous onset than control gilts (25.5%) whereas IM5FTAI gilts had an intermediate percentage (31.4%; p > 0.05). The IOEO tended to be shorter (p = 0.06) in VS2.5FTAI (30.2 ± 1.4 h) than in control (34.7 ± 1.4 h) gilts, but there was no difference (p > 0.05) between control and IM5FTAI (32.8 ± 1.4 h) gilts. Farrowing rate was not different (p > 0.05) among treatments. Total born piglets (TB) was lower (p < 0.05) in VS2.5FTAI (12.3 ± 0.4) than in control gilts (14.1 ± 0.4), whereas intermediate TB was observed in IM5FTAI gilts (13.3 ± 0.4). Due to the advancement of ovulation, reduction of the hormonal dose and the ease of application, the vulvar submucosal route would be the best option for FTAI protocols, but their negative impact on litter size remains to be elucidated. Taking into account the good fertility results obtained in IM5FTAI gilts whose ovulation was not advanced, the possibility of a single FTAI without any hormonal treatment should be further investigated, to establish reliable FTAI protocols for gilts.     

Calcium channels in planar lipid bilayers: insights into mechanisms of ion permeation and gating

Electrophysiological recordings were used to analyze single calcium channels in planar lipid bilayers after membranes from bovine cardiac sarcolemmal vesicles had been incorporated into the bilayer. In these cell-free conditions, channels in the bilayer showed unitary barium or calcium conductances, gating kinetics, and pharmacological responses that were similar to dihydropyridine-sensitive calcium channels in intact cells. The open channel current varied in a nonlinear manner with voltage under asymmetric (that is, physiological) ionic conditions. However, with identical solutions on both sides of the bilayer, the current-voltage relation was linear. In matched experiments, calcium channels from skeletal muscle T-tubules differed significantly from cardiac calcium channels in their conductance properties and gating kinetics.     

腺胃病变型鸡传染性支气管炎的诊断研究

１９９５年以来在江苏一些市县养鸡场和养鸡专业户蛋鸡群流行以生长阻滞,剖检表现为腺胃显著肿大为特征的一种原因不明的“鸡传染性腺胃病“。通过病原分离、鉴定,血清学检测,病原免疫原基因分离、鉴定,病料组织核酸点杂交等方面诊断研究,证明本病是一种新近报道的腺胃病变型为特征的禽传染性支气管炎。     

Adrenaline: mechanism of action on the pacemaker potential in cardiac Purkinje fibers

The pacemaker potential in Purkinje fibers is generated by a slow fall in potassium current which allows the inward background currents to depolarize the membrane. Adrenaline shifts the relation between activation of the potassium current and membrane potential in a depolarizing direction. Consequently, during the pacemaker potential, the potassium current falls more rapidly to lower values and the inward currents then depolarize the membrane more quickly. The shift in the potassium activation curve produced by adrenaline is large compared to that produced by calcium ions. The molecular action of adrenaline may involve either a large change in the surface charge of the membrane or a change in the dependence of the potassium permeability on the local electric field.     

Fas preassociation required for apoptosis signaling and dominant inhibition by pathogenic mutations

Heterozygous mutations encoding abnormal forms of the death receptor Fas dominantly interfere with Fas-induced lymphocyte apoptosis in human autoimmune lymphoproliferative syndrome. This effect, rather than depending on ligand-induced receptor oligomerization, was found to stem from ligand- independent interaction of wild-type and mutant Fas receptors through a specific region in the extracellular domain. Preassociated Fas complexes were found in living cells by means of fluorescence resonance energy transfer between variants of green fluorescent protein. These results show that formation of preassociated receptor complexes is necessary for Fas signaling and dominant interference in human disease.     

Generation of calcium oscillations in fibroblasts by positive feedback between calcium and IP3

A wide variety of nonexcitable cells generate repetitive transient increases in cytosolic calcium ion concentration ([Ca2+]i) when stimulated with agonists that engage the phosphoinositide signalling pathway. Current theories regarding the mechanisms of oscillation disagree on whether Ca2+ inhibits or stimulates its own release from internal stores and whether inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DG) also undergo oscillations linked to the Ca2+ spikes. In this study, Ca2+ was found to stimulate its own release in REF52 fibroblasts primed by mitogens plus depolarization. However, unlike Ca2+ release in muscle and nerve cells, this amplification was insensitive to caffeine or ryanodine and required hormone receptor occupancy and functional IP3 receptors. Oscillations in [Ca2+]i were accompanied by oscillations in IP3 concentration but did not require functional protein kinase C. Therefore, the dominant feedback mechanism in this cell type appears to be Ca2+ stimulation of phospholipase C once this enzyme has been activated by hormone receptors.