Genome-wide identification and expression analysis of the OSCA gene family in Pyrus bretschneideri

Responses to osmotic change are critical for plant survival, development, and reproduction. Hyperosmolality-induced cytosolic free calcium concentration [(Ca2+)i] increase (OSCAs) proteins have been described as osmosensors in plants and animals. To investigate functional roles of OSCA genes (PbrOSCAs) in pear (Pyrus bretschneideri Rehd.), bioinformatics, and expression analyses of the PbrOSCAs were performed. Sixteen PbrOSCA members were identified in the pear genome. PbrOSCA family members were classified into four clades by sequence alignment and phylogenetic analysis. Moreover, protein structure analysis indicated that the 16 PbrOSCA members shared similar structures with their homologues in Arabidopsis and rice. Multi-transmembrane patterns and ion transport pore sites of PbrOSCAs were conserved, and expression profiles of PbrOSCA varied among tissues and with osmotic stress conditions. In particular, expression levels of six PbrOSCAs gradually increased with time during osmotic stress, suggesting that PbrOSCAs may play regulatory roles in plant osmotic stress responses.

The Lectin Chaperone Calnexin Is Involved in the Endoplasmic Reticulum Stress Response by Regulating Ca2+ Homeostasis in Aspergillus nidulans

ABSTRACT The Ca2+-mediated signaling pathway is crucial for environmental adaptation in fungi. Here we show that calnexin, a molecular chaperone located in the endoplasmic reticulum (ER), plays an important role in regulating the cytosolic free calcium concentration ([Ca2+]c) in Aspergillus nidulans. Inactivation of calnexin (ClxA) in A. nidulans caused severe defects in hyphal growth and conidiation under ER stress caused by the ER stress-inducing agent dithiothreitol (DTT) or high temperature. Importantly, defects in the ΔclxA mutant were restored by the addition of extracellular calcium. Furthermore, the CchA/MidA complex (the high-affinity Ca2+ channels), calcineurin (calcium/calmodulin-dependent protein phosphatase), and PmrA (secretory pathway Ca2+ ATPase) were required for extracellular calcium-based restoration of the DTT/thermal stress sensitivity in the ΔclxA mutant. Interestingly, the ΔclxA mutant exhibited markedly reduced conidium formation and hyphal growth defects under the low-calcium condition, which is similar to defects caused by mutations in MidA/CchA. Moreover, the phenotypic defects were further exacerbated in the ΔclxA ΔmidA ΔcchA mutant, which suggested that ClxA and MidA/CchA are both required under the calcium-limiting condition. Using the calcium-sensitive photoprotein aequorin to monitor [Ca2+]c in living cells, we found that ClxA and MidA/CchA complex synergistically coordinate transient increase in [Ca2+]c in response to extracellular calcium. Moreover, ClxA, in particular its luminal domain, plays a role in mediating the transient [Ca2+]c in response to DTT-induced ER stress in the absence of extracellular calcium, indicating ClxA may mediate calcium release from internal calcium stores. Our findings provide new insights into the role of calnexin in the regulation of calcium-mediated response in fungal ER stress adaptation. IMPORTANCE Calnexin is a well-known molecular chaperone conserved from yeast to humans. Although it contains calcium binding domains, little is known about the role of calnexin in Ca2+ regulation. In this study, we demonstrate that calnexin (ClxA) in the filamentous fungus Aspergillus nidulans, similar to the high-affinity calcium uptake system (HACS), is required for normal growth and conidiation under the calcium-limiting condition. The ClxA dysfunction decreases the transient cytosolic free calcium concentration ([Ca2+]c) induced by a high extracellular calcium or DTT-induced ER stress. Our findings provide the direct evidence that calnexin plays important roles in regulating Ca2+ homeostasis in addition to its role as a molecular chaperone in fungi. These results provide new insights into the roles of calnexin and expand knowledge of fungal stress adaptation.

Genomic and nongenomic stimulatory effect of aldosterone on H+-ATPase in proximal S3 segments

The genomic and nongenomic effects of aldosterone on the intracellular pH recovery rate (pHirr) via H+-ATPase and on cytosolic free calcium concentration ([Ca2+]i) were investigated in isolated proximal S3 segments of rats during superfusion with an Na+-free solution, by using the fluorescent probes BCECF-AM and FLUO-4-AM, respectively. The pHirr, after cellular acidification with a NH4Cl pulse, was 0.064 ± 0.003 pH units/min ( n = 17/74) and was abolished with concanamycin. Aldosterone (10−12, 10−10, 10−8, or 10−6 M with 1-h or 15- or 2-min preincubation) increased the pHirr. The baseline [Ca2+]i was 103 ± 2 nM ( n = 58). After 1 min of aldosterone preincubation, there was a transient and dose-dependent increase in [Ca2+]i and after 6-min preincubation there was a new increase in [Ca2+]i that persisted after 1 h. Spironolactone [mineralocorticoid (MR) antagonist], actinomycin D, or cycloheximide did not affect the effects of aldosterone (15- or 2-min preincubation) on pHirr and on [Ca2+]i but inhibited the effects of aldosterone (1-h preincubation) on these parameters. RU 486 [glucocorticoid (GR) antagonist] and dimethyl-BAPTA (Ca2+ chelator) prevented the effect of aldosterone on both parameters. The data indicate a genomic (1 h, via MR) and a nongenomic action (15 or 2 min, probably via GR) on the H+-ATPase and on [Ca2+]i. The results are compatible with stimulation of the H+-ATPase by increases in [Ca2+]i (at 10−12-10−6 M aldosterone) and inhibition of the H+-ATPase by decreases in [Ca2+]i (at 10−12 or 10−6 M aldosterone plus RU 486).

In Vitro Activity of Acanthamoeba castellanii on Human Platelets and Erythrocytes

ABSTRACT The effect of Acanthamoeba on human platelets and erythrocytes has not been fully elucidated. This paper reports that cell-free supernatants prepared from A. castellanii can activate human platelets, causing both a significant increase in the cytosolic free-calcium concentration and platelet aggregation. In addition, we demonstrated that platelet activation depends on the activity of ADP constitutively secreted into the medium by trophozoites. This study also showed that A. castellanii can affect human red blood cells, causing hemolysis, and provided evidence that hemolysis occurs in both contact-dependent and contact-independent ways; there are differences in kinetics, hemolytic activity, and calcium dependency between the contact-dependent and contact-independent mechanisms. Partial characterization of contact-independent hemolysis indicated that ADP does not affect the plasma membrane permeability of erythrocytes and that heat treatment of amoebic cell-free supernatant abolishes its hemolytic activity. These findings suggest that some heat-labile molecules released by A. castellanii trophozoites are involved in this phenomenon. Finally, our data suggest that human platelets and erythrocytes may be potential cell targets during Acanthamoeba infection.

Vitamin D derivatives acutely reduce endothelium-dependent contractions in the aorta of the spontaneously hypertensive rat

The available evidence suggests that vitamin D has cardiovascular effects besides regulating calcium homeostasis. To examine the effect of 1,25-dihydroxyvitamin D3, the major metabolite of vitamin D, on endothelium-dependent contractions, aortic rings of spontaneously hypertensive rats (SHR) were suspended in organ chambers for isometric force measurements. Rings were incubated with Nω-nitro-l-arginine methyl ester (l-NAME) and then exposed to increasing concentrations of acetylcholine, ATP, or the calcium ionophore to trigger contractions. This was done in the absence or presence of 1,25-dihydroxyvitamin D3. The release of prostacyclin after acetylcholine or A-23187 stimulation was also measured. The cytosolic-free calcium concentration was measured by confocal microscopy after incubation with the fluorescent dyes fluo-4 and fura red. The presence of vitamin D receptors was confirmed using immunohistochemistry. Acetylcholine- and ATP-induced endothelium-dependent contractions were significantly reduced compared with those obtained in the absence of the drug. This effect was not present if A-23187 was used as an agonist. The acetylcholine- but not the A-23187-induced release of prostacyclin was reduced by the acute administration of 1,25-dihydroxyvitamin D3. Exposure to 1,25-dihydroxyvitamin D3 reduced the increase in cytosolic-free calcium concentration caused by acetylcholine but not by A-23187 in cells. Vitamin D receptors were densely distributed in the endothelium. Inecalcitol (19-nor-14-epi-23-yne-1,25-dihydroxyvitamin D3), a synthetic analog of vitamin D, caused a comparable depression of endothelium-dependent contractions as 1,25-dihydroxyvitamin D3. These results demonstrate that vitamin D3 modulates vascular tone by reducing calcium influx into the endothelial cells and hence decreasing the production of endothelium-derived contracting factors.