Real-time, in vivo intracellular recordings of caterpillar-induced depolarization waves in sieve elements using aphid electrodes.

Plants propagate electrical signals in response to artificial wounding. However, little is known about the electrophysiological responses of the phloem to wounding, and whether natural damaging stimuli induce propagating electrical signals in this tissue. Here, we used living aphids and the direct current (DC) version of the electrical penetration graph (EPG) to detect changes in the membrane potential of Arabidopsis sieve elements (SEs) during caterpillar wounding. Feeding wounds in the lamina induced fast depolarization waves in the affected leaf, rising to maximum amplitude (c. 60 mV) within 2 s. Major damage to the midvein induced fast and slow depolarization waves in unwounded neighbor leaves, but only slow depolarization waves in non-neighbor leaves. The slow depolarization waves rose to maximum amplitude (c. 30 mV) within 14 s. Expression of a jasmonate-responsive gene was detected in leaves in which SEs displayed fast depolarization waves. No electrical signals were detected in SEs of unwounded neighbor leaves of plants with suppressed expression of GLR3.3 and GLR3.6. EPG applied as a novel approach to plant electrophysiology allows cell-specific, robust, real-time monitoring of early electrophysiological responses in plant cells to damage, and is potentially applicable to a broad range of plant-herbivore interactions.

Protease modulation of the activity of the epithelial sodium channel expressed in Xenopus oocytes.

We have investigated the effect of extracellular proteases on the amiloride-sensitive Na+ current (INa) in Xenopus oocytes expressing the three subunits alpha, beta, and gamma of the rat or Xenopus epithelial Na+ channel (ENaC). Low concentrations of trypsin (2 microg/ml) induced a large increase of INa within a few minutes, an effect that was fully prevented by soybean trypsin inhibitor, but not by amiloride. A similar effect was observed with chymotrypsin, but not with kallikrein. The trypsin-induced increase of INa was observed with Xenopus and rat ENaC, and was very large (approximately 20-fold) with the channel obtained by coexpression of the alpha subunit of Xenopus ENaC with the beta and gamma subunits of rat ENaC. The effect of trypsin was selective for ENaC, as shown by the absence of effect on the current due to expression of the K+ channel ROMK2. The effect of trypsin was not prevented by intracellular injection of EGTA nor by pretreatment with GTP-gammaS, suggesting that this effect was not mediated by G proteins. Measurement of the channel protein expression at the oocyte surface by antibody binding to a FLAG epitope showed that the effect of trypsin was not accompanied by an increase in the channel protein density, indicating that proteolysis modified the activity of the channel present at the oocyte surface rather than the cell surface expression. At the single channel level, in the cell-attached mode, more active channels were observed in the patch when trypsin was present in the pipette, while no change in channel activity could be detected when trypsin was added to the bath solution around the patch pipette. We conclude that extracellular proteases are able to increase the open probability of the epithelial sodium channel by an effect that does not occur through activation of a G protein-coupled receptor, but rather through proteolysis of a protein that is either a constitutive part of the channel itself or closely associated with it.

Mechanism of urinary calcium regulation by urinary magnesium and pH.

Urinary magnesium and pH are known to modulate urinary calcium excretion, but the mechanisms underlying these relationships are unknown. In this study, the data from 17 clinical trials in which urinary magnesium and pH were pharmacologically manipulated were analyzed, and it was found that the change in urinary calcium excretion is directly proportional to the change in magnesium excretion and inversely proportional to the change in urine pH; a regression equation was generated to relate these variables (R(2) = 0.58). For further exploration of these relationships, intravenous calcium chloride, magnesium chloride, or vehicle was administered to rats. Magnesium infusion significantly increased urinary calcium excretion (normalized to urinary creatinine), but calcium infusion did not affect magnesium excretion. Parathyroidectomy did not prevent this magnesium-induced hypercalciuria. The effect of magnesium loading on calciuria was still observed after treatment with furosemide, which disrupts calcium and magnesium absorption in the thick ascending limb, suggesting that the effect may be mediated by the distal nephron. The calcium channel TRPV5, normally present in the distal tubule, was expressed in Xenopus oocytes. Calcium uptake by TRPV5 was directly inhibited by magnesium and low pH. In summary, these data are compatible with the hypothesis that urinary magnesium directly inhibits renal calcium absorption, which can be negated by high luminal pH, and that this regulation likely takes place in the distal tubule.

The deubiquitinating enzyme AMSH3 is required for intracellular trafficking and vacuole biogenesis in Arabidopsis thaliana.

Ubiquitination, deubiquitination, and the formation of specific ubiquitin chain topologies have been implicated in various cellular processes. Little is known, however, about the role of ubiquitin in the development of cellular organelles. Here, we identify and characterize the deubiquitinating enzyme AMSH3 from Arabidopsis thaliana. AMSH3 hydrolyzes K48- and K63-linked ubiquitin chains in vitro and accumulates both ubiquitin chain types in vivo. amsh3 mutants fail to form a central lytic vacuole, accumulate autophagosomes, and mis-sort vacuolar protein cargo to the intercellular space. Furthermore, AMSH3 is required for efficient endocytosis of the styryl dye FM4-64 and the auxin efflux facilitator PIN2. We thus present evidence for a role of deubiquitination in intracellular trafficking and vacuole biogenesis.

Application of 1-methylcyclopropene, calcium chloride and calcium amino acid chelate on fresh-cut cantaloupe muskmelon

The objective of this work was to determine the effects of postharvest application of 1-methylcyclopropene (1-MCP) and two calcium salts, applied individually or combined, on firmness and visual quality of fresh-cut muskmelon stored in air, for 18 days. Two sets of fruits, one of them exposed to 1-MCP at 300 nL L-1, were cut into cubes, dipped in deionized water, or in 1% Ca solutions as CaCl2, or in calcium amino acid chelate (Ca-chelate), placed in clamshell containers, and stored in air at 5±1ºC and 90±5% RH, for 18 days. The assay was conducted using an entirely randomized design, with three replications, in a split plot array. Evaluation of visual appearance, color, flesh firmness, total soluble solids, titratable acidity, and pH was performed right after treatments, and every period of three days, up to eighteen days. Application of 1-MCP at 300 nL L-1, calcium chloride or Ca-chelate, or the combination 1-MCP and calcium, preserved initial freshness and reduced softening of the samples. Ca-chelate synergistically enhanced the effect of 1-MCP on firmness after nine days of storage, while calcium chloride improved firmness of the samples throughout storage. Ca-chelate may serve as an alternative for shelf life extension of cantaloupe fresh-cut muskmelon.

The CD8 beta polypeptide is required for the recognition of an altered peptide ligand as an agonist.

T cell activation is triggered by the specific recognition of cognate peptides presented by MHC molecules. Altered peptide ligands are analogs of cognate peptides which have a high affinity for MHC molecules. Some of them induce complete T cell responses, i.e. they act as agonists, whereas others behave as partial agonists or even as antagonists. Here, we analyzed both early (intracellular Ca2+ mobilization), and late (interleukin-2 production) signal transduction events induced by a cognate peptide or a corresponding altered peptide ligand using T cell hybridomas expressing or not the CD8 alpha and beta chains. With a video imaging system, we showed that the intracellular Ca2+ response to an altered peptide ligand induces the appearance of a characteristic sustained intracellular Ca2+ concentration gradient which can be detected shortly after T cell interaction with antigen-presenting cells. We also provide evidence that the same altered peptide ligand can be seen either as an agonist or a partial agonist, depending on the presence of CD8beta in the CD8 co-receptor dimers expressed at the T cell surface.

Mineralocorticoid receptor is essential for corticosteroid-induced up-regulation of L-type calcium currents in cultured neonatal cardiomyocytes.

Despite the fact that mineralocorticoid receptor (MR) antagonist drugs such as spironolactone and eplerenone reduce the mortality in heart failure patients, there is, thus far, no unambiguous demonstration of a functional role of MR in cardiac cells. The aim of this work was to investigate the activation pathway(s) mediating corticosteroid-induced up-regulation of cardiac calcium current (ICa). In this study, using neonatal cardiomyocytes from MR or glucocorticoid receptor (GR) knockout (KO) mice, we show that MR is essential for corticosteroid-induced up-regulation of ICa. This study provides the first direct and unequivocal evidence for MR function in the heart.

Effects of pioglitazone on renal calcium excretion.

Context: Glitazones increase fracture risk in long-term users and in postmenopausal women. Studies have demonstrated deleterious effects of glitazones on bone metabolism. Glitazones also have direct renal tubular effects increasing sodium reabsorption. We hypothesized that glitazones may also regulate renal calcium excretion. Design: In this double-blind, randomized, placebo-controlled, four-way, crossover study, we examined the effects of pioglitazone (45 mg/d for 6 wk) or placebo on renal calcium and phosphate excretion and PTH levels during different sodium intakes in 16 individuals (eight with type 2 diabetes and eight with essential hypertension). Results: Pioglitazone had no effect on corrected plasma calcium and phosphate levels but decreased significantly the alkaline phosphatase and PTH levels. Pioglitazone induced on average a 45% increase in urinary calcium excretion. The fractional excretion of calcium rose to the same extent, suggesting a glomerular filtration rate-independent effect. Sodium intake did not influence the calciuric effect of pioglitazone. Changes in diurnal and nocturnal calciuria were similar. There was no effect of pioglitazone on phosphate excretion. Conclusion: Pioglitazone decreases PTH levels and increases urinary calcium excretion, independently from changes in glomerular filtration rate and from the sodium load, suggesting an inhibitory effect of pioglitazone on the tubular reabsorption of calcium. These effects may contribute to the increased fracture risk with glitazone treatment.

Relationship between macrofauna, mineralogy and exchangeable calcium and magnesium in Cerrado Oxisols under pasture

The objective of this work was to assess the relationship between macrofauna, mineralogy and exchangeable calcium and magnesium in Cerrado Oxisols under pasture. Twelve collection points were chosen in the Distrito Federal and in Formosa municipality, Goiás state, Brazil, representing four soil groups with varied levels of calcium + magnesium and kaolinite/(kaolinite + gibbsite) ratios. Soil macrofauna was collected in triplicate at each collection point, and identified at the level of taxonomic groups. Macrofauna density showed correlation with contents of kaolinite, gibbsite and exchangeable Ca + Mg in the soils. Mineralogy and exchangeable Ca + Mg had significant effects on taxonomic groups and relative density of soil macrofauna. The termites (Isoptera) were more abundant in soils with low exchangeable Ca + Mg; earthworms (Oligochaeta), in soils with high levels of kaolinite; and Hemiptera and Coleoptera larvae were more abundant in gibbsitic soils with higher contents of total carbon.

Revisiting spatial distribution and biochemical composition of calcium-containing crystals in human osteoarthritic articular cartilage.

INTRODUCTION: Calcium-containing (CaC) crystals, including basic calcium phosphate (BCP) and calcium pyrophosphate dihydrate (CPP), are associated with destructive forms of osteoarthritis (OA). We assessed their distribution and biochemical and morphologic features in human knee OA cartilage. METHODS: We prospectively included 20 patients who underwent total knee replacement (TKR) for primary OA. CaC crystal characterization and identification involved Fourier-transform infra-red spectrometry and scanning electron microscopy of 8 to 10 cartilage zones of each knee, including medial and lateral femoral condyles and tibial plateaux and the intercondyle zone. Differential expression of genes involved in the mineralization process between cartilage with and without calcification was assessed in samples from 8 different patients by RT-PCR. Immunohistochemistry and histology studies were performed in 6 different patients. RESULTS: Mean (SEM) age and body mass index of patients at the time of TKR was 74.6 (1.7) years and 28.1 (1.6) kg/m², respectively. Preoperative X-rays showed joint calcifications (chondrocalcinosis) in 4 cases only. The medial femoro-tibial compartment was the most severely affected in all cases, and mean (SEM) Kellgren-Lawrence score was 3.8 (0.1). All 20 OA cartilages showed CaC crystals. The mineral content represented 7.7% (8.1%) of the cartilage weight. All patients showed BCP crystals, which were associated with CPP crystals for 8 joints. CaC crystals were present in all knee joint compartments and in a mean of 4.6 (1.7) of the 8 studied areas. Crystal content was similar between superficial and deep layers and between medial and femoral compartments. BCP samples showed spherical structures, typical of biological apatite, and CPP samples showed rod-shaped or cubic structures. The expression of several genes involved in mineralization, including human homolog of progressive ankylosis, plasma-cell-membrane glycoprotein 1 and tissue-nonspecific alkaline phosphatase, was upregulated in OA chondrocytes isolated from CaC crystal-containing cartilages. CONCLUSIONS: CaC crystal deposition is a widespread phenomenon in human OA articular cartilage involving the entire knee cartilage including macroscopically normal and less weight-bearing zones. Cartilage calcification is associated with altered expression of genes involved in the mineralisation process.

Regulation of the transforming growth factor beta-responsive transcription factor CTF-1 by calcineurin and calcium/calmodulin-dependent protein kinase IV.

Transforming growth factor beta (TGF-beta) is a pluripotent peptide hormone that regulates various cellular activities, including growth, differentiation, and extracellular matrix protein gene expression. We previously showed that TGF-beta induces the transcriptional activation domain (TAD) of CTF-1, the prototypic member of the CTF/NF-I family of transcription factors. This induction correlates with the proposed role of CTF/NF-I binding sites in collagen gene induction by TGF-beta. However, the mechanisms of TGF-beta signal transduction remain poorly understood. Here, we analyzed the role of free calcium signaling in the induction of CTF-1 transcriptional activity by TGF-beta. We found that TGF-beta stimulates calcium influx and mediates an increase of the cytoplasmic calcium concentration in NIH3T3 cells. TGF-beta induction of CTF-1 is inhibited in cells pretreated with thapsigargin, which depletes the endoplasmic reticulum calcium stores, thus further arguing for the potential relevance of calcium mobilization in TGF-beta action. Consistent with this possibility, expression of a constitutively active form of the calcium/calmodulin-dependent phosphatase calcineurin or of the calcium/calmodulin-dependent kinase IV (DeltaCaMKIV) specifically induces the CTF-1 TAD and the endogenous mouse CTF/NF-I proteins. Both calcineurin- and DeltaCaMKIV-mediated induction require the previously identified TGF-beta-responsive domain of CTF-1. The immunosuppressants cyclosporin A and FK506 abolish calcineurin-mediated induction of CTF-1 activity. However, TGF-beta still induces the CTF-1 TAD in cells treated with these compounds or in cells overexpressing both calcineurin and DeltaCaMKIV, suggesting that other calcium-sensitive enzymes might mediate TGF-beta action. These results identify CTF/NF-I as a novel calcium signaling pathway-responsive transcription factor and further suggest multiple molecular mechanisms for the induction of CTF/NF-I transcriptional activity by growth factors.

Fastidious intracellular bacteria as causal agents of community-acquired pneumonia.

Intracellular bacteria are common causes of community-acquired pneumonia that grow poorly or not at all on standard culture media and do not respond to beta-lactam antibiotic therapy. Apart from well-established agents of pneumonia such as Legionella pneumophila, Mycoplasma pneumoniae, Chlamydia pneumoniae, Chlamydia psittaci and Coxiella burnetii, some new emerging pathogens have recently been recognized, mainly Parachlamydia acanthamoebae and Simkania negevensis, two Chlamydia-related bacteria. Most of them are causes of benign and self-limited infections. However, they may cause severe pneumonia in some cases (i.e., Legionnaires' disease) and they may cause outbreaks representing a public health problem deserving prompt recognition and appropriate therapy. Although extrapulmonary manifestations are often present, no clinical features allow them to be distinguished from classical bacterial agents of pneumonia such as Streptococcus pneumoniae. Thus, specific molecular diagnostic tools are very helpful for early recognition of the offending bacteria, whereas serology often only allows retrospective or late diagnosis. Macrolides remain the best empirical treatment of intracellular respiratory pathogens, although some observational studies suggest that quinolones may be superior for the treatment of legionellosis.

A gain-of-function allele of TPC1 activates oxylipin biogenesis after leaf wounding in Arabidopsis.

Jasmonates, potent lipid mediators of defense gene expression in plants, are rapidly synthesized in response to wounding. These lipid mediators also stimulate their own production via a positive feedback circuit, which depends on both JA synthesis and JA signaling. To date, molecular components regulating the activation of jasmonate biogenesis and its feedback loop have been poorly characterized. We employed a genetic screen capable of detecting the misregulated activity of 13-lipoxygenase, which operates at the entry point of the jasmonate biosynthesis pathway. Leaf extracts from the Arabidopsis fou2 (fatty acid oxygenation upregulated 2) mutant displayed an increased capacity to catalyze the synthesis of lipoxygenase (LOX) metabolites. Quantitative oxylipin analysis identified less than twofold increased jasmonate levels in healthy fou2 leaves compared to wild-type; however, wounded fou2 leaves strongly increased jasmonate biogenesis compared to wounded wild-type. Furthermore, the plants displayed enhanced resistance to the fungus Botrytis cinerea. Higher than wild-type LOX activity and enhanced resistance in the fou2 mutant depend fully on a functional jasmonate response pathway. The fou2 mutant carries a missense mutation in the putative voltage sensor of the Two Pore Channel 1 gene (TPC1), which encodes a Ca(2+)-permeant non-selective cation channel. Patch-clamp analysis of fou2 vacuolar membranes showed faster time-dependent conductivity and activation of the mutated channel at lower membrane potentials than wild-type. The results indicate that cation fluxes exert strong control over the positive feedback loop whereby JA stimulates its own synthesis.

Identification of neuronal network properties from the spectral analysis of calcium imaging signals in neuronal cultures

Neuronal networks in vitro are prominent systems to study the development of connections in living neuronal networks and the interplay between connectivity, activity and function. These cultured networks show a rich spontaneous activity that evolves concurrently with the connectivity of the underlying network. In this work we monitor the development of neuronal cultures, and record their activity using calcium fluorescence imaging. We use spectral analysis to characterize global dynamical and structural traits of the neuronal cultures. We first observe that the power spectrum can be used as a signature of the state of the network, for instance when inhibition is active or silent, as well as a measure of the network's connectivity strength. Second, the power spectrum identifies prominent developmental changes in the network such as GABAA switch. And third, the analysis of the spatial distribution of the spectral density, in experiments with a controlled disintegration of the network through CNQX, an AMPA-glutamate receptor antagonist in excitatory neurons, reveals the existence of communities of strongly connected, highly active neurons that display synchronous oscillations. Our work illustrates the interest of spectral analysis for the study of in vitro networks, and its potential use as a network-state indicator, for instance to compare healthy and diseased neuronal networks.

Effets des supplémentations en calcium et vitamine D sur la maladie cardiovasculaire. [Effects of calcium and vitamin D supplementations on cardiovascular disease: review article].

Studies exploring the effect of calcium supplementation on cardiovascular risk suggest that systolic blood pressure decreases with supplementation. A lower calcium intake has been associated with an increased risk of stroke. By contrast, calcium supplementation may increase the risk of myocardial infarction. The effect of vitamin D supplementation on blood pressure is still unclear and no effect of vitamin D supplementation on coronary heart disease or stroke has been clearly demonstrated. There is a lack of randomized clinical trials primarily addressing the effect of these parameters on CVD. Currently, the use of calcium and vitamin D supplementations for the prevention of cardiovascular disease is not justified.