Signal transduction by the cloned glucagon-like peptide-1 receptor: comparison with signaling by the endogenous receptors of beta cell lines.

Glucagon-like peptide-1 (GLP-1) is a gastrointestinal hormone that potentiates glucose-induced insulin secretion by pancreatic beta cells. The mechanisms of interaction between GLP-1 and glucose signaling pathways are not well understood. Here we studied the coupling of the cloned GLP-1 receptor, expressed in fibroblasts or in COS cells, to intracellular second messengers and compared this signaling with that of the endogenous receptor expressed in insulinoma cell lines. Binding of GLP-1 to the cloned receptor stimulated formation of cAMP with the same dose dependence and similar kinetics, compared with the endogenous receptor of insulinoma cells. Compared with forskolin-induced cAMP accumulation, that induced by GLP-1 proceeded with the same initial kinetics but rapidly reached a plateau, suggesting fast desensitization of the receptor. Coupling to the phospholipase C pathway was assessed by measuring inositol phosphate production and variations in the intracellular calcium concentration. No GLP-1-induced production of inositol phosphates could be measured in the different cell types studied. A rise in the intracellular calcium concentration was nevertheless observed in transfected COS cells but was much smaller than that observed in response to norepinephrine in cells also expressing the alpha 1B-adrenergic receptor. Importantly, no such increase in the intracellular calcium concentration could be observed in transfected fibroblasts or insulinoma cells, which, however, responded well to thrombin or carbachol, respectively. Together, our data show that interaction between GLP-1 and glucose signaling pathways in beta cells may be mediated uniquely by an increase in the intracellular cAMP concentration, with the consequent activation of protein kinase A and phosphorylation of elements of the glucose-sensing apparatus or of the insulin granule exocytic machinery.

Postnatal expression pattern of calcium-binding proteins in organotypic thalamic cultures and in the dorsal thalamus in vivo.

The present study describes the postnatal expression of calbindin, calretinin and parvalbumin and glutamic acid decarboxylase (GAD) and microtubule-associated protein 2 (MAP2) in organotypic monocultures of rat dorsal thalamus compared to the thalamus in vivo. Cultures were maintained for up to 7 weeks. Cortex-conditioned medium improved the survival of thalamic cultures. MAP2-immunoreactive material was present in somata and dendrites of small and large-sized neurons throughout the cultures. Parvalbumin immunoreactivity was present in larger multipolar or bitufted neurons along the edge of a culture. These neurons also displayed strong parvalbumin mRNA and GAD mRNA expression, and GABA immunoreactivity. They likely corresponded to cells of the nucleus reticularis thalami. Parvalbumin mRNA, but neither parvalbumin protein nor GAD mRNA, was expressed in neurons with large somata within the explant. They likely represented relay cells. GAD mRNA, but not parvalbumin mRNA, was expressed in small neurons within the explants. Small neurons also displayed calbindin- and calretinin-immunoreactivity. The small neurons likely represented local circuit neurons. The time course of expression of the calcium-binding proteins revealed that all were present at birth with the predicted molecular weights. A low, but constant parvalbumin expression was observed in vitro without the developmental increase seen in vivo, which most likely represented parvalbumin from afferent sources. In contrast, the explantation transiently downregulated the calretinin and calbindin expression, but the neurons recovered the expression after 14 and 21 days, respectively. In conclusion, thalamic monocultures older than three weeks represent a stable neuronal network containing well differentiated neurons of the nucleus reticularis thalami, relay cells and local circuit neurons.

Phosphate solubilization and synergism between P-solubilizing and arbuscular mycorrhizal fungi

The objective of this work was to evaluate the ability of several P-solubilizing fungi to solubilize aluminum phosphate and Araxá apatite as well as the synergism between the P-solubilizing fungus, PSF 7, and arbuscular mycorrhizal fungi to promote clover growth amended with aluminum phosphate. Two experiments were carried out, the first under laboratory conditions and the second in a controlled environmental chamber. In the first experiment, PSF 7, PSF 9, PSF 21 and PSF 22 isolates plus control were incubated in liquid medium at 28ºC for eight days. On the 2nd, 4th and 8th day of incubation, pH and soluble P were determined. In the second experiment, clover was sowed in plastic pots containing 300 g of sterilized substrate amended with aluminum phosphate, 3 g L-1, in presence and absence of PSF 7 isolate and arbuscular mycorrhizal fungi. A completely randomized design, in factorial outline 2x2 (presence and absence of PSF 7 and arbuscular mycorrhizal fungi) and five replicates were used. In the first experiment, higher P content was detected in the medium containing aluminum phosphate. PSF 7 is the best fungi isolate which increases aluminum solubilization with major tolerance to Al3+. Clover growth was stimulated by presence of PSF 7 and arbuscular mycorrhizal fungi. There is synergism between microorganisms utilized to improve plant nutrition.

Systemic exposure to tobramycin after local antibiotic treatment with calcium sulphate as carrier material.

INTRODUCTION: Osteoset(®) T is a calcium sulphate void filler containing 4% tobramycin sulphate, used to treat bone and soft tissue infections. Despite systemic exposure to the antibiotic, there are no pharmacokinetic studies in humans published so far. Based on the observations made in our patients, a model predicting tobramycin serum levels and evaluating their toxicity potential is presented. METHODS: Following implantation of Osteoset(®) T, tobramycin serum concentrations were monitored systematically. A pharmacokinetic analysis was performed using a non-linear mixed effects model based on a one compartment model with first-degree absorption. RESULTS: Data from 12 patients treated between October 2006 and March 2008 were analysed. Concentration profiles were consistent with the first-order slow release and single-compartment kinetics, whilst showing important variability. Predicted tobramycin serum concentrations depended clearly on both implanted drug amount and renal function. DISCUSSION AND CONCLUSION: Despite the popularity of aminoglycosides for local antibiotic therapy, pharmacokinetic data for this indication are scarce, and not available for calcium sulphate as carrier material. Systemic exposure to tobramycin after implantation of Osteoset(®) T appears reassuring regarding toxicity potential, except in case of markedly impaired renal function. We recommend in adapting the dosage to the estimated creatinine clearance rather than solely to the patient's weight.

Functional expression of PHO1 to the Golgi and trans-Golgi network and its role in export of inorganic phosphate.

Arabidopsis thaliana PHO1 is primarily expressed in the root vascular cylinder and is involved in the transfer of inorganic phosphate (Pi) from roots to shoots. To analyze the role of PHO1 in transport of Pi, we have generated transgenic plants expressing PHO1 in ectopic A. thaliana tissues using an estradiol-inducible promoter. Leaves treated with estradiol showed strong PHO1 expression, leading to detectable accumulation of PHO1 protein. Estradiol-mediated induction of PHO1 in leaves from soil-grown plants, in leaves and roots of plants grown in liquid culture, or in leaf mesophyll protoplasts, was all accompanied by the specific release of Pi to the extracellular medium as early as 2-3 h after addition of estradiol. Net Pi export triggered by PHO1 induction was enhanced by high extracellular Pi and weakly inhibited by the proton-ionophore carbonyl cyanide m-chlorophenylhydrazone. Expression of a PHO1-GFP construct complementing the pho1 mutant revealed GFP expression in punctate structures in the pericycle cells but no fluorescence at the plasma membrane. When expressed in onion epidermal cells or in tobacco mesophyll cells, PHO1-GFP was associated with similar punctate structures that co-localized with the Golgi/trans-Golgi network and uncharacterized vesicles. However, PHO1-GFP could be partially relocated to the plasma membrane in leaves infiltrated with a high-phosphate solution. Together, these results show that PHO1 can trigger Pi export in ectopic plant cells, strongly indicating that PHO1 is itself a Pi exporter. Interestingly, PHO1-mediated Pi export was associated with its localization to the Golgi and trans-Golgi networks, revealing a role for these organelles in Pi transport.

Economics of Using Calcium Chloride vs. Sodium Chloride for Deicing/Anti-Icing, TR-488, 2008

The use of chemicals is a critical part of a pro-active winter maintenance program. However, ensuring that the correct chemicals are used is a challenge. On the one hand, budgets are limited, and thus price of chemicals is a major concern. On the other, performance of chemicals, especially at lower pavement temperatures, is not always assured. Two chemicals that are used extensively by the Iowa Department of Transportation (Iowa DOT) are sodium chloride (or salt) and calcium chloride. While calcium chloride can be effective at much lower temperatures than salt, it is also considerably more expensive. Costs for a gallon of salt brine are typically in the range of $0.05 to $0.10, whereas calcium chloride brine may cost in the range of $1.00 or more per gallon. These costs are of course subject to market forces and will thus change from year to year. The idea of mixing different winter maintenance chemicals is by no means new, and in general discussions it appears that many winter maintenance personnel have from time to time mixed up a jar of chemicals and done some work around the yard to see whether or not their new mix “works.” There are many stories about the mixture turning to “mayonnaise” (or, more colorfully, to “snot”) suggesting that mixing chemicals may give rise to some problems most likely due to precipitation. Further, the question of what constitutes a mixture “working” in this context is a topic of considerable discussion. In this study, mixtures of salt brine and calcium chloride brine were examined to determine their ice melting capability and their freezing point. Using the results from these tests, a linear interpolation model of the ice melting capability of mixtures of the two brines has been developed. Using a criterion based upon the ability of the mixture to melt a certain thickness of ice or snow (expressed as a thickness of melt-water equivalent), the model was extended to develop a material cost per lane mile for the full range of possible mixtures as a function of temperature. This allowed for a comparison of the performance of the various mixtures. From the point of view of melting capacity, mixing calcium chloride brine with salt brine appears to be effective only at very low temperatures (around 0° F and below). However, the approach described herein only considers the material costs, and does not consider application costs or other aspects of the mixture performance than melting capacity. While a unit quantity of calcium chloride is considerably more expensive than a unit quantity of sodium chloride, it also melts considerably more ice. In other words, to achieve the same result, much less calcium chloride brine is required than sodium chloride brine. This is important in considering application costs, because it means that a single application vehicle (for example, a brine dispensing trailer towed behind a snowplow) can cover many more lane miles with calcium chloride brine than with salt brine before needing to refill. Calculating exactly how much could be saved in application costs requires an optimization of routes used in the application of liquids in anti-icing, which is beyond the scope of the current study. However, this may be an area that agencies wish to pursue for future investigation. In discussion with winter maintenance personnel who use mixtures of sodium chloride and calcium chloride, it is evident that one reason for this is because the mixture is much more persistent (i.e. it stays longer on the road surface) than straight salt brine. Operationally this persistence is very valuable, but at present there are not any established methods to measure the persistence of a chemical on a pavement. In conclusion, the study presents a method that allows an agency to determine the material costs of using various mixtures of salt brine and calcium chloride brine. The method is based upon the requirement of melting a certain quantity of snow or ice at the ice-pavement interface, and on how much of a chemical or of a mixture of chemicals is required to do that.

Prévalence et déterminants de l'hypophosphatémie sévère dans un service de Médecine interne

L'hypophosphatémie sévère définie comme une phosphatémie plasmatique < 0.32 mmol/l (Norme : 0.8-1-4 mmol/l) est associé à une morbidité et mortalité accrues. Il s'agit d'un trouble électrolytique dont la prévalence a été évaluée entre 0.24-0.42 % dans une population d'un hôpital général. Un nombre considérable de maladies et de situations cliniques ont été identifiées comme étant associées à une hypophosphatémie. Méthodologie Etude rétrospective chez les patients du service de médecine interne du Centre Hospitalier Universitaire Vaudois (CHUV, Lausanne) au cours de la période 2008-2010 qui ont présenté au moins un épisode de hypophosphatémie sévère définie comme une phosphatémie ≤ 0.35 mmol/l. Nous avons obtenu les données sur l'âge, le sexe, et les taux plasmatiques de : calcium, albumine, créatinine, bicarbonate veineux, glucose et acide urique.Nous avons étudié la prévalence de l'hypophosphatémie sévère et les pathologies associées. Comparaisondans une analyse cas-contrôles des caractéristiques des patients avec hypophosphatémie sévère et ceux ayant des valeurs de phosphate plasmatiques normales(0.8-1.4 mmol/l). Résultats La prévalence des patients ayant présenté au moins une valeur de phosphatémie ≤0.35 mmol/l pendant l'hospitalisation est de 1.3% sur les trois années cumulées. Chez les 84 patients avec hypophosphatémie sévère, la majorité présente plusieurs causes concomitantes. Nous avons retrouvé les étiologies suivantes : dénutrition sévère, 59.5% ; dialyse, 34.5% ; diarrhées chroniques, 23.8% ; consommation chronique d'alcool, 21.4% ; syndrome de renutrition inappropriée (refeeding syndrome), 20.3% et hyperparathyroïdisme primaire, 4.8%. L'hypokaliémie, l'hypocalcémie et l'hypomagnésémie sont positivement et significativement associées à l'hypophosphatémie. Conclusion La prévalence est rare dans un service de médecine interne, mais reste très probablement sous- diagnostiquée. Les patients à risque de développer ce trouble électrolytique sont les patients souffrant de dénutrition avec son pendant le syndrome de renutritioninappropriée et la consommation chronique d'alcool, et les patients sous dialyse.

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.

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.

Surface modification of biomaterials for conjugation with human fetal osteoblasts.

Surface functionalization of hydroxyapatite (HA) and beta-tricalcium phosphate (TCP) bioceramics with chemical ligands containing a pyrrogallol moiety was developed to improve the adhesion of bone cell precursors to the biomaterials. Fast and biocompatible copper-free click reaction with azido-modified human fetal osteoblasts resulted in improved cell binding to both HA and TCP bioceramics, opening the way for using this methodology in the preparation of cell-engineered bone implants.

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.

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.

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.

Dissection of local and systemic transcriptional responses to phosphate starvation in Arabidopsis.

Phosphate is a crucial and often limiting nutrient for plant growth. To obtain inorganic phosphate (P(i) ), which is very insoluble, and is heterogeneously distributed in the soil, plants have evolved a complex network of morphological and biochemical processes. These processes are controlled by a regulatory system triggered by P(i) concentration, not only present in the medium (external P(i) ), but also inside plant cells (internal P(i) ). A 'split-root' assay was performed to mimic a heterogeneous environment, after which a transcriptomic analysis identified groups of genes either locally or systemically regulated by P(i) starvation at the transcriptional level. These groups revealed coordinated regulations for various functions associated with P(i) starvation (including P(i) uptake, P(i) recovery, lipid metabolism, and metal uptake), and distinct roles for members in gene families. Genetic tools and physiological analyses revealed that genes that are locally regulated appear to be modulated mostly by root development independently of the internal P(i) content. By contrast, internal P(i) was essential to promote the activation of systemic regulation. Reducing the flow of P(i) had no effect on the systemic response, suggesting that a secondary signal, independent of P(i) , could be involved in the response. Furthermore, our results display a direct role for the transcription factor PHR1, as genes systemically controlled by low P(i) have promoters enriched with P1BS motif (PHR1-binding sequences). These data detail various regulatory systems regarding P(i) starvation responses (systemic versus local, and internal versus external P(i) ), and provide tools to analyze and classify the effects of P(i) starvation on plant physiology.

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.