Calcitonin gene related peptide mediates cardioprotection by remote preconditioning

Excitation of sensory nerves and activation of myocardial protein kinase C (PKC) epsilon contribute to the transduction of remote preconditioning (RPC) to the heart. Since calcitonin gene related peptide (CGRP) is an important mediator of sensory neurons we tried to delineate whether CGRP a) protects the heart from ischemic injury, b) is involved in cardioprotection after RPC, and c) leads to an activation of myocardial PKCepsilon. RPC was achieved by brief mesenteric artery occlusion followed by reperfusion. Myocardial infarct size (IS) was measured by TTC staining after temporary coronary artery occlusion (CAO) in rats. CGRP plasma levels were determined by radioimmunoassay and PKCepsilon was measured by quantitative immunoblotting. CGRP infusion reduced infarct size by 57%, an action that was abolished after co-treatment with the PKC inhibitor chelerythrine. RPC significantly increased CGRP plasma levels, reduced infarct size, and activated myocardial PKCepsilon. Infarct size reduction was abolished and PKCepsilon activation was significantly attenuated by CGRP(8-37), a specific CGRP receptor antagonist. Ganglion blockade with hexamethonium did not influence CGRP release by RPC but abolished CGRP mediated myocardial PKCepsilon activation. In conclusion, CGRP protects the heart from ischemic injury and is involved in RPC, presumably by activating myocardial PKCepsilon.     

Effects of high dietary fibre diets formulated from by-products from vegetable and agricultural industries on plasma metabolites in gestating sows

The aim of the present study was to examine the biochemical influence of feeding high dietary fibre (DF) diets formulated from by-products from the vegetable and agricultural industries to sows during early to mid-gestation. The effect of feeding frequency (once vs. twice daily) on diurnal plasma metabolites patterns was also examined. The study included a total of 48 gestating sows from four blocks (12 gestating sows in each block). The sows were fed four different diets containing varying levels of starch (304-519 g/kg dry matter (DM)) and DF (171-404 g/kg DM) but with equal amounts of net energy. The low-DF diet (control) was based on barley and wheat, and the three high-DF diets formulated by replacing barley and wheat by pectin residue, sugar beet pulp and potato pulp, respectively. The experimental design comprised two periods of 4 weeks each. Half the sows were fed once daily at 08:00 h in the first period and twice daily at 08:00 and 15:00 h during the second period, and vice versa for the other half of the sows. Plasma samples from vena jugularis were collected by venipuncture at 07:00, 09:00, 12:00 and 19:00 h. Feeding high-DF increased plasma short-chain fatty acids (p = 0.02) and non-esterified fatty acids (p < 0.001). However, there was no clear effect of DF on glucose and insulin responses. A negative correlation between amount of DF in the diets and plasma creatine (R2 = 1.00; diet effect: p = 0.02) suggested that plasma creatine concentrations was an indicator for the level of glucose-glycogen interchange. Furthermore, an explorative approach using nuclear magnetic resonance spectroscopy-based metabonomics identified betaine (p < 0.001), dimethyl sulfone (DMSO2; p < 0.001) and scyllo-inositol (p < 0.001) as biomarkers for the different by-products; pectin residue was related to high plasma levels of DMSO2, sugar beet pulp to plasma betaine, DMSO2 and scyllo-inositol, and potato pulp to plasma DMSO2 and scyllo-inositol. In conclusion, replacing starch by DF affected surprisingly few metabolites in peripheral plasma. No negative effects were found in feeding pectin residue, sugar beet pulp or potato pulp for gestating sows as judged from the minor metabolic changes.     

Unlocking the Bottleneck in Forward Genetics Using Whole-Genome Sequencing and Identity by Descent to Isolate Causative Mutations

Forward genetics screens with N-ethyl-N-nitrosourea (ENU) provide a powerful way to illuminate gene function and generate mouse models of human disease; however, the identification of causative mutations remains a limiting step. Current strategies depend on conventional mapping, so the propagation of affected mice requires non-lethal screens; accurate tracking of phenotypes through pedigrees is complex and uncertain; out-crossing can introduce unexpected modifiers; and Sanger sequencing of candidate genes is inefficient. Here we show how these problems can be efficiently overcome using whole-genome sequencing (WGS) to detect the ENU mutations and then identify regions that are identical by descent (IBD) in multiple affected mice. In this strategy, we use a modification of the Lander-Green algorithm to isolate causative recessive and dominant mutations, even at low coverage, on a pure strain background. Analysis of the IBD regions also allows us to calculate the ENU mutation rate (1.54 mutations per Mb) and to model future strategies for genetic screens in mice. The introduction of this approach will accelerate the discovery of causal variants, permit broader and more informative lethal screens to be used, reduce animal costs, and herald a new era for ENU mutagenesis.     

Cutting edge: the ontogeny and function of Va14Ja18 natural T lymphocytes require signal processing by protein kinase C theta and NF-kappa B

The rapid and robust immunoregulatory cytokine response of Va14Ja18 natural T (iNKT) cells to glycolipid Ags determines their diverse functions. Unlike conventional T cells, iNKT lymphocyte ontogeny absolutely requires NF-kappa B signaling. However, the precise role of NF-kappa B in iNKT cell function and the identity of upstream signals that activate NF-kappa B in this T cell subset remain unknown. Using mice in which iNKT cell ontogeny has been rescued despite inhibition of NF-kappa B signaling, we demonstrate that iNKT cell function requires NF-kappa B in a lymphocyte-intrinsic manner. Furthermore, the ontogeny of functional iNKT cells requires signaling through protein kinase C theta, which is dispensable for conventional T lymphocyte development. The unique requirement of protein kinase C theta implies that signals emanating from the TCR activate NF-kappa B during iNKT cell development and function. Thus, we conclude that NF-kappa B signaling plays a crucial role at distinct levels of iNKT cell biology.     

Catabolic and anabolic periarticular bone changes in patients with rheumatoid arthritis: a computed tomography study on the role of age,disease duration and bone markers

Introduction

The aim of this study was to determine the factors, including markers of bone resorption and bone formation, which determine catabolic and anabolic periarticular bone changes in patients with rheumatoid arthritis (RA).

Methods

Forty RA patients received high-resolution peripheral quantitative computed tomography (HR-pQCT) analysis of the metacarpophalangeal joints II and III of the dominantly affected hand at two sequential time points (baseline, one year follow-up). Erosion counts and scores as well as osteophyte counts and scores were recorded. Simultaneously, serum markers of bone resorption (C-terminal telopeptide of type I collagen (CTX I), tartrate-resistant acid phosphatase 5b (TRAP5b)), bone formation (bone alkaline phosphatase (BAP), osteocalcin (OC)) and calcium homeostasis (parathyroid hormone (PTH), 25-hydroxyvitamin D3 (Vit D)) were assessed. Bone biomarkers were correlated to imaging data by partial correlation adjusting for various demographic and disease-specific parameters. Additionally, imaging data were analyzed by mixed linear model regression.

Results

Partial correlation analysis showed that TRAP5b levels correlate significantly with bone erosions, whereas BAP levels correlate with osteophytes at both time points. In the mixed linear model with erosions as the dependent variable, disease duration (P <0.001) was the key determinant for these catabolic bone changes. In contrast, BAP (P = 0.001) as well as age (P = 0.018), but not disease duration (P = 0.762), were the main determinants for the anabolic changes (osteophytes) of the periarticular bone in patients with RA.

Conclusions

This study shows that structural bone changes assessed with HR-pQCT are accompanied by alterations in systemic markers of bone resorption and bone formation. Besides, it can be shown that bone erosions in RA patients depend on disease duration, whereas osteophytes are associated with age as well as serum level of BAP. Therefore, these data not only suggest that different variables are involved in formation of bone erosions and osteophytes in RA patients, but also that periarticular bone changes correlate with alterations in systemic markers of bone metabolism, pointing out BAP as an important parameter.     

Behavioral analyses of sugar processing in choice, feeding, and learning in larval Drosophila

Gustatory stimuli have at least 2 kinds of function: They can support immediate, reflexive responses (such as substrate choice and feeding) and they can drive internal reinforcement. We provide behavioral analyses of these functions with respect to sweet taste in larval Drosophila. The idea is to use the dose-effect characteristics as behavioral "fingerprints" to dissociate reflexive and reinforcing functions. For glucose and trehalose, we uncover relatively weak preference. In contrast, for fructose and sucrose, preference responses are strong and the effects on feeding pronounced. Specifically, larvae are attracted to, and feeding is stimulated most strongly for, intermediate concentrations of either sugar: Using very high concentrations (4 M) results in weakened preference and suppression of feeding. In contrast to such an optimum function regarding choice and feeding, an asymptotic dose-effect function is found for reinforcement learning: Learning scores reach asymptote at 2 M and remain stable for a 4-M concentration. A similar parametric discrepancy between the reflexive (choice and feeding) and reinforcing function is also seen for sodium chloride (Niewalda T, Singhal S, Fiala A, Saumweber T, Wegener S, Gerber B, in preparation). We discuss whether these discrepancies are based either on inhibition from high-osmolarity sensors upon specifically the reflexive pathways or whether different sensory pathways, with different effective dose-response characteristics, may have preferential access to drive either reflex responses or modulatory neurons mediating internal reinforcement, respectively.     

Metabolic profiling identifies trehalose as an abundant and diurnally fluctuating metabolite in the microalga <Emphasis Type="Italic">Ostreococcus tauri</Emphasis>

Introduction

The picoeukaryotic alga Ostreococcus tauri (Chlorophyta) belongs to the widespread group of marine prasinophytes. Despite its ecological importance, little is known about the metabolism of this alga.

Objectives

In this work, changes in the metabolome were quantified when O. tauri was grown under alternating cycles of 12 h light and 12 h darkness.

Methods

Algal metabolism was analyzed by gas chromatography-mass spectrometry. Using fluorescence-activated cell sorting, the bacteria associated with O. tauri were depleted to below 0.1% of total cells at the time of metabolic profiling.

Results

Of 111 metabolites quantified over light–dark cycles, 20 (18%) showed clear diurnal variations. The strongest fluctuations were found for trehalose. With an intracellular concentration of 1.6 mM in the dark, this disaccharide was six times more abundant at night than during the day. This fluctuation pattern of trehalose may be a consequence of starch degradation or of the synchronized cell cycle. On the other hand, maltose (and also sucrose) was below the detection limit (~10 μM). Accumulation of glycine in the light is in agreement with the presence of a classical glycolate pathway of photorespiration. We also provide evidence for the presence of fatty acid methyl and ethyl esters in O. tauri.

Conclusions

This study shows how the metabolism of O. tauri adapts to day and night and gives new insights into the configuration of the carbon metabolism. In addition, several less common metabolites were identified.

     

Ca2+-calmodulin-dependent facilitation and Ca2+ inactivation of Ca2+ release-activated Ca2+ channels

In non-excitable cells, one major route for Ca2+ influx is through store-operated Ca2+ channels in the plasma membrane. These channels are activated by the emptying of intracellular Ca2+ stores, and in some cell types store-operated influx occurs through Ca2+ release-activated Ca2+ (CRAC) channels. Here, we report that intracellular Ca2+ modulates CRAC channel activity through both positive and negative feedback steps in RBL-1 cells. Under conditions in which cytoplasmic Ca2+ concentration can fluctuate freely, we find that store-operated Ca2+ entry is impaired either following overexpression of a dominant negative calmodulin mutant or following whole-cell dialysis with a calmodulin inhibitory peptide. The peptide had no inhibitory effect when intracellular Ca2+ was buffered strongly at low levels. Hence, Ca2+-calmodulin is not required for the activation of CRAC channels per se but is an important regulator under physiological conditions. We also find that the plasma membrane Ca2+ATPase is the dominant Ca2+ efflux pathway in these cells. Although the activity of the Ca2+ pump is regulated by calmodulin, the store-operated Ca2+ entry is more sensitive to inhibition by the calmodulin mutant than by Ca2+ extrusion. Hence, these two plasmalemmal Ca2+ transport systems may differ in their sensitivities to endogenous calmodulin. Following the activation of Ca2+ entry, the rise in intracellular Ca2+ subsequently feeds back to further inhibit Ca2+ influx. This slow inactivation can be activated by a relatively brief Ca2+ influx (30-60 s); it reverses slowly and is not altered by overexpression of the calmodulin mutant. Hence, the same messenger, intracellular Ca2+, can both facilitate and inactivate Ca2+ entry through store-operated CRAC channels and through different mechanisms.