Dynamic ¹³C/¹ H NMR imaging uncovers sugar allocation in the living seed

Seed growth and accumulation of storage products relies on the delivery of sucrose from the maternal to the filial tissues. The transport route is hidden inside the seed and has never been visualized in vivo. Our approach, based on high‐field nuclear magnetic resonance and a custom made ¹³C/¹H double resonant coil, allows the non‐invasive imaging and monitoring of sucrose allocation within the seed. The new technique visualizes the main stream of sucrose and determines its velocity during the grain filling in barley (Hordeum vulgare L.). Quantifiable dynamic images are provided, which allow observing movement of ¹³C‐sucrose at a sub‐millimetre level of resolution. The analysis of genetically modified barley grains (Jekyll transgenic lines, seg8 and Risø13 mutants) demonstrated that sucrose release via the nucellar projection towards the endosperm provides an essential mean for the control of seed growth by maternal organism. The sucrose allocation was further determined by structural and metabolic features of endosperm. Sucrose monitoring was integrated with an in silico flux balance analysis, representing a powerful platform for non‐invasive study of seed filling in crops.     

Gustatory feedback affects feeding related motor pattern generation in starved 3rd instar larvae of Calliphora vicina

Gustatory feedback allows animals to distinguish between edible and noxious food and adapts centrally generated feeding motor patterns to environmental demands. In reduced preparations obtained from starved Calliphora larvae, putatively appetitive (ethanol), aversive (sodium acetate) and neutral (glucose) gustatory stimuli were applied to the anterior sense organs. The resulting sensory response was recorded from the maxillary- and antennal nerves. All three stimuli increased the neural activity in both nerves. Recordings obtained from the antennal nerve to monitor the activation pattern of the cibarial dilator muscles, demonstrated an effect of gustatory input on the central pattern generator for feeding. Ethanol consistently enhanced the rhythmic activity of the CDM motor neurons either by speeding up the rhythm or by increasing the burst duration. Ethanol also had an enhancing effect on the motor patterns of a protractor muscle which moves the cephalopharyngeal skeleton relative to the body. Sodium acetate showed a state dependent effect: in preparations without spontaneous CDM activity it initiated rhythmic motor patterns, while an ongoing CDM rhythm was inhibited. Surprisingly glucose had an enhancing effect which was less pronounced than that of ethanol. Gustatory feedback therefore can modify and adapt the motor output of the multifunctional central pattern generator for feeding.     

Subcellular heterogeneity of ryanodine receptor properties in ventricular myocytes with low T-tubule density

Rationale

In ventricular myocytes of large mammals, not all ryanodine receptor (RyR) clusters are associated with T-tubules (TTs); this fraction increases with cellular remodeling after myocardial infarction (MI).

Objective

To characterize RyR functional properties in relation to TT proximity, at baseline and after MI.

Methods

Myocytes were isolated from left ventricle of healthy pigs (CTRL) or from the area adjacent to a myocardial infarction (MI). Ca²⁺ transients were measured under whole-cell voltage clamp during confocal linescan imaging (fluo-3) and segmented according to proximity of TTs (sites of early Ca²⁺ release, F>F₅₀ within 20 ms) or their absence (delayed areas). Spontaneous Ca²⁺ release events during diastole, Ca²⁺ sparks, reflecting RyR activity and properties, were subsequently assigned to either category.

Results

In CTRL, spark frequency was higher in proximity of TTs, but spark duration was significantly shorter. Block of Na⁺/Ca²⁺ exchanger (NCX) prolonged spark duration selectively near TTs, while block of Ca²⁺ influx via Ca²⁺ channels did not affect sparks properties. In MI, total spark mass was increased in line with higher SR Ca²⁺ content. Extremely long sparks (>47.6 ms) occurred more frequently. The fraction of near-TT sparks was reduced; frequency increased mainly in delayed sites. Increased duration was seen in near-TT sparks only; Ca²⁺ removal by NCX at the membrane was significantly lower in MI.

Conclusion

TT proximity modulates RyR cluster properties resulting in intracellular heterogeneity of diastolic spark activity. Remodeling in the area adjacent to MI differentially affects these RyR subpopulations. Reduction of the number of sparks near TTs and reduced local NCX removal limit cellular Ca²⁺ loss and raise SR Ca²⁺ content, but may promote Ca²⁺ waves.     

Leishmania major: analysis of lymphocyte and macrophage cellular phenotypes during infection of susceptible and resistant mice

Genetically susceptible BALB/c and resistant C57BL/6 mice were infected with Leishmania major and the phenotypes of the responding cells in the draining lymph nodes and cutaneous lesions were analyzed. As early as 1 week, significantly increased numbers of L3T4+ cells as compared to Lyt-2+ cells were present in BALB/c mice lymph nodes (P less than 0.005). Increases in L3T4+ and Lyt-2+ cells were comparable in C57BL/6 mice, resulting in threefold lower L3T4/Lyt-2 ratio than in BALB/c mice. T cell subsets were activated in both strains to express interleukin-2 receptor (IL2R) above resting values, although greater numbers of activated L3T4+ cells were present in the draining lymph nodes from BALB/c at 1 and 3 weeks of infection than in C57BL/6 (P = 0.02). Despite the presence of activated L3T4+ cells in both strains, macrophages differed in the expression of immunologically important surface molecules during infection. Tissue macrophages from BALB/c mice were IgG1/G2b Fc receptor (FcR)+ and Ia- late in disease, whereas macrophages in C57BL/6 became FcR and Ia during healing. BALB/c mice, treated with monoclonal antibody GK1.5 to transiently deplete L3T4+ cells, became resistant to subsequent infection and developed a macrophage phenotype that was FcR- and Ia+. These differences in macrophage phenotype were closely linked to susceptibility during infection with L. major and may play a role in the pathophysiology of murine leishmaniasis.     

Interleukin 12 and the regulation of CD4+ T-cell subset responses during murine Leishmaniasis

Interleukin 12 is unique among cytokines in that it is capable of protecting genetically susceptible mice against progressive infection with Leishmania major. Because of the probable causal relationships between CD4(+) T-cell differentiation, cytokine production and disease outcome, this cytokine may prove useful as a component of cytokine-bosed therapies and Thl-selective vaccines, as discussed here by Frederick Heinzel.     

Ca2+/calmodulin-dependent cytochrome c reductase activity of brain nitric oxide synthase.

Nitric oxide acts as a widespread signal molecule and represents the endogenous activator of soluble guanylyl cyclase. In endothelial cells and brain tissue, NO is enzymatically formed from L-arginine by Ca2+/calmodulin-regulated NO synthases which require NADPH, tetrahydrobiopterin, and molecular oxygen as cofactors. Here we show that purified brain NO synthase binds to cytochrome c-agarose and exhibits superoxide dismutase-insensitive cytochrome c reductase activity with a Vmax of 10.2 mumol x mg-1 x min-1 and a Km of 34.1 microM. Cytochrome c reduction was largely dependent on Ca2+/calmodulin and cochromatographed with L-citrulline formation during gel filtration. When reconstituted with cytochrome P450, NO synthase induced a moderate Ca(2+)-independent hydroxylation of N-ethylmorphine. NO synthase also reduced the artificial electron acceptors nitro blue tetrazolium and 2,6-dichlorophenolindophenol. Cytochrome c, 2,6-dichlorophenolindophenol, and nitro blue tetrazolium inhibited NO synthase activity determined as formation of L-citrulline from 0.1 mM L-arginine in a concentration-dependent manner with half-maximal effects at 166, 41, and 7.3 microM, respectively. These results suggest that NO synthase may participate in cellular electron transfer processes and that a variety of electron-acceptors may interfere with NO formation due to the broad substrate specificity of the reductase domain of NO synthase.