d-Glycerate Transport by the Pea Chloroplast Glycolate Carrier : Studies on [1-C]d-Glycerate Uptake and d-Glycerate Dependent O(2) Evolution

Rapid direct conversion of exogenously supplied [¹⁴C]aspartate to [¹⁴C] asparagine and to tricarboxylic cycle acids was observed in alfalfa (Medicago sativa L.) nodules. Aspartate aminotransferase activity readily converted carbon from exogenously applied [¹⁴C]aspartate into the tricarboxylic acid cycle with subsequent conversion to the organic acids malate, succinate, and fumarate. Aminooxyacetate, an inhibitor of aminotransferase activity, reduced the flow of carbon from [¹⁴C]aspartate into tricarboxylic cycle acids and decreased ¹⁴CO₂ evolution by 99%. Concurrently, maximum conversion of aspartate to asparagine was observed in aminooxyacetate treated nodules (30 nanomoles asparagine per gram fresh weight per hour. Metabolism of [¹⁴C]aspartate and distribution of nodulefixed ¹⁴CO₂ suggest that two pools of aspartate occur in alfalfa nodules: (a) one involved in asparagine biosynthesis, and (b) another supplying a malate/aspartate shuttle. Conversion of [¹⁴C]aspartate to [¹⁴C]asparagine was not inhibited by methionine sulfoximine, a glutamine synthetase inhibitor, or azaserine, a glutmate synthetase, inhibitor. The data did not indicate that asparagine biosynthesis in alfalfa nodules has an absolute requirement for glutamine. Radioactivity in the xylem sap, derived from nodule ¹⁴CO₂ fixation, was markedly decreased by treating nodulated roots with aminooxyacetate, methionine sulfoximine, and azaserine. Inhibitors decreased the [¹⁴C]aspartate and [¹⁴]asparagine content of xylem sap by greater than 80% and reduced the total amino nitrogen content of xylem sap (including nonradiolabeled amino acids) by 50 to 80%. Asparagine biosynthesis in alfalfa nodules and transport in xylem sap are dependent upon continued aminotransferase activity and an uninterrupted assimilation of ammonia via the glutamine synthetase/glutamate synthase pathway. Continued assimilation of ammonia apparently appears crucial to continued root nodule CO₂ fixation in alfalfa.     

Glucagon signalling in the dorsal vagal complex is sufficient and necessary for high‐protein feeding to regulate glucose homeostasis in vivo

High‐protein feeding acutely lowers postprandial glucose concentration compared to low‐protein feeding, despite a dichotomous rise of circulating glucagon levels. The physiological role of this glucagon rise has been largely overlooked. We here first report that glucagon signalling in the dorsal vagal complex (DVC) of the brain is sufficient to lower glucose production by activating a Gcgr–PKA–ERK–K_ATP channel signalling cascade in the DVC of rats in vivo. We further demonstrate that direct blockade of DVC Gcgr signalling negates the acute ability of high‐ vs. low‐protein feeding to reduce plasma glucose concentration, indicating that the elevated circulating glucagon during high‐protein feeding acts in the brain to lower plasma glucose levels. These data revise the physiological role of glucagon and argue that brain glucagon signalling contributes to glucose homeostasis during dietary protein intake.     

Measurement of proton-linked transport activities in pyranine loaded chloroplast inner envelope vesicles

A method has been devised for loading chloroplast inner envelope vesicles prepared from pea (Pisum sativum L. var Progress No. 9) or spinach (Spinacia oleracea L.) with 8-hydroxypyrene-1,3,6-trisulfonate (pyranine), a membrane impermeant, fluorescent pH indicator. Two known proton-linked transport activities of the inner envelope, glycolate/H⁺ co-transport and phosphate/phosphoglycerate exchange have been shown to cause quenching of the internal pyranine fluorescence. This represents the first demonstration that these vesicles are sealed and competent for transport measurements. The technique, as it now stands, is essentially qualitative. It does, however, offer advantages over transport measurements with intact chloroplasts, for example compatibility with rapid mixing techniques and accessibility of the transport proteins to antibodies.     

Design and synthesis of compounds that extend yeast replicative lifespan

This past decade has seen the identification of numerous conserved genes that extend lifespan in diverse species, yet the number of compounds that extend lifespan is relatively small. A class of compounds called STACs, which were identified as activators of Sir2/SIRT1 NAD+-dependent deacetylases, extend the lifespans of multiple species in a Sir2-dependent manner and can delay the onset of age-related diseases such as cancer, diabetes and neurodegeneration in model organisms. Plant-derived STACs such as fisetin and resveratrol have several liabilities, including poor stability and relatively low potency as SIRT1 activators. To develop improved STACs, stilbene derivatives with modifications at the 4' position of the B ring were synthesized using a Horner-Emmons-based synthetic route or by hydrolyzing deoxyrhapontin. Here, we describe synthetic STACs with lower toxicity toward human cells, and higher potency with respect to SIRT1 activation and lifespan extension in Saccharomyces cerevisiae. These studies show that it is possible to improve upon naturally occurring STACs based on a number of criteria including lifespan extension.     

Solubilization, partial purification, and reconstitution of the glycolate/glycerate transporter from chloroplast inner envelope membranes

The glycolate/glycerate transporter of spinach (Spinacia oleracea L.) chloroplast inner envelope membranes was solubilized by treatment of the membranes with sodium cholate. Mixtures of the cholate extracts and soy asolectin were subjected to gel filtration to remove the detergent. The reconstituted vesicles were frozen, thawed, and sonicated in a buffer that contained 10 millimolar d-glycerate and, usually, [³H]sucrose as an internal space indicator. The dilution of the vesicles into a medium that contained 0.4 millimolar [¹⁴C]d-glycerate resulted in a rapid accumulation of labeled glycerate, followed by a much slower loss of [¹⁴C]d-glycerate from the vesicles. This behavior is characteristic of counterflow. The accumulation of [¹⁴C]d-glycerate was strongly inhibited by HgCl₂, which blocks glycolate/glycerate transport in intact chloroplasts. In the absence of proton ionophores, the extent of [¹⁴C]glycolate accumulation under similar conditions was much greater than that of [¹⁴C]d-glycerate. External glycolate inhibited d-glycerate counterflow and external d-glycerate inhibited glycolate counterflow. The external pH dependence of the efflux of [¹⁴C]d-glycerate accumulated in vesicles by counterflow and its inhibition by external l-mandelate are characteristics displayed by glycolate transport in intact chloroplasts. Partial purification of the transporter was achieved by glycerol gradient centrifugation. The solubilized glycolate and glycerate counterflow activities, assayed by reconstitution into vesicles, were found to sediment similarly.     

Eicosapentaenoic acid and docosahexaenoic acid reduce interleukin-1β-mediated cartilage degradation

Introduction  

In inflammatory joint disease, such as osteoarthritis (OA), there is an increased level of proinflammatory cytokines, such as interleukin (IL)-1β. These cytokines stimulate the production of matrix metalloproteinases (MMPs), which leads to the degradation of the cartilage extracellular matrix and the loss of key structural components such as sulphated glycosaminoglycan (sGAG) and collagen II. The aim of this study was to examine the therapeutic potential of n-3 polyunsaturated fatty acids (PUFAs) in an in vitro model of cartilage inflammation.     

Xenohormesis: sensing the chemical cues of other species

Many plant molecules interact with and modulate key regulators of mammalian physiology in ways that are beneficial to health, but why? We propose that heterotrophs (animals and fungi) are able to sense chemical cues synthesized by plants and other autotrophs in response to stress. These cues provide advance warning about deteriorating environmental conditions, allowing the heterotrophs to prepare for adversity while conditions are still favorable.