Contents of Volume 53

Plant Molecular Biology -     

Compartmentation of glutamate metabolism in brain. Evidence for the existence of two different tricarboxylic acid cycles in brain

1. (14)C from [1-(14)C]glucose injected intraperitoneally into mice is incorporated into glutamate, aspartate and glutamine in the brain to a much greater extent than (14)C from [2-(14)C]glucose. This difference for [1-(14)C]glucose and [2-(14)C]glucose increases with time. The amount of (14)C in C-1 of glutamate increases steadily with time with both precursors. It is suggested that a large part of the glutamate and aspartate pools in brain are in close contact with intermediates of a fast-turning tricarboxylic acid cycle. 2. (14)C from [1-(14)C]acetate and [2-(14)C]acetate is incorporated to a much larger extent into glutamine than into glutamate. An examination of the time-course of (14)C incorporated into glutamine and glutamate reveals that glutamine is not formed from the glutamate pool, labelled extensively by glucose, but from a small glutamate pool. This small glutamate pool is not derived from an intermediate of a fast-turning tricarboxylic acid cycle. 3. It is proposed that two different tricarboxylic acid cycles exist in brain.     

Effects of different branched-chain amino acids supplementation protocols on the inflammatory response of LPS-stimulated RAW 264.7 macrophages

Although branched-chain amino acids (BCAA) are commonly used as a strategy to recover nutritional status of critically ill patients, recent findings on their role as immunonutrients have been associated with unfavorable outcomes, especially in obese patients. The present study aimed to explore the effects of different BCAA supplementation protocols in the inflammatory response of LPS-stimulated RAW 264.7 macrophages. Cell cultures were divided into five groups, with and without BCAA supplementation, (2 mmol/L of each amino acid). Then, cell cultures followed three different treatment protocols, consisting of a pretreatment (PT), an acute treatment (AT), and a chronic treatment (CT) with BCAA and LPS stimulation (1 µg/mL). Cell viability was analyzed by MTT assay, NO production was assessed by the Griess reaction and IL-6, IL-10, TNF-α and PGE2 synthesis, was evaluated by ELISA. BCAA significantly increased cell viability in AT and CT protocols, and NO and IL-10 synthesis in all treatment protocols. IL-6 synthesis was only increased in PT and CT protocols. TNF-α and PGE2 synthesis were not altered in any of the protocols and groups. BCAA supplementation was able to increase both pro and anti-inflammatory mediators synthesis by RAW 264.7 macrophages, which was influenced by the protocol applied. Moreover, these parameters were significantly increased by isoleucine supplementation, highlighting a potential research field for future studies.

     

Diversity,structure and function of the coiled‐coil domains of plant NLR immune receptors

Plant nucleotide‐binding, leucine‐rich repeat receptors (NLRs) perceive pathogen avirulence effectors and activate defense responses. Nucleotide‐binding, leucine‐rich repeat receptors are classified into coiled‐coil (CC)‐containing and Toll/interleukin‐1 receptor (TIR)‐containing NLRs. Recent advances suggest that NLR CC domains often function in signaling activation, especially for induction of cell death. In this review, we outline our current understanding of NLR CC domains, including their diversity/classification and structure, their roles in cell death induction, disease resistance, and interaction with other proteins. Furthermore, we provide possible directions for future work.     

Alpha-Helical Destabilization of the Bcl-2-BH4-Domain Peptide Abolishes Its Ability to Inhibit the IP3 Receptor

The anti-apoptotic Bcl-2 protein is the founding member and namesake of the Bcl-2-protein family. It has recently been demonstrated that Bcl-2, apart from its anti-apoptotic role at mitochondrial membranes, can also directly interact with the inositol 1,4,5-trisphosphate receptor (IP₃R), the primary Ca²⁺-release channel in the endoplasmic reticulum (ER). Bcl-2 can thereby reduce pro-apoptotic IP₃R-mediated Ca²⁺ release from the ER. Moreover, the Bcl-2 homology domain 4 (Bcl-2-BH4) has been identified as essential and sufficient for this IP₃R-mediated anti-apoptotic activity. In the present study, we investigated whether the reported inhibitory effect of a Bcl-2-BH4 peptide on the IP ₃R1 was related to the distinctive α-helical conformation of the BH4 domain peptide. We therefore designed a peptide with two glycine “hinges” replacing residues I14 and V15, of the wild-type Bcl-2-BH4 domain (Bcl-2-BH4-IV/GG). By comparing the structural and functional properties of the Bcl-2-BH4-IV/GG peptide with its native counterpart, we found that the variant contained reduced α-helicity, neither bound nor inhibited the IP ₃R1 channel, and in turn lost its anti-apoptotic effect. Similar results were obtained with other substitutions in Bcl-2-BH4 that destabilized the α-helix with concomitant loss of IP₃R inhibition. These results provide new insights for the further development of Bcl-2-BH4-derived peptides as specific inhibitors of the IP₃R with significant pharmacological implications.     

Comparing two fish sampling standards over time: largely congruent results but with caveats

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Modulation of cytosolic calcium signaling by protein kinase A-mediated phosphorylation of inositol 1,4,5-trisphosphate receptors

Calcium release via intracellular Ca2+ release channels is a central event underpinning the generation of numerous, often divergent physiological processes. In electrically non-excitable cells, this Ca2+ release is brought about primarily through activation of inositol 1,4,5-trisphosphate receptors and typically takes the form of calcium oscillations. It is widely believed that information is carried in the temporal and spatial characteristics of these signals. Furthermore, stimulation of individual cells with different agonists can generate Ca2+ oscillations with dramatically different spatial and temporal characteristics. Thus, mechanisms must exist for the acute regulation of Ca2+ release such that agonist-specific Ca2+ signals can be generated. One such mechanism by which Ca2+ signals can be modulated is through simultaneous activation of multiple second messenger pathways. For example, activation of both the InsP3 and cAMP pathways leads to the modulation of Ca2+ release through protein kinase A mediated phosphoregulation of the InsP3R. Indeed, each InsP3R subtype is a potential substrate for PKA, although the functional consequences of this phosphorylation are not clear. This review will focus on recent advances in our understanding of phosphoregulation of InsP3R, as well as the functional consequences of this modulation in terms of eliciting specific cellular events.