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.

     

Cyclosporin A does not protect the disruption of the inner mitochondrial membrane potential induced by potassium ionophores in intact K562 cells

Mitochondrial dysfunction has been widely associated with programmed cell death. Studies of intact cells are important for the understanding of the process of cell death and its relation to mitochondrial physiology. Using cytofluorometric approaches we studied the mitochondrial behavior in an erythroleukemic cell line. The effects of protonophore carbonyl cyanide m-chlorophenylhydrazone (CCCP), potassium exchanger (nigericin), potassium ionophore (valinomycin), Na+K+-ATPase inhibitor (ouabain) and mitochondrial permeability transition pore inhibitor (cyclosporin A) were evaluated. Cyclosporin A (CSA) was very effective in attenuating the disruption of inner mitochondrial membrane potential induced by CCCP. However, CSA failed to protect the loss of inner mitochondrial membrane potential induced by potassium intracellular flux manipulation. Our findings suggest that mitochondrial cyclophilin is not involved in the cell events mediated by deregulation of potassium flux, underlining the need for further studies in intact tumor cells for a better understanding of the involvement of mitochondria physiology in cell death events.     

Genome based identification and analysis of the pre-replicative complex of Arabidopsis thaliana

Eukaryotic DNA replication requires an ordered and regulated machinery to control G1/S transition. The formation of the pre-replicative complex (pre-RC) is a key step involved in licensing DNA for replication. Here, we identify all putative components of the full pre-RC in the genome of the model plant Arabidopsis thaliana. Different from the other eukaryotes, Arabidopsis houses in its genome two putative homologs of ORC1, CDC6 and CDT1. Two mRNA variants of AtORC4 subunit, with different temporal expression patterns, were also identified. Two-hybrid binary interaction assays suggest a primary architectural organization of the Arabidopsis ORC, in which AtORC3 plays a central role in maintaining the complex associations. Expression profiles differ among pre-RC components suggesting the existence of various forms of the complex, possibly playing different roles during development. In addition, the expression of the putative pre-RC genes in non-proliferating plant tissues suggests that they might have roles in processes other than DNA replication licensing.