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.

     

Murine interferon-gamma inducible protein-10 (IP-10) secreted by Lactococcus lactis chemo-attracts human CD3+ lymphocytes

Chemokines are members of the super family of cytokines necessary for leukocyte recruitment in tissues and lymphoid organs. The interferon-gamma inducible protein-10 (IP-10) chemo-attracts CXCR3-expressing cells, such as activated T lymphocytes and monocytes. We have genetically engineered a strain of Lactococcus lactis to secrete a biologically active murine IP-10 that interacts with human CXCR3, its homolog receptor, and chemo-attracts human CD3+ T lymphocytes.     

Differential localization of α’ and β subunits of protein kinase CK2 during rat spermatogenesis

Protein kinase CK2 is a serine/threonine kinase expressed in organisms from yeast to human and is composed of a catalytic subunit (α or α’) and a regulatory subunit (β) forming a holoenzyme with the possible subunit combinations α₂β₂, α’₂β₂, or αα’β₂. This kinase has been shown to be involved in embryonic development and gametogenesis. We have studied the expression of the CK2α’ and CK2β subunits during the first wave of spermatogenesis and in adult testis in the rat. Western blot analyses have demonstrated that both CK2α’ and CK2β are expressed in testes from birth to adulthood. A more detailed study of the protein localization of CK2α’ and CK2β by immunohistochemistry suggests that CK2α’ and CK2β are localized in the nuclei of Sertoli cells in 5-day-old rats, whereas they appear to have a cytoplasmic localization in older animals. In adult testes, CK2α’ and CK2β subunits are present in spermatocytes. Both subunits exhibit a similar expression pattern with the highest level in spermatocytes at stages VIII-XIV. Interestingly, CK2β is highly expressed in spermatogonia, whereas CK2α’ is barely detectable. Mature epididymal spermatozoa express CK2α’ in the acrosome and CK2β in the flagellum. This new evidence therefore indicates that protein kinase CK2 has a possible role at various stages during mammalian spermatogenesis, a process that involves proliferation, meiosis, apoptosis, and differentiation. CK2 might thus emerge as a new pivotal control enzyme at various levels in mammalian spermatogenesis.     

Essential oil composition of Bothriochloa Kuntze (Poaceae) from South America and their chemotaxonomy

The essential oils compositions of the 13 taxa (twelve species and two varities) of Bothriochloa have been studied. Two entities are endemics: Bothriochloa eurylemma and Bothriochloa meridionalis. The other nine taxa have disjunt distribution between North and South America and are representatives of two complexes: the Bothriochloa barbinodis complex and the Bothriochloa saccharoides complex. Multivariated statistical analysis (Principal Component Analysis, Hierarchical Cluster Analysis) applied to GC–MS data seems to have systematic significance for the delimitation of the species, independently of their morphological characteristics. However, some affinities with chromosome complement and mode of reproduction (amphimixis or sexuality) can be recognized.     

Evolutionarily conserved proteins MnmE and GidA catalyze the formation of two methyluridine derivatives at tRNA wobble positions

The wobble uridine of certain bacterial and mitochondrial tRNAs is modified, at position 5, through an unknown reaction pathway that utilizes the evolutionarily conserved MnmE and GidA proteins. The resulting modification (a methyluridine derivative) plays a critical role in decoding NNG/A codons and reading frame maintenance during mRNA translation. The lack of this tRNA modification produces a pleiotropic phenotype in bacteria and has been associated with mitochondrial encephalomyopathies in humans. In this work, we use in vitro and in vivo approaches to characterize the enzymatic pathway controlled by the Escherichia coli MnmE•GidA complex. Surprisingly, this complex catalyzes two different GTP- and FAD-dependent reactions, which produce 5-aminomethyluridine and 5-carboxymethylamino-methyluridine using ammonium and glycine, respectively, as substrates. In both reactions, methylene-tetrahydrofolate is the most probable source to form the C5-methylene moiety, whereas NADH is dispensable in vitro unless FAD levels are limiting. Our results allow us to reformulate the bacterial MnmE•GidA dependent pathway and propose a novel mechanism for the modification reactions performed by the MnmE and GidA family proteins.     

Dissection of a β‐barrel motif leads to a functional dimer: The case of the intestinal fatty acid binding protein

A lingering issue in the area of protein engineering is the optimal design of β motifs. In this regard, the framework provided by intestinal fatty acid binding protein (IFABP) was successfully chosen to explore the consequences on structure and function of the redesign of natural motifs. A truncated form of IFABP (Δ98Δ) served to illustrate the nonintuitive notion that the integrity of the β‐barrel can indeed be compromised with no effect on the ability to attain a native‐like fold. This is most likely the outcome of the key role played by the preservation of essential core residues. In the search for the minimal structural determinants of this fold, Δ98Δ offered room for further intervention. A dissection of this protein leads to a new abridged variant, Δ78Δ, containing 60% of the amino acids of IFABP. Spectroscopic analyses indicate that Δ78Δ retains substantial β‐sheet content and preserves tertiary interactions, displaying cooperative unfolding and binding activity. Most strikingly, this construct adopts a remarkably stable dimeric structure in solution. This phenomenon takes advantage of the inherent structural plasticity of this motif, likely profitting from edge‐to‐edge interactions between β‐sheets, whereas avoiding the most commonly occurring outcome represented by aggregation.     

Discovery and optimization of CRTH2 and DP dual antagonists

A series of phenylacetic acid derivatives was discovered as CRTH2 antagonists. Modification of the series led to compounds that are also antagonists of DP. Since activation of CRTH2 and DP are believed to play key roles in mediating responses of asthma and other immune diseases, this series was optimized to increase the dual antagonistic activities and improve pharmacokinetic properties. These efforts led to selection of AMG 009 as a clinical candidate.     

Optimization of a series of quinazolinone-derived antagonists of CXCR3

The evaluation of the CXCR3 antagonist AMG 487 in clinic trials was complicated due to the formation of an active metabolite. In this Letter, we will discuss the further optimization of the quinazolinone series that led to the discovery of compounds devoid of the formation of the active metabolite that was seen with AMG 487. In addition, these compounds also feature increased potency and good pharmacokinetic properties. We will also discuss the efficacy of the lead compound 34 in a mouse model of cellular recruitment induced by bleomycin.