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.

     

Protein Malnutrition Induces Bone Marrow Mesenchymal Stem Cells Commitment to Adipogenic Differentiation Leading to Hematopoietic Failure

Protein malnutrition (PM) results in pathological changes that are associated with peripheral leukopenia, bone marrow (BM) hypoplasia and alterations in the BM microenvironment leading to hematopoietic failure; however, the mechanisms involved are poorly understood. In this context, the BM mesenchymal stem cells (MSCs) are cells intimately related to the formation of the BM microenvironment, and their differentiation into adipocytes is important because adipocytes are cells that have the capability to negatively modulate hematopoiesis. Two-month-old male Balb/c mice were subjected to protein-energy malnutrition with a low-protein diet containing 2% protein, whereas control animals were fed a diet containing 12% protein. The hematopoietic parameters and the expression of CD45 and CD117 positive cells in the BM were evaluated. MSCs were isolated from BM, and their capability to produce SCF, IL-3, G-CSF and GM-CSF were analyzed. The expression of PPAR-γ and C/EBP-α as well as the expression of PPAR-γ and SREBP mRNAs were evaluated in MSCs together with their capability to differentiate into adipocytes in vitro. The malnourished animals had anemia and leukopenia as well as spleen and bone marrow hypoplasia and a reduction in the expression of CD45 and CD117 positive cells from BM. The MSCs of the malnourished mice presented an increased capability to produce SCF and reduced production of G-CSF and GM-CSF. The MSCs from the malnourished animals showed increased expression of PPAR-γ protein and PPAR-γ mRNA associated with an increased capability to differentiate into adipocytes. The alterations found in the malnourished animals allowed us to conclude that malnutrition committed MSC differentiation leading to adipocyte decision and compromised their capacity for cytokine production, contributing to an impaired hematopoietic microenvironment and inducing the bone marrow failure commonly observed in protein malnutrition states.     

Synthesis of the same two proteins prior to larval diapause and pupation in the spruce budworm,Choristoneura fumiferana

Spruce budworm larvae produce large quantities of two proteins (Choristoneura fumiferana diapause associated proteins 1 and 2, CfDAP1 and CfDAP2) that are diapause related. These proteins appeared soon after hatching and increased in abundance, reaching maximum levels by four days into the 1st instar, and they remained at high levels until three days after the termination of diapause. These two proteins were purified to homogeneity and their NH2-terminal sequences were obtained. Oligonucleotide primers designed on the basis of these NH2-terminal sequences were used in RT-PCR to isolate the cDNA fragments coding for these proteins. These PCR fragments were then used as probes to isolate the cDNAs that contained the complete coding region. The 2.5kb mRNAs coding for these proteins started to appear 24hr after hatching and large quantities of these mRNAs were detected in 1st instar and 2nd instar larvae until the 2nd instar larvae entered diapause. Low levels of these mRNAs were detected in the 2nd instar larvae that were preparing to enter diapause, in those that were in diapause as well as in those that terminated diapause. Low levels of CfDAP1 mRNA were also detected on days 1 and 2 after ecdysis to the 3rd instar. However, no CfDAP1 and CfDAP2 mRNAs could be detected during the 4th and 5th instar larval stages. The mRNAs reappeared 24hr after the 5th instar larvae molted into the 6th instar and increased to reach maximum levels by 60hr after ecdysis. The mRNA levels remained high until 156hr after ecdysis into the 6th instar (36-48hr before pupal ecdysis), after which they disappeared once again. Immunocytochemical analyses showed that CfDAP1 protein was present in 2nd and 6th instar larval fat body but not in 5th instar larval fat body. Thus, the same two genes were expressed for the first time before C. fumiferana larvae entered diapause and for a 2nd time before pupation.     

TRANSFER CELLS IN THE PLACENTAL PAD AND CARYOPSIS COAT OF PAPPOPHORUM SUBBULBOSUM ARECH. (POACEAE)

During caryopsis development the layers of the pericarp, integuments, and nucellus all contribute to the formation of the caryopsis coat. The coat consists of a layer of outer pericarp epidermal transfer cells, a collapsed and senescent layer of middle pericarp cells, and a discontinuous layer of inner pericarp epidermal transfer cells. The latter is not present across the placental pad. The integuments are present as a collapsed dense layer, the nucellus is discontinuous and cellular. The placental pad occurs at the ventral surface of the caryopsis, opposite the scutellum and coleorhiza. It consists of 15–20 collapsed cell layers, including the pigment strand and placental vascular bundle. From the inside several partially collapsed cell layers of the nucellar projection occur which contain transfer-cell walls. The middle dense layers, the pigment strand, consist of the middle pericarp remnant, plus the remains of the placental vascular bundle. The pericarp inner epidermis does not extend across the pad. The aleurone layer is a continuous uniseriate layer around the entire caryopsis except at the placental pad; here it is crushed and contains the remnant of a transfer-cell wall. The outer pericarp epidermis is a continuous layer of transfer cells across the pad. These cells contain membranous inclusions suggesting that they may be functional during germination.     

Technical evaluation of nested PCR for the diagnosis of experimental sporotrichosis

BackgroundSporotrichosis caused by the dimorphic fungus Sporothrix schenckii can presents in a variety of clinical forms. Routine diagnosis is made by mycology and serology studies. Few investigations have been focused on the evaluation of the molecular diagnosis.AimTo determine the value of the nested PCR technique for the diagnosis of experimental sporotrichosis in organs of mice, and to compare the results with the established laboratory diagnostic procedures.MethodsBALB/c mice were inoculated with growing concentrations of the 2 morphological phases of the fungus. The infected animals were sacrificed one month later and specimens from liver, spleen, lung and testicle were obtained to perform wet mount, culture and molecular diagnosis by the nested PCR technique. Blood samples were obtained for determination of specific antibodies against S. schenckii by the double immunodiffusion procedure.ResultsThe pathogenicity observed with the different concentrations of the fungus inoculated and its isolation by culture, showed scarce differences in the study of specimens from organs infected with the 2 morphological phases of S. schenckii. Specimens from organs of mice inoculated with the mycelial phase when studied by wet mount and culture, showed a higher positivity (100 and 37.5%) than those from mice inoculated with the yeast phase (73 and 2%). However, diagnosis by the nested PCR molecular technique applied to the latter specimens showed a higher percentage of positivity (75%) and 43% of positive results coming from animals infected with the mycelial phase. Specific antibody detection was positive in 100% all groups of infected mice.ConclusionsIn the study of experimental sporotrichosis in mice, the culture, as well as the antibody detection, was an effective diagnostic procedure, while the nested PCR and microscopic studies had a lower diagnostic value.     

Influence of the H‐site residue 108 on human glutathione transferase P1‐1 ligand binding: Structure‐thermodynamic relationships and thermal stability

The effect of the Y108V mutation of human glutathione S‐transferase P1‐1 (hGST P1‐1) on the binding of the diuretic drug ethacrynic acid (EA) and its glutathione conjugate (EASG) was investigated by calorimetric, spectrofluorimetric, and crystallographic studies. The mutation Tyr 108 → Val resulted in a 3D‐structure very similar to the wild type (wt) enzyme, where both the hydrophobic ligand binding site (H‐site) and glutathione binding site (G‐site) are unchanged except for the mutation itself. However, due to a slight increase in the hydrophobicity of the H‐site, as a consequence of the mutation, an increase in the entropy was observed. The Y108V mutation does not affect the affinity of EASG for the enzyme, which has a higher affinity (K_d ～ 0.5 μM) when compared with those of the parent compounds, K ～ 13 μM, K ～ 25 μM. The EA moiety of the conjugate binds in the H‐site of Y108V mutant in a fashion completely different to those observed in the crystal structures of the EA or EASG wt complex structures. We further demonstrate that the ΔC_p values of binding can also be correlated with the potential stacking interactions between ligand and residues located in the binding sites as predicted from crystal structures. Moreover, the mutation does not significantly affect the global stability of the enzyme. Our results demonstrate that calorimetric measurements maybe useful in determining the preference of binding (the binding mode) for a drug to a specific site of the enzyme, even in the absence of structural information.     

Isolation of Sporothrix schenckii from environmental samples in Venezuela

The dimorphic fungus Sporothrix schenckii is the etiological agent of sporotrichosis, a subcutaneous mycosis frequently found in Latin America. The isolation of this fungus from the environment and other sources has been widely reported. Nevertheless, to our knowledge this fungus has not been isolated from the endemic areas of Venezuela. In studies related to a clinical case of sporotrichosis in "Colonia Tovar", produced by traumatism after manipulating soil samples, the fungus was isolated from the soil of that particular area. This is the first report of the isolation of S. schenckii from environmental sources in an endemic area of Venezuela.     

Studies of the Nucleopolyhedrovirus Infection Process in Insects by Using the Green Fluorescence Protein as a Reporter

A recombinant Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) expressing the green fluorescence protein (GFP) under the control of the AcMNPV polyhedrin promoter was constructed to study the spatial and temporal regulation of baculovirus infection in a permissive host. Larvae that ingested AcMNPV-GFP showed localized expression of GFP in the midgut epithelial cells, as well as hemocytes, at 24 h postinfection. The presence of fluorescence in these tissues indicated not only that the virus was replicating but also that the very late viral proteins were being synthesized. Secondary infection occurred within the tracheal cells throughout the body cavity, confirming earlier reports, and these foci of infection allowed entry of the virus into other tissues, such as the epidermis and the fat body.