Clock genes determination in whole blood in goats housed under a long light cycle.

An innate 24 h circadian clock drives various behavioral processes via expression of clock genes that regulate circadian rhythmicity and temporal signals. Elucidating the gene expression in goats may contribute to improving the knowledge of the regulation of circadian rhythms in this species. Five nonpregnant and nonlactating Maltese goats with no evidence of disease were kept in an indoor pen under the natural long photoperiod (05:05-20:56 h) and natural environmental temperature (23°C and 60% RH). They were fed an Alfalfa hay and concentrate mixture provided twice a day; water was available ad libitum. Blood samples were collected every 4 h over a 48 h period into PAX gene Blood RNA Tubes and stored at -80°C until processing. Clock genes (Clock; Cry1; Cry2; Per2; Per3) were determined using real-time quantitative polymerase chain reaction. During the experimental period, locomotor activity was monitored by an actigraphy-based data logger that records a digitally integrated measure of motor activity as a means to assess indices of discomfort during study and stability of the circadian rhythm. All of the tested genes showed daily rhythmicity in their expression in whole blood. Differences in their circadian parameters were observed. Mesor and amplitude were statistically different among the tested gene (Mesor: F(4.30) = 205.30; p < .0001; amplitude: F(4.30) = 104.80; p < .0001), with each gene showing its acrophase at a different time of day (F(4.30) = 81.17; p < .0001), and differences were observed between the two days of monitoring (F(1.30) = 10.25; p = .003). The application of two-way analysis of variance (ANOVA) on robustness of rhythm values did not show statistical differences among the tested genes (F(4.30) = 1.83; p = .14) and between the two days of monitoring (F(1.30) = 1.16; p = .28). Locomotor activity data recording were in accordance with the data reported in literature, indicating the absence of discomfort or alteration of circadian rhythms during the experimental period. Our results support the presence of a cyclic transcription of clock genes in whole blood of healthy goats housed under a long light natural photoperiod and natural environmental conditions.

Differentiation and characterization of rat adipose tissue mesenchymal stem cells into endothelial-like cells.

In this study, mesenchymal stem cells were isolated from rat adipose tissue (AD-MSCs) to characterize and differentiate them into endothelial-like cells. AD-MSCs were isolated by mechanical and enzymatic treatments, and their identity was verified by colony-forming units (CFU) test and by differentiation into cells of mesodermal lineages. The endothelial differentiation was induced by plating another aliquot of cells in EGM-2 medium, enriched with specific endothelial growth factors. Five subcultures were performed. The expression of stemness genes (OCT4, SOX2 and NANOG) was investigated. The presence of CD90 and the absence of the CD45 were evaluated by flow cytometry. The endothelial-like cells were characterized by the evaluation of morphological changes and gene expression analysis for endothelial markers (CD31, CD144, CD146). Characterization of AD-MSCs showed their ability to form clones, to differentiate in vitro and the OCT-4, SOX-2, NANOG genes expression. Immunophenotypic characterization showed the CD90 presence and the CD45 absence. The endothelial-like cells showed morphological changes, the expression of CD31, CD144, CD146 genes and the presence of CD31 membrane receptor. Matrigel assay showed their ability to form network and vessels-like structures. This study lays the foundations for future evaluation of the potential AD-MSCs pro-angiogenic and therapeutic role.

The effect of gamma-linolenic acid on clinical status, red cell fatty acid composition and membrane microviscosity in infants with atopic dermatitis.

A double blind placebo-controlled study of two doses of gamma-linolenic acid, provided by evening primrose oil (EPO, Epogam, Searle, U.K.), in children with atopic dermatitis was performed: 1) to examine the effect of gamma-linolenic acid administration on the clinical status of children with atopic dermatitis and abnormalities of IgE-mediated immune responses compared to those without such IgE abnormalities; 2) to investigate the effect of gamma-linolenic acid on red cell fatty acid composition and 3) to assess whether treatment with gamma-linolenic acid induced changes in red cell membrane microviscosity. A significant improvement in the overall severity of the clinical condition was seen in children treated with gamma-linolenic acid, independent of whether the children had manifestations of IgE-mediated allergy. Furthermore, gamma-linolenic acid treatment increased the percentage content of n-6 fatty acids in erythrocyte cell membrane; this increase was more marked in the membranes of children treated with high doses of EPO. In the high dose group a significant increase in dihomogamma-linolenic acid (DGLA) occurred. This may be of particular relevance because of the potential importance of DGLA as a precursor of antiinflammatory prostanoids. Red cell membrane microviscosity did not change in any group after treatment with EPO, even in high doses, despite a significant increase in the proportion of long chain polyunsaturated fatty acids.