IL-10 is excluded from the functional cytokine memory of human CD4+ memory T lymphocytes

Epigenetic modifications, including DNA methylation, profoundly influence gene expression of CD4(+) Th-specific cells thereby shaping memory Th cell function. We demonstrate here a correlation between a lacking fixed potential of human memory Th cells to re-express the immunoregulatory cytokine gene IL10 and its DNA methylation status. Memory Th cells secreting IL-10 or IFN-gamma were directly isolated ex vivo from peripheral blood of healthy volunteers, and the DNA methylation status of IL10 and IFNG was assessed. Limited difference in methylation was found for the IL10 gene locus in IL-10-secreting Th cells, as compared with Th cells not secreting IL-10 isolated directly ex vivo or from in vitro-established human Th1 and Th2 clones. In contrast, in IFN-gamma(+) memory Th cells the promoter of the IFNG gene was hypomethylated, as compared with IFN-gamma-nonsecreting memory Th cells. In accordance with the lack of epigenetic memory, almost 90% of ex vivo-isolated IL-10-secreting Th cells lacked a functional memory for IL-10 re-expression after restimulation. Our data indicate that IL10 does not become epigenetically marked in human memory Th cells unlike effector cytokine genes such as IFNG. The exclusion of IL-10, but not effector cytokines, from the functional memory of human CD4(+) T lymphocytes ex vivo may reflect the need for appropriate regulation of IL-10 secretion, due to its potent immunoregulatory potential.     

Procedure for Human Saphenous Veins Ex Vivo Perfusion and External Reinforcement

The mainstay of contemporary therapies for extensive occlusive arterial disease is venous bypass graft. However, its durability is threatened by intimal hyperplasia (IH) that eventually leads to vessel occlusion and graft failure. Mechanical forces, particularly low shear stress and high wall tension, are thought to initiate and to sustain these cellular and molecular changes, but their exact contribution remains to be unraveled. To selectively evaluate the role of pressure and shear stress on the biology of IH, an ex vivo perfusion system (EVPS) was created to perfuse segments of human saphenous veins under arterial regimen (high shear stress and high pressure). Further technical innovations allowed the simultaneous perfusion of two segments from the same vein, one reinforced with an external mesh. Veins were harvested using a no-touch technique and immediately transferred to the laboratory for assembly in the EVPS. One segment of the freshly isolated vein was not perfused (control, day 0). The two others segments were perfused for up to 7 days, one being completely sheltered with a 4 mm (diameter) external mesh. The pressure, flow velocity, and pulse rate were continuously monitored and adjusted to mimic the hemodynamic conditions prevailing in the femoral artery. Upon completion of the perfusion, veins were dismounted and used for histological and molecular analysis. Under ex vivo conditions, high pressure perfusion (arterial, mean = 100 mm Hg) is sufficient to generate IH and remodeling of human veins. These alterations are reduced in the presence of an external polyester mesh.     

ICER-1gamma overexpression drives palmitate-mediated connexin36 down-regulation in insulin-secreting cells

Channels formed by the gap junction protein connexin36 (Cx36) contribute to the proper control of insulin secretion. We investigated the impact of chronic hyperlipidemia on Cx36 expression in pancreatic beta-cells. Prolonged exposure to the saturated free fatty acid palmitate reduced the expression of Cx36 in several insulin-secreting cell lines and isolated mouse islets. The effect of palmitate was fully blocked upon protein kinase A (PKA) inhibition by H89 and (Rp)-cAMP, indicating that the cAMP/PKA pathway is involved in the control of Cx36 expression. Palmitate treatment led to overexpression of the inducible cAMP early repressor (ICER-1gamma), which bound to a functional cAMP-response element located in the promoter of the CX36 gene. Inhibition of ICER-1gamma overexpression prevented the Cx36 decrease, as well as the palmitate-induced beta-cell secretory dysfunction. Finally, freshly isolated islets from mice undergoing a long term high fat diet expressed reduced Cx36 levels and increased ICER-1gamma levels. Taken together, these data demonstrate that chronic exposure to palmitate inhibits the Cx36 expression through PKA-mediated ICER-1gamma overexpression. This Cx36 down-regulation may contribute to the reduced glucose sensitivity and altered insulin secretion observed during the pre-diabetic stage and in the metabolic syndrome.     

Alkaline cooking and tortilla quality in maize grains from the humid,tropical lands of Mexico

Maize (Zea mays L.) tortilla is the major staple food for the Mexican population. Nine tropical maize genotypes were evaluated. All samples had white grains, a common characteristic in tropical maize, and therefore they were appropriate for nixtamalized flour industry. Grain, flour, masa and tortilla characteristics of each maize genotype were evaluated. Length, width, thickness, weight of 1000 grains and hardness of grain were determined. Moisture content, proteins, fat, ash, mean particle size, water absorption index, enthalpy, and flour temperature were also evaluated. Adhesiveness and cohesiveness were evaluated in masa. Moisture content, protein, capacity to puff up, roll making, tension and cutting strength were determined in tortillas. There were significant differences (p≤0.05) in most of the evaluated characteristics. Grain length values varied between 9.26 and 11.02 mm for populations 23 and 22, respectively. Grain hardness oscillated between 11.17 (population 32) and 14.75 (landrace Mejen). According to the weight of 1000 grains most genotypes had small grains. The minimum and maximum moisture values of flour and tortillas were 8.33-9.99% and 46.20-50.36%, respectively. The texture of tortillas elaborated from population 32 and landrace Mejen had the lowest tension and cutting strength, resulting the best genotypes for making tortilla.     

The phylogenetic potential of entire 26S rDNA sequences in plants

18S ribosomal RNA genes are the most widely used nuclear sequences for phylogeny reconstruction at higher taxonomic levels in plants. However, due to a conservative rate of evolution, 18S rDNA alone sometimes provides too few phylogenetically informative characters to resolve relationships adequately. Previous studies using partial sequences have suggested the potential of 26S or large-subunit (LSU) rDNA for phylogeny retrieval at taxonomic levels comparable to those investigated with 18S rDNA. Here we explore the patterns of molecular evolution of entire 26S rDNA sequences and their impact on phylogeny retrieval. We present a protocol for PCR amplification and sequencing of entire (approximately 3.4 kb) 26S rDNA sequences as single amplicons, as well as primers that can be used for amplification and sequencing. These primers proved useful in angiosperms and Gnetales and likely have broader applicability. With these protocols and primers, entire 26S rDNA sequences were generated for a diverse array of 15 seed plants, including basal eudicots, monocots, and higher eudicots, plus two representatives of Gnetales. Comparisons of sequence dissimilarity indicate that expansion segments (or divergence domains) evolve 6.4 to 10.2 times as fast as conserved core regions of 26S rDNA sequences in plants. Additional comparisons indicate that 26S rDNA evolves 1.6 to 2.2 times as fast as and provides 3.3 times as many phylogenetically informative characters as 18S rDNA; compared to the chloroplast gene rbcL, 26S rDNA evolves at 0.44 to 1.0 times its rate and provides 2.0 times as many phylogenetically informative characters. Expansion segment sequences analyzed here evolve 1.2 to 3.0 times faster than rbcL, providing 1.5 times the number of informative characters. Plant expansion segments have a pattern of evolution distinct from that found in animals, exhibiting less cryptic sequence simplicity, a lower frequency of insertion and deletion, and greater phylogenetic potential.      

Bacterial species and evolution: Theoretical and practical perspectives

A discussion of the species problem in modern evolutionary biology serves as the point of departure for an exploration of how the basic science aspects of this problem relate to efforts to map bacterial diversity for practical pursuits—for prospecting among the bacteria for useful genes and gene-products. Out of a confusing array of species concepts, the Cohesion Species Concept seems the most appropriate and useful for analyzing bacterial diversity. Techniques of allozyme analysis and DNA fingerprinting can be used to put this concept into practice to map bacterial genetic diversity, though the concept requires minor modification to encompass cases of complete asexuality. Examples from studies of phenetically definedBacillus species provide very partial maps of genetic population structure. A major conclusion is that such maps frequently reveal deep genetic subdivision within the phenetically defined specles; divisions that in some cases are clearly distinct genetic species. Knowledge of such subdivisions is bound to make prospecting within bacterial diversity more effective. Under the general concept of genetic cohesion a hypothetical framework for thinking about the full range of species conditions that might exist among bacteria is developed and the consequences of each such model for species delineation, and species identification are discussed. Modes of bacterial evolution, and a theory of bacterial speciation with and without genetic recombination, are examined. The essay concludes with thoughts about prospects for very extensive mapping of bacterial diversity in the service of future efforts to find useful products. In this context, evolutionary biology becomes the handmaiden of important industrial activities. A few examples of past success in commercializing bacterial gene-products from species ofBacillus and a few other bacteria are reviewed.     

Arterial branching in man and monkey

Vessel diameters and branching angles are measured from a large number of arterial bifurcations in the retina of a normal human subject and in that of a rhesus monkey. The results are compared with each other and with theoretical results on this subject.