Solid-phase synthesis of a combinatorial library of dihydroceramide analogues and its activity in human alveolar epithelial cells

Solid-phase synthesis of a small combinatorial library of dihydroceramide analogues as mixtures of erythro and threo diastereomers is described. Some dihydroceramide analogues cause growth arrest and apoptosis in a dose-dependent manner in human alveolar epithelial cells. This activity is likely due to the threo isomers, as evidenced by cellular studies with a pair of diastereomerically pure N-acyldihydrosphingosines. The apoptotic activity reported in this work provides information for the design of new compounds that may provide the basis for the generation of biochemical tools for the study of different pathologies where ceramide and/or dihydroceramide are involved.     

Phytosterols, but not pectin, added to a high-saturated-fat diet modify saturated fatty acid excretion in relation to chain length

The main objective of this article was to study how the excretion of saturated fatty acids (SFA) is modified after the consumption of a high-saturated-fat diet that was supplemented with phytosterol and pectin. We present the results of a longitudinal 4-week study on guinea pigs. Diets were supplemented with 0.33% of cholesterol and differed in the content of pectin (three levels) and of phytosterols (three levels). Seventy-two female Dunkin Hartley guinea pigs were randomly assigned to the treatment groups (8 animals/group). Addition of phytosterol resulted in a decrease of lauric (12:0) and myristic (14:0) excretions and in an increase of arachidic (20:0) and behenic (22:0) excretions. Palmitic (16:0) and stearic (18:0) acids did not show a clear change after phytosterol supplementation. Addition of pectin resulted in a decreased excretion of all SFA, although this was not significant. These results suggest that phytosterols added to a high-saturated-fat diet enhance the absorption of the most atherogenic fatty acids (lauric and myristic) after 1 week of treatment, as compared with the high-saturated-fat diet alone.     

Effect of low-temperature fermentation on yeast nitrogen metabolism

The aim of this study was to analyse the influence of low-temperature wine fermentation on nitrogen consumption and nitrogen regulation. Synthetic grape must was fermented at 25 and 13°C. Low-temperature decreased both the fermentation and the growth rates. Yeast cells growing at low-temperature consumed less nitrogen than at 25°C. Specifically, cells at 13°C consumed less ammonium and glutamine, and more tryptophan. Low-temperature seemed to relax the nitrogen catabolite repression (NCR) as deduced from the gene expression of ammonium and amino acid permeases (MEP2 and GAP1) and the uptake of some amino acids subjected to NCR (i.e. arginine and glutamine). Low-temperature influences the quantity and the quality of yeast nitrogen requirements. Nitrogen-deficient grape musts and low temperature are two of the main prevalent causes of sluggish fermentations and, therefore, the effects of both growth conditions on yeast metabolism are of considerable interest for wine making.     

The role of MAPK signaling in patterning and establishing axial symmetry in the gastropod Haliotis asinina

Gastropods are members of the Spiralia, a diverse group of invertebrates that share a common early developmental program, which includes spiral cleavage and a larval trochophore stage. The spiral cleavage program results in the division of the embryo into four quadrants. Specification of the dorsal (D) quadrant is intimately linked with body plan organization and in equally cleaving gastropods occurs when one of the vegetal macromeres makes contact with overlying micromeres and receives an inductive signal that activates a MAPK signaling cascade. Following the induction of the 3D macromere, the embryo begins to gastrulate and assumes a bilateral cleavage pattern. Here we inhibit MAPK activation in 3D with U0126 and examine its effect on the formation and patterning of the trochophore, using a suite of territory-specific markers. The head (pretrochal) region appears to maintain quadri-radial symmetry in U0126-treated embryos, supporting a role for MAPK signaling in 3D in establishing dorsoventral polarity in this region. Posterior (posttrochal) structures - larval musculature, shell and foot - fail to develop in MAPK inhibited trochophores. Inhibition of 3D specification by an alternative method - monensin treatment - yields similar abnormal trochophores. However, genes that are normally expressed in the ectodermal structures (shell and foot) are detected in U0126- and monensin-perturbed larvae in patterns that suggest that this region has latent dorsoventral polarity that is manifested even in the absence of D quadrant specification.     

Overexpression of the cytosolic form of phosphoenolpyruvate carboxykinase (GTP) in skeletal muscle repatterns energy metabolism in the mouse

Transgenic mice, containing a chimeric gene in which the cDNA for phosphoenolpyruvate carboxykinase (GTP) (PEPCK-C) (EC 4.1.1.32) was linked to the alpha-skeletal actin gene promoter, express PEPCK-C in skeletal muscle (1-3 units/g). Breeding two founder lines together produced mice with an activity of PEPCK-C of 9 units/g of muscle (PEPCK-C(mus) mice). These mice were seven times more active in their cages than controls. On a mouse treadmill, PEPCK-C(mus) mice ran up to 6 km at a speed of 20 m/min, whereas controls stopped at 0.2 km. PEPCK-C(mus) mice had an enhanced exercise capacity, with a VO(2max) of 156 +/- 8.0 ml/kg/min, a maximal respiratory exchange ratio of 0.91 +/- 0.03, and a blood lactate concentration of 3.7 +/- 1.0 mm after running for 32 min at a 25 degrees grade; the values for control animals were 112 +/- 21 ml/kg/min, 0.99 +/- 0.08, and 8.1 +/- 5.0 mm respectively. The PEPCK-C(mus) mice ate 60% more than controls but had half the body weight and 10% the body fat as determined by magnetic resonance imaging. In addition, the number of mitochondria and the content of triglyceride in the skeletal muscle of PEPCK-C(mus) mice were greatly increased as compared with controls. PEPCK-C(mus) mice had an extended life span relative to control animals; mice up to an age of 2.5 years ran twice as fast as 6-12-month-old control animals. We conclude that overexpression of PEPCK-C repatterns energy metabolism and leads to greater longevity.     

CD8+ T cells from SIV elite controller macaques recognize Mamu-B*08-bound epitopes and select for widespread viral variation

Background

It is generally accepted that CD8⁺ T cell responses play an important role in control of immunodeficiency virus replication. The association of HLA-B27 and -B57 with control of viremia supports this conclusion. However, specific correlates of viral control in individuals expressing these alleles have been difficult to define. We recently reported that transient in vivo CD8⁺ cell depletion in simian immunodeficiency virus (SIV)-infected elite controller (EC) macaques resulted in a brief period of viral recrudescence. SIV replication was rapidly controlled with the reappearance of CD8⁺ cells, implicating that these cells actively suppress viral replication in ECs.

Methods and Findings

Here we show that three ECs in that study made at least seven robust CD8⁺ T cell responses directed against novel epitopes in Vif, Rev, and Nef restricted by the MHC class I molecule Mamu-B*08. Two of these Mamu-B*08-positive animals subsequently lost control of SIV replication. Their breakthrough virus harbored substitutions in multiple Mamu-B*08-restricted epitopes. Indeed, we found evidence for selection pressure mediated by Mamu-B*08-restricted CD8⁺ T cells in all of the newly identified epitopes in a cohort of chronically infected macaques.

Conclusions

Together, our data suggest that Mamu-B*08-restricted CD8⁺ T cell responses effectively control replication of pathogenic SIV_mac239. All seven regions encoding Mamu-B*08-restricted CD8⁺ T cell epitopes also exhibit amino acid replacements typically seen only in the presence of Mamu-B*08, suggesting that the variation we observe is indeed selected by CD8⁺ T cell responses. SIV_mac239 infection of Indian rhesus macaques expressing Mamu-B*08 may therefore provide an animal model for understanding CD8⁺ T cell-mediated control of HIV replication in humans.     

Divergent selection and drift shape the genomes of two avian sister species spanning a saline–freshwater ecotone

The role of species divergence due to ecologically based divergent selection—or ecological speciation—in generating and maintaining biodiversity is a central question in evolutionary biology. Comparison of the genomes of phylogenetically related taxa spanning a selective habitat gradient enables discovery of divergent signatures of selection and thereby provides valuable insight into the role of divergent ecological selection in speciation. Tidal marsh ecosystems provide tractable opportunities for studying organisms' adaptations to selective pressures that underlie ecological divergence. Sharp environmental gradients across the saline–freshwater ecotone within tidal marshes present extreme adaptive challenges to terrestrial vertebrates. Here, we sequence 20 whole genomes of two avian sister species endemic to tidal marshes—the saltmarsh sparrow (Ammospiza caudacutus) and Nelson's sparrow (A. nelsoni)—to evaluate the influence of selective and demographic processes in shaping genome‐wide patterns of divergence. Genome‐wide divergence between these two recently diverged sister species was notably high (genome‐wide F_ST = 0.32). Against a background of high genome‐wide divergence, regions of elevated divergence were widespread throughout the genome, as opposed to focused within islands of differentiation. These patterns may be the result of genetic drift resulting from past tidal march colonization events in conjunction with divergent selection to different environments. We identified several candidate genes that exhibited elevated divergence between saltmarsh and Nelson's sparrows, including genes linked to osmotic regulation, circadian rhythm, and plumage melanism—all putative candidates linked to adaptation to tidal marsh environments. These findings provide new insights into the roles of divergent selection and genetic drift in generating and maintaining biodiversity.     

Genome-wide Linkage Scan Reveals Three Putative Breast-Cancer-Susceptibility Loci

Despite all the research efforts made during the last few decades, most of the cases of families with breast cancer remain unexplained. Mutations in BRCA1 and BRCA2, and in other breast-cancer-susceptibility genes, account for about 25% of familial breast cancer. Linkage studies have failed to identify other breast-cancer-susceptibility genes. The selection criteria of the families, differences in the population background, or clinical and genetic heterogeneity, among other factors, might determine the power to detect the linkage signal. We have performed a SNP-based linkage scan with a total of 6000 SNP markers across the genome in 41 breast-cancer Spanish families, with an average of four breast-cancer cases per family not associated with BRCA1 or BRCA2 germline mutations. In addition, we have included three BRCA-positive families to test the power in linkage detection from a low-complexity family in which a high-penetrance mutation segregates. We have identified three regions of interest, located on 3q25, 6q24, and 21q22. The two former regions showed a suggestive linkage signal (HLOD scores 3.01 and 2.26, respectively), and the latter region showed a significant linkage signal (HLOD score 3.55). Moreover, we found that a subset of 13 families with bilateral breast cancer presented a HLOD of 3.13 on the 3q25 region. Our results suggest that several variables must be taken into account before performing a linkage study in familial breast cancer because of the high heterogeneity within non-BRCA1/2 families. Phenotypic and geographic homogeneity could be the most important factors.