Phenotypic Switching Induced by Damaged Matrix Is Associated with DNA Methyltransferase 3A (DNMT3A) Activity and Nuclear Localization in Smooth Muscle Cells (SMC)

Extracellular matrix changes are often crucial inciting events for fibroproliferative disease. Epigenetic changes, specifically DNA methylation, are critical factors underlying differentiated phenotypes. We examined the dependency of matrix-induced fibroproliferation and SMC phenotype on DNA methyltransferases. The cooperativity of matrix with growth factors, cell density and hypoxia was also examined. Primary rat visceral SMC of early passage (0–2) were plated on native collagen or damaged/heat-denatured collagen. Hypoxia was induced with 3% O₂ (balanced 5% CO₂ and 95% N₂) over 48 hours. Inhibitors were applied 2–3 hours after cells were plated on matrix, or immediately before hypoxia. Cells were fixed and stained for DNMT3A and smooth muscle actin (SMA) or smooth muscle myosin heavy chain. Illumina 450 K array of CpG sites was performed on bisulfite-converted DNA from smooth muscle cells on damaged matrix vs native collagen. Matrix exquisitely regulates DNMT3A localization and expression, and influences differentiation in SMCs exposed to denatured matrix +/− hypoxia. Analysis of DNA methylation signatures showed that Matrix caused significant DNA methylation alterations in a discrete number of CpG sites proximal to genes related to SMC differentiation. Matrix has a profound effect on the regulation of SMC phenotype, which is associated with altered expression, localization of DNMTs and discrete changes DNA methylation.     

Amino acid starvation sensitizes cancer cells to proteasome inhibition

We explored the crosstalk between protein degradation and synthesis in cancer cells. The tumorigenic cell line, MCF7, showed enhanced proteasome activity compared to the nontumorigenic line, MCF10A. Although there was no difference in the sensitivity of MCF7 and MCF10A cells to proteasome inhibition in complete growth medium, combining proteasome inhibition with amino acid deprivation led to reduced protein synthesis and survival of MCF7 cells, with a lesser effect on MCF10A cells. Additional cancer cell lines (including CAG and A431) could be strongly sensitized to proteasome inhibition by concomitant amino acid deprivation, whereas others were completely resistant to proteasome inhibition. We hypothesize that protein catabolism contributes to the pool of free amino acids available for protein synthesis, leading to a crucial role of the proteasome in cell survival during amino acid depletion, in some tumor cell lines.     

Novel method for the synthesis of urea backbone cyclic peptides using new Alloc‐protected glycine building units

Cyclization of bioactive peptides, utilizing functional groups serving as natural pharmacophors, is often accompanied with loss of activity. The backbone cyclization approach was developed to overcome this limitation and enhance pharmacological properties. Backbone cyclic peptides are prepared by the incorporation of special building units, capable of forming amide, disulfide and coordinative bonds. Urea bridge is often used for the preparation of cyclic peptides by connecting two amine functionalized side chains. Here we present urea backbone cyclization as an additional method for the preparation of backbone cyclic peptide libraries. A straightforward method for the synthesis of crystalline Fmoc‐N^α [ω‐amino(Alloc)‐alkyl] glycine building units is presented. A set of urea backbone cyclic Glycogen Synthase Kinase 3 analogs was prepared and assessed for protein kinase B inhibition as anticancer leads. Copyright © 2010 European Peptide Society and John Wiley & Sons, Ltd.     

Reduced fertility of female mice lacking CD81

In somatic cells, the tetraspanins CD81 and CD9 associate with each other, with additional tetraspanins and with non-tetraspanin molecules to form proteolipidic complexes. Here we show that CD81 is expressed on the surface of oocytes where it associates with tetraspanin-enriched membrane structures. A major CD9 and CD81 partner, CD9P-1, is also expressed by oocytes. Deletion of CD81 gene in mice results in a 40% reduction of female fertility. In vitro insemination indicated that this infertility is due to a deficiency of oocytes to fuse with sperm. While the fertility of CD9-/- mice is severely but not completely impaired, double knock-out CD9-/- CD81-/- mice were completely infertile indicating that CD9 and CD81 play complementary roles in sperm-egg fusion. Finally, a fraction of CD9 was transferred from CD81-/- oocytes to sperm present in the perivitelline space indicating that the defect of fusion of CD81-/- oocytes does not result from an impaired initial gamete interaction.     

Mating disruption method against the vine mealybug,Planococcus ficus: effect of sequential treatment on infested vines

The vine mealybug, Planococcus ficus (Signoret) (Hemiptera: Pseudococcidae), is a major pest of vineyards. Here, we tested the efficacy of the mating disruption method against the pest when applied during one or two successive years in high and low infestation levels. Following 1 year of treatment, at low initial infestation levels a shutdown of pheromone traps was observed, along with a significant reduction in infested vines. With initially high infestation levels, a gradual reduction in infested vines was observed, with a trap shutdown seen only after the second year of pheromone application. We discuss the implications of the male mating disruption method for this pest in which the wingless females are aggregated with limited movement among vines, offering multiple mating opportunities for the flying male.     

Effects of the marine unicellular alga Nannochloropsis sp. to reduce the plasma and liver cholesterol levels in male rats fed on diets with cholesterol

The effects of Nannochloropsis were studied on rats consuming hypercholesterolemic diets. The whole biomass and the hexane/ethanol extract increased the plasma and hepatic eicosapentaenoic and docosahexaenoic acids levels, and reduced the cholesterol levels. We also observed a higher level of propionate, and a lower ratio between acetate and propionate. These data suggest the efficacy of Nannochloropsis in reducing cholesterol levels.     

Regenerative Surface Plasmon Resonance (SPR) biosensor: real-time measurement of fibrinogen in undiluted human serum using the competitive adsorption of proteins

Epidemiological studies suggest that elevated plasma fibrinogen levels are associated with an increased risk of cardiovascular disorders. Normal fibrinogen level is in the range of 1.5-4.5mg/mL, depending upon both genetic (intrinsic) and environmental (extrinsic) factors. An increase of 0.25mg/mL from the normal level can often be correlated with a high risk of cardiovascular disease. Thus, it is useful to monitor fibrinogen level in serum of a patient for clinical diagnosis. We report a regenerative biosensor that measures real-time fibrinogen levels in undiluted serum. The biosensor uses Surface Plasmon Resonance (SPR), highly sensitive optical technique. The biosensor does not use bio-receptors (i.e., antibodies, enzymes, DNA, etc.) unlike conventional biosensors, and deploys the nature of competitive adsorption of proteins to achieve selective detection of fibrinogen. We measured fibrinogen-spiked serum samples with a concentration of 1.5-4.5 mg/mL, and repeated six measurement trials to obtain statistical distribution of the measurements using the regeneration method of the sensing surface. The SPR biosensor has a sensitivity of 42 mDeg/(mg/mL) for a fibrinogen concentration in the range of 0.5-2.5 mg/mL, whereas it was hard to correlate the measurements to the spiked-fibrinogen samples of above 2.5 mg/mL.     

Predator effects on aquatic community assembly: disentangling the roles of habitat selection and post-colonization processes

Top predators are known to play an important role in the assembly of communities via two mechanisms: (1) by altering the colonization (or emigration) patterns of prey through behavioral habitat selection, and (2) by altering vital rates (e.g. mortality, birth) of prey after colonization. While both these mechanisms act to determine assembly, research has focused on either their combined overall effects (confounding them), or examined them singly. As a result, it remains unclear how these mechanisms act to sequentially shape community structure. In this study, we experimentally disentangle habitat selection and post‐colonization effects of predaceous fish to test their independent and combined influence on the assembly of insect and larval amphibian communities in experimental freshwater habitats. Specifically, we ask, ‘do the behavioral choices of colonists continue to structure aquatic communities even after post‐colonization processes have occurred?’ Like previous studies, we found that colonization was strongly reduced by the presence of fish cues. More importantly, these effects of fish on prey colonization behavior combined independently with post‐colonization processes to determine the overall effect of predators on community assembly. Although habitat selection and predation both reduced abundance and biomass of most taxa in the post‐colonization communities, these factors had qualitatively different effects on aspects of trophic structure. Habitat selection altered the ratio of secondary to primary consumer abundance and biomass, while post‐colonization predation drove strong trophic cascades not observed in response to habitat selection. Our results suggest that behavioral choices regarding habitat selection can have lasting and unique effects on the structure of aquatic communities.