Inducing Ni sensitivity in the Ni hyperaccumulator plant <Emphasis Type="Italic">Alyssum inflatum</Emphasis> Nyárády (Brassicaceae) by transforming with <Emphasis Type="Italic">CAX1</Emphasis>, a vacuolar membrane calcium transporter

The importance of calcium in nickel tolerance was studied in the nickel hyperaccumulator plant Alyssum inflatum by gene transformation of CAX1, a vacuolar membrane transporter that reduces cytosolic calcium. CAX1 from Arabidopsis thaliana with a CaMV35S promoter accompanying a kanamycin resistance gene was transferred into A. inflatum using Agrobacterium tumefaciens. Transformed calli were sub-cultured three times on kanamycin-rich media and transformation was confirmed by PCR using a specific primer for CAX1. At least 10 callus lines were used as a pool of transformed material. Both transformed and untransformed calli were treated with varying concentrations of either calcium (1–15 mM) or nickel (0–500 µM) to compare their responses to those ions. Increased external calcium generally led to increased callus biomass, however, the increase was greater for untransformed callus. Further, increased external calcium led to increased callus calcium concentrations. Transformed callus was less nickel tolerant than untransformed callus: under increasing nickel concentrations callus relative growth rate was significantly less for transformed callus. Transformed callus also contained significantly less nickel than untransformed callus when exposed to the highest external nickel concentration (200 µM). We suggest that transformation with CAX1 decreased cytosolic calcium and resulted in decreased nickel tolerance. This in turn suggests that, at low cytosolic calcium concentrations, other nickel tolerance mechanisms (e.g., complexation and vacuolar sequestration) are insufficient for nickel tolerance. We propose that high cytosolic calcium is an important mechanism that results in nickel tolerance by nickel hyperaccumulator plants.     

An investigation into the role of different constituents in damage accumulation in arterial tissue and constitutive model development

Carotid atherosclerotic plaque rupture is one of the leading causes of stroke. Treatments for atherosclerosis can induce tissue damage during the deployment of an intravascular device or through external tissue clamping during surgery. In this paper, a constituent specific study was performed to investigate the role of the ground matrix and collagen fibres of arterial tissue in response to supra-physiological loads. Cyclic mechanical tests were conducted on intact and collagenase-digested strips of porcine common carotid arteries. Using these tests, four passive damage-relevant phenomena were studied, namely (i) Mullins effect, (ii) hysteresis, (iii) permanent set and (iv) matrix failure and fibre rupture. A constitutive model was also developed to capture all of these damage-relevant phenomena using a continuum damage mechanics approach. The implemented constitutive model was fit to experimental results for both intact and digested samples. The results of this work demonstrate the important role of the ground matrix in the permanent deformation of the arterial tissue under high loads. Supra-physiological load-induced tissue damage may play a key role in vascular remodelling in arteries with atherosclerosis or following interventional procedures.     

3D QSAR studies,pharmacophore modeling,and virtual screening of diarylpyrazole–benzenesulfonamide derivatives as a template to obtain new inhibitors,using human carbonic anhydrase II as a model protein

A 3D-QSAR modeling was performed on a series of diarylpyrazole-benzenesulfonamide derivatives acting as inhibitors of the metalloenzyme carbonic anhydrase (CA, EC 4.2.1.1). The compounds were collected from two datasets with the same scaffold, and utilized as a template for a new pharmacophore model to screen the ZINC database of commercially available derivatives. The datasets were divided into training, test, and validation sets. As the first step, comparative molecular field analysis (CoMFA), CoMFA region focusing and comparative molecular similarity indices analysis (CoMSIA) in parallel with docking studies were applied to a set of 41 human (h) CA II inhibitors. The validity and the prediction capacity of the resulting models were evaluated by leave-one-out (LOO) cross-validation approach. The reliability of the model for the prediction of possibly new CA inhibitors was also tested.     

Intra-erythrocyte Magnesium Is Associated with Gamma-Glutamyl Transferase in Obese Children and Adolescents

This study aims at determining the association between markers of hepatic injury and serum, urinary, and intra-erythrocyte magnesium concentrations and dietary magnesium intake in obese children and adolescents. In a case–control study, 42 obese children and adolescents (8–18 years) and 42 sex- and puberty-matched controls were studied. Serum, urinary, and intra-erythrocyte magnesium levels, indices of insulin sensitivity, and liver enzymes were measured. Dietary magnesium intake was assessed using a food frequency questionnaire. Obese children and adolescents exhibited insulin resistance as determined by a higher fasting insulin and the HOMA-IR (p < 0.001) and lower QUICKI indices (p = 0.001); in addition these subjects had significantly higher intra-erythrocyte magnesium (IEM) concentrations, than non-obese ones (3.99 ± 1.05 vs. 3.35 ± 1.26 mg/dL of packed cell; p = 0.015). Among liver enzymes, only gamma-glutamyl transferase (GGT) was significantly higher in obese than in non-obese subjects (22.7 ± 9.4 vs. 17.1 ± 7.9 U/l; p = 0.002). A positive association was found between GGT and IEM in both groups; however in multivariate analysis, in obese subjects, only GGT (p = 0.026) and, in non-obese subjects, only age (p = 0.006) remained as significant predictors of IEM. In conclusion, increased IEM concentration was seen in insulin-resistant obese children and adolescents; furthermore, serum GGT was associated with IEM, independently of body mass index and HOMA-IR.     

Lectins influence chondrogenesis and osteogenesis in limb bud mesenchymal cells

The role of cell surface glycoproteins in cell behavior can be characterized by their interactions with plant lectins. This study was designed to identify the effects of lectins on chondrogenesis and osteogenesis in limb bud mesenchymal cells in vitro. Limb bud mesenchymal cells from mouse embryos were cultured in high-density micromass culture. Wheat germ agglutinin (WGA), concanavalin A (ConA), peanut agglutinin (PNA), Dolichos biflorus agglutinin (DBA) and Ricinus communis agglutinin (RCA) were added separately to the culture media. Cells were cultured for 5 or 9 days, and cell viability was assayed by neutral red on day 5. The micromasses were stained with alcian blue, alizarin red S and Von Kossa stains, and alkaline phosphatase assays were also done. Dolichos biflorus agglutinin induced an increase in chondrogenesis, calcium precipitation and proteoglycan production. ConA and PNA did not affect chondrocyte differentiation but induced chondrocytes to produce more proteoglycan. Wheat germ agglutinin reduced chondrification and ossification but induced mesenchymal cells to store lipid droplets. Ricinus communis agglutinin 1 was toxic and significantly reduced cell survival. In conclusion, DBA was the most effective inducer of ossification and chondrification. Wheat germ agglutinin induced adipogenesis instead. These assays showed that lectins play important roles in limb bud development.     

Salicylic acid affects growth,essential oil and chemical compositions of thyme (Thymus daenensis Celak.) under reduced irrigation

Salicylic acid (SA) may reduce the negative impact of water deficit on growth and metabolite yield of Thymus daenensis Celak subsp. daenensis Celak. The effect of foliar application of SA and reduced irrigation on growth, oil yield, chemical components, and antibacterial and antioxidant activities of T. daenensis in field condition were investigated. Treatments comprised 0.0, 1.5 and 3.0 M SA applied to plants under normal irrigation and stressed conditions. Results indicated that irrigation regime had a significant effect on growing degree days (GDD) required to reach early and full flowering. Foliar application of SA influenced GDD from early growing stage to 50 % and full flowering, minimum radius and canopy diameter. The highest values of oil content (3.2 % v/w) and yield (14.9 g m^?2) were obtained from application of 3.0 M SA. Percentage of some chemical constituents in the essential oil extracted from the plants under stress was higher than non-stressed plants. Thymol content was significantly reduced under stressed conditions. Foliar application of SA significantly improved carvacrol, α-thujene, α-pinene and p-cymene contents in the oils, but reduced thymol and, β-caryophyllene amounts. Our results showed that foliar application of SA reduced the negative effect of water deficit on thymol content in the essential oil of T. daenensis. The essential oils of T. daenensis exhibited antioxidant and antibacterial activities when plants were sprayed with 1.5 and 3.0 M SA, respectively.     

Protein Microarray Characterization of the S-Nitrosoproteome

Nitric oxide (NO) mediates a substantial part of its physiologic functions via S-nitrosylation, however the cellular substrates for NO-mediated S-nitrosylation are largely unknown. Here we describe the S-nitrosoproteome using a high-density protein microarray chip containing 16,368 unique human proteins. We identified 834 potentially S-nitrosylated human proteins. Using a unique and highly specific labeling and affinity capture of S-nitrosylated proteins, 138 cysteine residues on 131 peptides in 95 proteins were determined, defining critical sites of NO''s actions. Of these cysteine residues 113 are novel sites of S-nitrosylation. A consensus sequence motif from these 834 proteins for S-nitrosylation was identified, suggesting that the residues flanking the S-nitrosylated cysteine are likely to be the critical determinant of whether the cysteine is S-nitrosylated. We identify eight ubiquitin E3 ligases, RNF10, RNF11, RNF41, RNF141, RNF181, RNF208, WWP2, and UBE3A, whose activities are modulated by S-nitrosylation, providing a unique regulatory mechanism of the ubiquitin proteasome system. These results define a new and extensive set of proteins that are susceptible to NO regulation via S-nitrosylation. Similar approaches could be used to identify other post-translational modification proteomes.It is known that NO regulates the majority of its physiologic function through S-nitrosylation (1). Protein-assisted or small molecule, S-nitrosoglutathione (GSNO)¹ trans-nitrosylation, oxidative S-nitrosation, and metalloprotein-catalyzed S-nitrosylation are the prominent cellular mechanisms that are utilized to S-nitrosylate proteins (2). A number of proteins are known to be S-nitrosylated and this post-translational modification can either activate or inactivate a protein''s biologic activity (1, 3). A number of attempts at probing tissue-specific S-nitrosoproteomes have been made, but the results of these are limited to proteins that are S-nitrosylated to a great degree and which are present at high concentrations (2, 4–6). Recently, to investigate determinants of S-nitrosylation, yeast and human target protein microarrays have been studied. However, these assay were limited because of the small number of proteins present on the chip (7). In addition, many proteins that are known to be S-nitrosylated have been studied through a targeted and biased approach (8). To overcome these shortcomings, we report the use of a 16,368 human protein microarray chip to better define the human S-nitrosoproteome.Ubiquitin is a 76-amino-acid long polypeptide that can be covalently added to lysine residues on targeted proteins either as single monomers or in chains. Ubiquitination of proteins can dramatically alter their function or localization depending on the number of ubiquitin attached and the nature of their linkages. The most well characterized ubiquitin-mediated process is targeting of the protein for degradation by the 26S proteasome, which occurs via poly-ubiquitination linked together through lysine 48 on the ubiquitin monomers. Ubiquitination occurs in a three-step enzymatic process in which the third enzyme, the ubiquitin protein ligase (E3) determines protein target specificity (9). NO S-nitrosylates the RING finger E3 ligases, parkin and XIAP, modifying their function (10, 11). In the case of parkin, S-nitrosylation transiently activates its E3 ligase activity, but ultimately inhibits its activity (12). In contrast, XIAP''s E3 ligase activity is unaffected by S-nitrosylation, but its anti-apoptotic function is compromised (11). Using the 16,368 human protein microarray, we identify a number of NO-regulated E3 ligases, the majority of which are activated by NO-dependent S-nitrosylation.     

Oligoprogenitor Cells Derived from Spermatogonia Stem Cells Improve Remyelination in Demyelination Model

Embryonic stem (ES) like cells-derived testis represents a possible alternative to replace of neurons and glia. Here, we differentiated spermatogonia cells to oligoprogenitor (OP) like cells and transplanted them to demyelination model and assess their recovery potential in a demyelinated corpus callosum model in rats. ES like cells were differentiated to OP like cells using appropriate inducers and were transplanted in situ to demyelinated corpus callosum. Cell integration as well as demyelination extension and myelination intensity changes were evaluated using histologic studies and immunocytochemistry after 2 and 4 weeks post transplantation. Investigation of Nestin, NF68, Olig2, and NG2 by immunocytochemical technique indicated the differentiation of ES like cells to neuroprogenitor and oligodendrocyte like cells in each induction stage. Histologic findings showed a significant decrease in demyelination extension and a significant increase in remyelination intensity in cell transplanted groups. Also on the base of PLP expression, differentiation of transplanted cells was confirmed to myelinogenic cells using immunocytochemistry technique. We conclude that ES like cells derived from spermatogonia cells can be differentiated to OP like cells that can form myelin after transplantation into the demyelination model in rat, this represents recovery potential of spermatogonia cells which opens new window for cell therapeutic approaches using spermatogonial stem cells.