TNF-alpha coupled to membrane of apoptotic cells favors the cross-priming to melanoma antigens

The cross-presentation of Ags derived from apoptotic cell processing contributes to peripheral tolerance. Environmental signals possibly modify this default outcome, favoring cross-priming. In this study, we anchored via a biotin-avidin-biotin bridge soluble TNF-alpha to the membrane of apoptotic melanoma cells and studied in vivo and in vitro the interaction with Ag-presenting phagocytes. TNF-alpha-coated apoptotic melanoma cells injected s.c. had a faster and more efficient access to draining lymph nodes, with cross-priming of melanoma-specific CTLs and delayed outgrowth of melanomas in all treated animals. Twenty percent of the animals, in the absence of further adjuvant, did not develop the tumor. Immature dendritic cells challenged with TNF-alpha-coated apoptotic melanoma cells secreted proinflammatory cytokines in an autocrine/paracrine fashion, efficiently matured, as assessed functionally and by flow cytometry and cross-presented with enhanced efficiency melanoma Ags to MHC class I- and II-restricted T cells. The results indicate that TNF-alpha targeted to apoptotic membranes, at concentrations that can be safely reached in growing tumors without undue systemic toxicity, influences the outcome of the disposal of dying cells and enhances tumor immunogenicity.     

Overexpression of homogeneous and active ABCG2 in insect cells

The multidrug transporter ABCG2, a membrane protein with six transmembrane segments, can be overexpressed with the baculovirus/insect cell system. However, ABCG2 is produced as two species with different migration behavior via SDS–PAGE. Evidences suggest that this is due to the accumulation of an immature ABCG2 species, since: (i) the upper species, with higher apparent molecular weight, was favored by treatments reducing the rate of protein synthesis; (ii) the lower species was accumulated in presence of an endoplasmic reticulum stress inducer, and could be converted into the upper species during electrophoresis with 9 M urea; (iii) each species was differently solubilized by detergents: the upper species was partially solubilized by non-ionic and zwitterionic detergents, whereas the lower one required stronger surfactants; (iv) membrane ATPase activity from infected insect cells was essentially associated to the upper species. Altogether, these results suggest that although the insect cell/baculovirus system is not ideally adapted to overexpress human ABCG2, it is able to produce appreciable amounts of purified protein and the addition of agents reducing the rate of protein synthesis improves the homogeneity, making it a suitable heterologous expression system.     

Predicting haplotype carriers from SNP genotypes in Bos taurus through linear discriminant analysis

Background

SNP (single nucleotide polymorphisms) genotype data are increasingly available in cattle populations and, among other things, can be used to predict carriers of specific haplotypes. It is therefore convenient to have a practical statistical method for the accurate classification of individuals into carriers and non-carriers. In this paper, we present a procedure combining variable selection (i.e. the selection of predictive SNPs) and linear discriminant analysis for the identification of carriers of a haplotype on BTA19 (Bos taurus autosome 19) known to be associated with reduced cow fertility. A population of 3645 Brown Swiss cows and bulls genotyped with the 54K SNP-chip was available for the analysis.

Results

The overall error rate for the prediction of haplotype carriers was on average very low (∼≤1%). The error rate was found to depend on the number of SNPs in the model and their density around the region of the haplotype on BTA19. The minimum set of SNPs to still achieve accurate predictions was 5, with a total test error rate of 1.59.

Conclusions

The paper describes a procedure to accurately identify haplotype carriers from SNP genotypes in cattle populations. Very few misclassifications were observed, which indicates that this is a very reliable approach for potential applications in cattle breeding.     

Biophysical functionality in polysaccharides: from Lego-blocks to nano-particles

The objective of the paper is to show the very important biophysical concepts that have been developed with polysaccharides. In particular, an attempt will be made to relate “a posteriori” the fundamental aspects, both experimental and theoretical, with some industrial applications of polysaccharide-based materials. The overview of chain conformational aspects includes relationships between topological features and local dynamics, exemplified for some naturally occurring carbohydrate polymers. Thus, by using simulation techniques and computational studies, the physicochemical properties of aqueous solutions of polysaccharides are interpreted. The relevance of conformational disorder–order transitions, chain aggregation, and phase separation to the underlying role of the ionic contribution to these processes is discussed. We stress the importance of combining information from analysis of experimental data with that from statistical–thermodynamic models for understanding the conformation, size, and functional stability of industrially important polysaccharides. The peculiar properties of polysaccharides in industrial applications are summarized for the particularly important example of nanoparticles production, a field of growing relevance and scientific interest.     

Characterization of nucleoside triphosphate diphosphohydrolase activity in Trichomonas gallinae and the influence of penicillin and streptomycin in extracellular nucleotide hydrolysis

Here we described an nucleoside triphosphate diphosphohydrolase (NTPDase) activity in living trophozoites of Trichomonas gallinae. The enzyme hydrolyzes a variety of purine and pyrimidine nucleoside di- and triphosphates in an optimum pH range of 6.0-8.0. This enzyme activity was activated by high concentrations of divalent cations, such as calcium and magnesium. Contaminant activities were ruled out because the enzyme was not inhibited by classical inhibitors of ATPases (ouabain, 5.0 mM sodium azide, oligomycin) and alkaline phosphatases (levamisole). A significant inhibition of ATP hydrolysis (38%) was observed in the presence of 20 mM sodium azide. Sodium orthovanadate inhibited ATP and ADP hydrolysis (24% and 78%), respectively. The apparent K(M) (Michaelis constant) values were 667.62+/-13 microM for ATP and 125+/-5.3 microM for ADP. V(max) (maximum velocity) values were 0.44+/-0.007 nmol Pi min(-1) per 10(6) trichomonads and 0.91+/-0.12 nmol Pi min(-1) per 10(6) trichomonads for ATP and ADP, respectively. Moreover, we showed a marked decrease in ATP, ADP and AMP hydrolysis when the parasites were grown in the presence of penicillin and streptomycin. The existence of an NTPDase activity in T. gallinae may be involved in pathogenicity, protecting the parasite from the cytolytic effects of the extracellular nucleotides.     

Cadmium induces acidosis in maize root cells

* Cadmium (Cd) stress increases cell metabolic demand for sulfur, reducing equivalents, and carbon skeletons, to sustain phytochelatin biosynthesis for Cd detoxification. In this condition the induction of potentially acidifying anaplerotic metabolism in root tissues may be expected. For these reasons the effects of Cd accumulation on anaplerotic metabolism, glycolysis, and cell pH control mechanisms were investigated in maize (Zea mays) roots. * The study compared root apical segments, excised from plants grown for 24 h in a nutrient solution supplemented, or not, with 10 microM CdCl(2), using physiological, biochemical and (31)P-nuclear magnetic resonance (NMR) approaches. * Cadmium exposure resulted in a significant decrease in both cytosolic and vacuolar pH of root cells and in a concomitant increase in the carbon fluxes through anaplerotic metabolism leading to malate biosynthesis, as suggested by changes in dark CO2 fixation, metabolite levels and enzyme activities along glycolysis, and mitochondrial alternative respiration capacity. This scenario was accompanied by a decrease in the net H(+) efflux from the roots, probably related to changes in plasma membrane permeability. * It is concluded that anaplerotic metabolism triggered by Cd detoxification processes might lead to an imbalance in H(+) production and consumption, and then to cell acidosis.     

Hyper-expression of Small Nucleolar RNAs (snoRNAs) in Female Inflorescences of Hazelnut (Corylus avellana L.) Supports rRNA Aggregation In vitro

The above article appeared     

A novel dendrimeric peptide with antimicrobial properties: structure-function analysis of SB056

The novel antimicrobial peptide with a dimeric dendrimer scaffold, SB056, was empirically optimized by high-throughput screening. This procedure produced an intriguing primary sequence whose structure-function analysis is described here. The alternating pattern of hydrophilic and hydrophobic amino acids suggests the possibility that SB056 is a membrane-active peptide that forms amphiphilic β-strands in a lipid environment. Circular dichroism confirmed that the cationic SB056 folds as β-sheets in the presence of anionic vesicles. Lipid monolayer surface pressure experiments revealed unusual kinetics of monolayer penetration, which suggest lipid-induced aggregation as a membranolytic mechanism. NMR analyses of the linear monomer and the dendrimeric SB056 in water and in 30% trifluoroethanol, on the other hand, yielded essentially unstructured conformations, supporting the excellent solubility and storage properties of this compound. However, simulated annealing showed that many residues lie in the β-region of the Ramachandran plot, and molecular-dynamics simulations confirmed the propensity of this peptide to fold as a β-type conformation. The excellent solubility in water and the lipid-induced oligomerization characteristics of this peptide thus shed light on its mechanism of antimicrobial action, which may also be relevant for systems that can form toxic β-amyloid fibrils when in contact with cellular membranes. Functionally, SB056 showed high activity against Gram-negative bacteria and some limited activity against Gram-positive bacteria. Its potency against Gram-negative strains was comparable (on a molar basis) to that of colistin and polymyxin B, with an even broader spectrum of activity than numerous other reference compounds.     

Adrenal glands and testes as steroidogenic tissue are affected by retinoylation reaction

This study was undertaken to better understand the physiological role of the retinoylation process in steroidogenic tissues. In adrenal gland mitochondria, the retinoylation extent was found equal to that of testes mitochondria but without ATP in the incubation buffer. We pointed out that the endogenous mitochondrial ATP in adrenal glands is much higher than in testes, about 1.3 x 10⁻² M and 5.2 x 10⁻⁸ M, respectively. In addition, less CoASH is required for the maximal acylation activity of the retinoyl moiety to protein(s) compared to testes. The fatty acid analysis revealed a different composition of mitochondrial membranes of these two tissues. Among the different values of fatty acids, it is important to note that adrenal glands contain a much higher amount of C18:0 and a much lower amount of C22:5 ω6 and C22:6 ω3 than testes in the mitochondrial membranes. In addition, there were also differences in arachidonic acid (ARA, C20:4 ω6) content between adrenal glands and testes mitochondria. These different values in the fatty acids composition should explain the different extent of the retinoylation process between the two organs.     

Hypertrophic cardiomyopathy: two homozygous cases with "typical" hypertrophic cardiomyopathy and three new mutations in cases with progression to dilated cardiomyopathy

About 10% of cases of hypertrophic cardiomyopathy (HCM) evolve into dilated cardiomyopathy (DCM) with unknown causes. We studied 11 unrelated patients (pts) with HCM who progressed to DCM (group A) and 11 who showed "typical" HCM (group B). Mutational analysis of the beta-myosin heavy chain (MYH7), myosin-binding protein C (MYBPC3), and cardiac troponin T (TNNT2) genes demonstrated eight mutations affecting MYH7 or MYBPC3 gene, five of which were new mutations. In group A-pts, the first new mutation occurred in the myosin head-rod junction and the second occurred in the light chain-binding site. The third new mutation leads to a MYBPC3 lacking titin and myosin binding sites. In group B, two pts with severe HCM carried two homozygous MYBPC3 mutations and one with moderate hypertrophy was a compound heterozygous for MYBPC3 gene. We identified five unreported mutations, potentially "malignant" defects as for the associated phenotypes, but no specific mutations of HCM/DCM.