Cholesterol oxidation products in the vascular remodeling due to atherosclerosis

Like the other oxidation products of the lipid moiety of plasma low density lipoproteins (LDL), cholesterol oxidation products are consistently found within the characteristic lesions of atherosclerosis, both in experimental animals and in man. A growing bulk of evidence suggests that oxysterols make a significant contribution to the vascular remodeling that occurs in atherosclerosis, being involved in various key steps of this complex process: endothelial cell dysfunction, adhesion of circulating blood cells, foam cell and fibrous cap formation, modulation of the extracellular matrix (ECM), vascular cell apoptosis and plaque’s instability. Moreover, oxysterols have been demonstrated to be at least one or two orders of magnitude more reactive than unoxidized cholesterol in exerting pro-inflammatory, pro-apoptotic, and pro-fibrogenic effects. Thus, a pathological level of cholesterol oxidation in the vasculature may be the missing molecular link between hypercholesterolemia and the formation of atherosclerotic lesions.     

Begging and cowbirds: brood parasites make hosts scream louder

Avian brood parasites have evolved striking begging abilitythat often allows them to prevail over the host progeny in competitionfor parental resources. Host young are therefore selected bybrood parasites to evolve behavioral strategies that reducethe cost of parasitism. We tested the prediction that the intensityof nestling begging displays functioning to attract parentalcare increases across species with the frequency of parasitismby the brown-headed cowbird (Molothrus ater). This was expectedbecause host young should try to prevail over highly competitiveparasitic broodmates in scramble interactions, act more selfishlywhen frequency of parasitism is high because brood parasitesoften affect more severely host condition than conspecific broodmates,and discount the kin selection costs of subtracting resourcesto unrelated parasites. Across 31 North American host species,begging loudness positively covaried with parasitism rate inPasserines, and such effect was stronger in species with smallcompared with large clutches. Begging loudness increased withbrood parasitism and nest predation among the most suitablehost species. These results held after controlling for concomitantecological factors and for common ancestry effects. Our resultssupport the hypothesis that avian brood parasitism has playeda role in the evolution of begging behavior of host young.     

Functional Complementation in Yeast Allows Molecular Characterization of Missense Argininosuccinate Lyase Mutations

Deficiency of argininosuccinate lyase (ASL) causes argininosuccinic aciduria, an urea cycle defect that may present with a severe neonatal onset form or with a late onset phenotype. To date phenotype-genotype correlations are still not clear because biochemical assays of ASL activity correlate poorly with clinical severity in patients. We employed a yeast-based functional complementation assay to assess the pathogenicity of 12 missense ASL mutations, to establish genotype-phenotype correlations, and to screen for intragenic complementation. Rather than determining ASL enzyme activity directly, we have measured the growth rate in arginine-free medium of a yeast ASL^null strain transformed with individual mutant ASL alleles. Individual haploid strains were also mated to obtain diploid, “compound heterozygous” yeast. We show that the late onset phenotypes arise in patients because they harbor individual alleles retaining high residual enzymatic activity or because of intragenic complementation among different mutated alleles. In these cases complementation occurs because in the hybrid tetrameric enzyme at least one active site without mutations can be formed or because the differently mutated alleles can stabilize each other, resulting in partial recovery of enzymatic activity. Functional complementation in yeast is simple and reproducible and allows the analysis of large numbers of mutant alleles. Moreover, it can be easily adapted for the analysis of mutations in other genes involved in urea cycle disorders.Argininosuccinic aciduria (ASAuria, MIM 207900)³ is an autosomal recessive disorder of the urea cycle caused by mutations of the ASL gene (hASL, MIM 608310), encoding argininosuccinate lyase (ASL; EC 4.3.2.1.) (1). This enzyme is ubiquitously expressed and catalyzes the reversible breakdown of argininosuccinate to arginine and fumarate. ASL belongs to a superfamily of hydrolases that includes adenylosuccinate lyase and fumarase, which share a homotetrameric structure and a similar catalytic mechanism. The tetrameric structure of ASL accounts for the phenomenon of intragenic complementation. This particular situation occurs when a multimeric protein is formed from subunits produced by differently mutated alleles of the same gene. On complementation, a partially functional hybrid protein is produced from the two distinct types of mutant subunits, neither of which individually has appreciable enzymatic activity (2).ASL participates to the urea cycle, and in humans it is essential for ammonia detoxification, whereas in lower organisms it is required for the biosynthesis of arginine. Saccharomyces cerevisiae strains harboring a deletion of the homolog of human ASL (ARG4) cannot grow on media lacking arginine (3).ASAuria is characterized by accumulation of argininosuccinic acid (ASA) in body fluids, and severe hyperammonaemia. The disease displays clinical heterogeneity with two main clinical phenotypes: the acute/neonatal onset form, with symptoms rapidly progressing to deep coma, apnea, and death (1), and the subacute/late onset type, which is diagnosed in infancy or childhood (4). Such patients may present simply with mental retardation or an epileptic disorder. In both types the diagnosis is established unambiguously by measuring plasma levels of ammonia (not always elevated in the late onset form), ASA, and its anhydrides by plasma amino acids assay (1). Over 40 mutations of the ASL gene have been reported, both amino acid substitutions and truncating variants, which are scattered throughout the gene (5, 6).We have previously reported the identification of novel mutations of the ASL gene in a cohort of Italian patients (7). In this study we employed a yeast model to validate the pathogenicity of missense ASL mutations found in our cohort, to study the effects of different allelic combinations, and to establish possible genotype-phenotype correlations.     

Amyloidogenic Potential of Transthyretin Variants: INSIGHTS FROM STRUCTURAL AND COMPUTATIONAL ANALYSES*

Human transthyretin (TTR) is an amyloidogenic protein whose mild amyloidogenicity is enhanced by many point mutations affecting considerably the amyloid disease phenotype. To ascertain whether the high amyloidogenic potential of TTR variants may be explained on the basis of the conformational change hypothesis, an aim of this work was to determine structural alterations for five amyloidogenic TTR variants crystallized under native and/or destabilizing (moderately acidic pH) conditions. While at acidic pH structural changes may be more significant because of a higher local protein flexibility, only limited alterations, possibly representing early events associated with protein destabilization, are generally induced by mutations. This study was also aimed at establishing to what extent wild-type TTR and its amyloidogenic variants are intrinsically prone to β-aggregation. We report the results of a computational analysis predicting that wild-type TTR possesses a very high intrinsic β-aggregation propensity which is on average not enhanced by amyloidogenic mutations. However, when located in β-strands, most of these mutations are predicted to destabilize the native β-structure. The analysis also shows that rat and murine TTR have a lower intrinsic β-aggregation propensity and a similar native β-structure stability compared with human TTR. This result is consistent with the lack of in vitro amyloidogenicity found for both murine and rat TTR. Collectively, the results of this study support the notion that the high amyloidogenic potential of human pathogenic TTR variants is determined by the destabilization of their native structures, rather than by a higher intrinsic β-aggregation propensity.Protein misfolding and aggregation are involved in the pathogenesis of particularly relevant human deposition diseases, known as amyloidoses. In such diseases, normally soluble proteins undergo misfolding and become insoluble, causing the extracellular deposition of fibrillar aggregates (for reviews, see Ref. 1, 2). To date, more than 40 distinct human proteins have been associated with amyloidoses. For some of such proteins, including transthyretin (TTR),⁴ lysozyme, gelsolin, ApoAI, and ApoAII, fibrinogen A α-chain and cystatin C, the amyloidogenic potential is induced, or is enhanced as in the case of TTR (see below), by specific mutations. The most frequent hereditary amyloidoses are caused by the genetic variants of human TTR (2).TTR is a homotetramer of about 55 kDa involved in the transport of thyroxine in the extracellular fluids and in the co-transport of vitamin A, by forming a macromolecular complex with retinol-binding protein, the specific plasma carrier of retinol (3–5). Its three-dimensional structure is known at high resolution (6, 7). The structure is characterized by a large predominance of β-strands, and its four monomers are arranged according to a 222 symmetry, where one of the 2-fold symmetry axes of the molecule coincides with a long channel that transverses the entire tetramer and harbors two symmetrical binding sites for the thyroid hormone thyroxine. Each monomer contains eight β-strands (A-H), arranged in a β-sandwich of two four-stranded β-sheets, with a short α-helix connecting two of the eight β-strands. In the tetramer, the four monomers are organized as a dimer of dimers. Two monomers are held together, forming a stable dimer through a net of H-bond interactions involving the two external β-strands H and F. The two dimers associate back to back and form the tetramer, by interacting mostly through hydrophobic contacts between residues of the AB and GH loops.Normal TTR possesses an inherent potential, albeit low, to generate amyloid fibrils, giving rise to Senile Systemic Amyloidosis (SSA) in ∼25% of the population aged over 80 years (8). More than 100 point mutations are described for human TTR. Most of them are involved in the hereditary amyloidoses known as familial amyloidotic polyneuropathy (FAP) or cardiomyopathy (FAC) (9). Single point mutations enhance the amyloidogenicity of TTR, so that patients show an earlier age of onset and a faster disease progression compared with SSA patients. The observation that single point mutations can drastically influence the disease phenotype is particularly relevant. In fact, the study of pathogenic TTR variants may provide clues to the mechanism of their abnormal behavior leading to amyloid formation. Although amyloidogenic proteins in general may be structurally unrelated to each other, and lead to various pathological phenotypes in humans, the amyloid fibrils originating from different proteins share the common cross-β structure, consisting in continuous β-sheets lying parallel to the longitudinal axis of the fibril, with the constituent β-strands running perpendicular to this axis. Therefore, the amyloidogenic proteins have to undergo structural alterations to be able to generate the cross-β structure, i.e. new β-pairing interactions have to be established on the way to fibril formation. However, the molecular mechanisms underlying protein misfolding and aggregation into highly ordered fibrillar structures are not clarified definitely, although significant progress is recently been made toward their elucidation (1, 10, 11).Based on the seminal observation that the rates of aggregation into amyloid fibrils in vitro correlate with simple physico-chemical amino acid features (12), several algorithms were introduced in recent years to predict, with good success, the intrinsic β-aggregation propensities of protein and peptide sequences (for a review, see Ref. 13). The intrinsic β-aggregation propensity is a measure of the tendency polypeptide chains may have to aggregate into the amyloid structure, provided that aggregation proceeds from unstructured monomers. The prediction of intrinsic propensities to β-aggregation for amyloidogenic or non-amyloidogenic variants of the same sequence was used to explain in several instances their relative ability to speed up/slow down in vitro fibrillogenesis or the enhancement/reduction of their amyloidogenic potential in vivo (14). However, a high intrinsic aggregation propensity may not result in an actual aggregation, due to the protecting role of the ordered native structure (15, 16). Therefore, the amyloidogenic potential in the TTR variants may depend further on the change of stability in the native TTR tetramer induced by mutations. In particular, it remains to be clarified to what extent human TTR possesses an intrinsic propensity to β-aggregation, and whether amyloidogenic mutations enhance such a propensity, or only destabilize the TTR tetramer, thereby facilitating the misfolding and misassembly of a protein which is in itself prone to β-aggregation.With regard to the pathway from native to misfolded TTR and to amyloid aggregation, the results of a number of in vitro studies are consistent with the rate-limiting dissociation of the TTR tetramer, followed by misfolding of TTR monomers and their downhill polymerization to generate pathological aggregates (17–25). The crystal structures of amyloidogenic TTR variants are generally well conserved (26–30). Accordingly, the functional properties of the variants, such as the ability to interact with retinol-binding protein (5), are maintained, being consistent with the fact that large conformational changes are not induced by amyloidogenic mutations, at least under native-like conditions (11). In vitro studies have shown that a moderately acidic medium (pH 4–5) facilitates TTR fibrillogenesis (17) and that the extent of fibril formation is remarkably enhanced for amyloidogenic TTR variants in comparison to wild-type TTR (31). Recently, it has been shown by x-ray analysis that an acidic pH (4.6) causes a large local conformational change in an amyloidogenic TTR variant (I84S) affecting two subunits within the tetramer, which probably destabilizes the TTR tetramer (32). In contrast, no significant structural changes for wild-type TTR at pH 4.6 and for I84S TTR at neutral pH were found, suggesting that conformational changes associated with a destabilization of the TTR native state may be induced or enhanced in amyloidogenic TTR variants by partially denaturing conditions (32). Pursuing these observations, we extend here our investigation to include other amyloidogenic TTR variants in comparison to the wild-type protein, with the aim to unravel structural alterations that are possibly associated with an enhanced amyloidogenic potential, according to the conformational change hypothesis (11). In addition, we report the results of a computational analysis of the mutational effects on both the intrinsic propensity to β-aggregation and the stability of the native β-structure. The same analysis is performed on murine and rat TTRs, whose structural organizations are very similar to that of the human protein (33, 34).     

Synthesis,cannabinoid receptor affinity,molecular modeling studies and in vivo pharmacological evaluation of new substituted 1-aryl-5-(1H-pyrrol-1-yl)-1H-pyrazole-3-carboxamides. 2. Effect of the 3-carboxamide substituent on the affinity and selectivity profile

New substituted 1-aryl-5-(1H-pyrrol-1-yl)-1H-pyrazole-3-carboxamides were synthesized by replacing the 2,4-dichlorobenzyl and cyclohexyl moieties at the 3-carboxamide nitrogen of the previously reported CB₁ receptor antagonists/inverse agonists 4 and 5. Several ligands showed potent affinity for the hCB₁ receptor, with K_i concentrations comparable to the reference compounds 1, 4 and 5, and exhibited CB₁ selectivity comparable to 1 and 2. Docking experiments and molecular dynamics (MD) simulations explained the potent hCB₁ binding affinity of compounds 31 and 37. According to our previous studies, 31 and 37 formed a H-bond with K3.28(192), which accounted for the high affinity for the receptor inactive state and the inverse agonist activity. The finding of inhibition of food intake following their acute administration to rats, supported the concept that the CB₁ selective compounds 4 and 52 act as antagonists/inverse agonists.     

CytokineDB: a database collecting biological information

Cytokines are subdivided in 12 sub-families and are described as multi-functional molecules that play an important biological activity in host defense system against pathogens, in homeostasis, tissue repair, cell growth and development. CytokineDB is an annotated database that collects biological information regarding the cytokines family in human and will be periodically updated by including new biological information. This database is freely available online and can be accessed at the URL: http://www.cro-m.eu/CytokineDB/     

On the hydrophobicity of nitrogen dioxide: Could there be a “lens” effect for NO2 reaction kinetics?

Solvent “lens” effects for the reaction kinetics of NO₂ can be evaluated on the basis of published Henry’s law constants for nitrogen dioxide in various solvents. Water-to-organic solvent partition coefficients were derived from Henry’s law constants and used to assess the tendencies of NO₂ toward fleeing the aqueous environments and concentrating in biological hydrophobic media. It is concluded, based only on the estimated aqueous medium-to-cell membrane partition coefficient for NO₂, that such tendencies will be relatively small, and that they may account for an acceleration of chemical reactions in biological hydrophobic media with reaction kinetics that are first order on NO₂ by a factor of approximately 3 ± 1. Thus, kinetic effects due to mass action will be relatively small but it is also important to recognize that because NO₂ will tend to dissolve in cell membranes, reactions with cell membrane components will not be hindered by lack of physical solubility at these loci. In comparison to other gases, nitrogen dioxide is less hydrophobic than NO, O₂ and N₂.     

An Outlook on Ovarian Cancer and Borderline Ovarian Tumors: Focus on Genomic and Proteomic Findings

Among the gynaecological malignancies, ovarian cancer is one of the neoplastic forms with the poorest prognosis and with the bad overall and disease-free survival rates than other gynaecological cancers. Ovarian tumors can be classified on the basis of the cells of origin in epithelial, stromal and germ cell tumors. Epithelial ovarian tumors display great histological heterogeneity and can be further subdivided into benign, intermediate or borderline, and invasive tumors. Several studies on ovarian tumors, have focused on the identification of both diagnostic and prognostic markers for applications in clinical practice. High-throughput technologies have accelerated the process of biomolecular study and genomic discovery; unfortunately, validity of these should be still demonstrated by extensive researches on sensibility and sensitivity of ovarian cancer novel biomarkers, determining whether gene profiling and proteomics could help differentiate between patients with metastatic ovarian cancer and primary ovarian carcinomas, and their potential impact on management. Therefore, considerable interest lies in identifying molecular and protein biomarkers and indicators to guide treatment decisions and clinical follow up. In this review, the current state of knowledge about the genoproteomic and potential clinical value of gene expression profiling in ovarian cancer and ovarian borderline tumors is discussed, focusing on three main areas: distinguishing normal ovarian tissue from ovarian cancers and borderline tumors, identifying different genotypes of ovarian tissue and identifying proteins linked to cancer or tumor development. By these targets, authors focus on the use of novel molecules, developed on the proteomics and genomics researches, as potential protein biomarkers in the management of ovarian cancer or borderline tumor, overlooking on current state of the art and on future perspectives of researches.Key Words: Ovarian cancer, borderline ovarian tumors, markers, genomics, proteomics, oncogenes.     

Increased Litter Build Up and Soil Organic Matter Stabilization in a Poplar Plantation After 6 Years of Atmospheric CO2 Enrichment (FACE): Final Results of POP-EuroFACE Compared to Other Forest FACE Experiments

Free air CO₂ enrichment (FACE) experiments in aggrading temperate forests and plantations have been initiated to test whether temperate forest ecosystems act as sinks for anthropogenic emissions of CO₂. These FACE experiments have demonstrated increases in net primary production and carbon (C) storage in forest vegetation due to increased atmospheric CO₂ concentrations. However, the fate of this extra biomass in the forest floor or mineral soil is less clear. After 6 years of FACE treatment in a short-rotation poplar plantation, we observed an additional sink of 32 g C m⁻² y⁻¹ in the forest floor. Mineral soil C content increased equally under ambient and increased CO₂ treatment during the 6-year experiment. However, during the first half of the experiment the increase in soil C was suppressed under FACE due to a priming effect, that is, the additional labile C increased the mineralization of older SOM, whereas during the second half of the experiment the increase in soil C was larger under FACE. An additional sink of 54 g C m⁻² y⁻¹ in the top 10 cm of the mineral soil was created under FACE during the second half of the experiment. Although, this FACE effect was not significant due to a combination of soil spatial variability and the low number of replicates that are inherent to the present generation of forest stand FACE experiments. Physical fractionation by wet sieving revealed an increase in the C and nitrogen (N) content of macro-aggregates due to FACE. Further fractionation by density showed that FACE increased C and N contents of the light iPOM and mineral associated intra-macro-aggregate fractions. Isolation of micro-aggregates from macro-aggregates and subsequent fractionation by density revealed that FACE increased C and N contents of the light iPOM, C content of the fine iPOM and C and N contents of the mineral associated intra-micro-aggregate fractions. From this we infer that the amount of stabilized C and N increased under FACE treatment. We compared our data with published results of other forest FACE experiments and infer that the type of vegetation and soil base saturation, as a proxy for bioturbation, are important factors related to the size of the additional C sinks of the forest floor–soil system under FACE. Author Contribution: MRH conceived of and designed the study, performed research, analyzed data, and wrote the paper; GES conceived of and designed the study and performed research.