Expression pattern of transglutaminases in the early differentiation stage of erupting rat incisor

Several studies demonstrated that transglutaminases play a key role in extracellular matrix stabilization needed for cell differentiation. We evaluated transglutaminase expression and activity in the pre-secretory stage of differentiation of the continuously erupting rat incisor. We observed that transglutaminase-mediated incorporation of monodansylcadaverine into protein substrates was specifically located in the apical loop, and along the basement membrane joining mesenchyme and inner dental epithelium in the odontogenic organ. Enzyme activity was associated with mRNAs for transglutaminase 1 and 2. Notably, labelling cells for these isoenzymes were observed in both mesenchymal and epithelial compartments, but not in the basement membrane, in the ameloblast facing pulp anterior region, where ameloblast and odontoblast differentiation begins. These findings demonstrate that transglutaminase 1 and transglutaminase 2 are expressed at a major extent in the pre-secretory stage of regenerating rat incisor, where they probably play complementary roles in cell signalling between mesenchyme and epithelium and extracellular matrix.     

Spatial-explicit assessment of current and future conservation options for the endangered Corsican Red Deer (Cervus elaphus corsicanus) in Sardinia

The Corsican red deer, a sub-species of the European red deer endemic to Sardinia and Corsica, was abundant on both islands at the beginning of 1900. It went extinct in Corsica and reached a minimum of 100 individuals in Sardinia by 1970. Numbers have recovered in Sardinia with more than 1,000 rutting males now present; in the 1980s the deer was reintroduced to Corsica, but the Sardinian population remains fragmented. We developed a potential distribution model in Sardinia using Ecological Niche Factor Analysis. To assess the deer’s protection status we compared the model with the existing and proposed conservation areas and investigated different conservation scenarios in relation to the expansion of its current range and resilience to future changes in land use and predicted trends of desertification. According to our results over 70% of Sardinia is unsuitable to the deer, nevertheless high suitability areas (Mediterranean forests away from main roads) are available throughout the island, particularly in the south and in the central-eastern part. Existing protected areas do not provide for the conservation of the deer but public owned forests, where hunting is prohibited, extend some level of protection, and the protected areas proposed by the Regional administration, if implemented, will be increasing this protection. Three main areas have emerged as conservation priorities to guarantee adequate conservation potential in the future. Our approach provides valuable data to inform conservation policy, and could be easily replicated in other parts of the Mediterranean.     

The 1-deoxy-d-xylulose 5-phosphate synthase gene co-localizes with a major QTL affecting monoterpene content in grapevine

Muscat flavor is a relevant trait both in winemaking and in fresh grape consumption. From a chemical point of view, it is strongly related to the accumulation of monoterpenes in berries. However, knowledge of the genetic mechanisms underlying its regulation is still limited. The objective of this study was to dissect the genetic determinism of aroma in grapevine by applying the analysis of quantitative trait loci (QTL) and the candidate gene (CG) approach. Two F₁ segregating progenies were evaluated through high-resolution gas chromatography–mass spectrometry (HRGC–MS) for the amounts of individual monoterpenes over 3 and 2 years. In the Italia × Big Perlon cross 34 CGs, chosen according to gene ontology (GO) terms, were placed on a complete map and tested for linkage with QTLs for linalool, nerol and geraniol levels. Two CGs mapped within a QTL for linalool content on LG 10. A third one co-localized with a major QTL for the level of the three monoterpenes on LG 5; this gene encodes 1-deoxy-d-xylulose 5-phosphate synthase (DXS), which is the first enzyme in the plastidial pathway of terpene biosynthesis. Depending on these findings, we report the first in silico analysis of grapevine DXS genes based on the whole genome sequence. Further research on the functional significance of these associations might help to understand the genetic control of Muscat flavor. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. J. Battilana and L. Costantini equally contributed to the work.     

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).     

Cytotoxic palladium complexes of bioreductive quinoxaline N1,N4-dioxide prodrugs

Four new palladium(II) complexes with the formula Pd(L)₂, where L are quinoxaline-2-carbonitrile N¹,N⁴-dioxide derivatives, were synthesized as a contribution to the chemistry and pharmacology of metal compounds with this class of pharmacologically interesting bioreductive prodrugs. Compounds were characterized by elemental, conductometric and thermogravimetric analyses, fast atom bombardment mass spectrometry (FAB-MS) and electronic, Fourier transform infrared (FTIR) and ¹H-nuclear magnetic resonance spectroscopies. The complexes were subjected to cytotoxic evaluation on V79 cells in hypoxic and aerobic conditions. In addition, a preliminary study on interaction with plasmid DNA in normoxia was performed. Complexes showed different in vitro biological behavior depending on the nature of the substituent on the quinoxaline ring. Pd(L1)₂ and Pd(L2)₂, where L1 is 3-aminoquinoxaline-2-carbonitrile N¹,N⁴-dioxide and L2 is 3-amino-6(7)-methylquinoxaline-2-carbonitrile N¹,N⁴-dioxide, showed non selective cytotoxicity, being cytotoxic either in hypoxic or in aerobic conditions. On the other hand, Pd(L3)₂, where L3 is 3-amino-6(7)-chloroquinoxaline-2-carbonitrile N¹,N⁴-dioxide, resulted in vitro more potent cytotoxin in hypoxia (P = 5.0 μM) than the corresponding free ligand (P = 9.0 μM) and tirapazamine (P = 30.0 μM), the first bioreductive cytotoxic drug introduced into clinical trials. In addition, it showed a very good selective cytotoxicity in hypoxic conditions, being non-cytotoxic in normoxia. Its hypoxic cytotoxicity relationship value, HCR, was of the same order than those of other hypoxia selective cytotoxins (i.e., Mitomycine C, Misonidazole and the N-oxide RB90740). Interaction of the complexes with plasmid DNA in normoxia showed dose dependent ability to relax the negative supercoiled forms via different mechanisms. Pd(L2)₂ introduced a scission event in supercoiled DNA yielding the circular relaxed form. Meanwhile, both Pd(L1)₂ and Pd(L3)₂ produced the loss of negative supercoils rendering a family of topoisomers with reduced electrophoretic mobility. Pd(L3)₂ showed a more marked effect than Pd(L1)₂. Indeed, for the highest doses assayed, Pd(L3)₂ was even able to introduce positive supercoils on the plasmid DNA.