Purification and characterization of the FeII- and alpha-ketoglutarate-dependent xanthine hydroxylase from Aspergillus nidulans

His6-tagged xanthine/alpha-ketoglutarate (alphaKG) dioxygenase (XanA) of Aspergillus nidulans was purified from both the fungal mycelium and recombinant Escherichia coli cells, and the properties of the two forms of the protein were compared. Evidence was obtained for both N- and O-linked glycosylation on the fungus-derived XanA, which aggregates into an apparent dodecamer, while bacterium-derived XanA is free of glycosylation and behaves as a monomer. Immunological methods identify phosphothreonine in both forms of XanA, with phosphoserine also detected in the bacterium-derived protein. Mass spectrometric analysis confirms glycosylation and phosphorylation of the fungus-derived sample, which also undergoes extensive truncation at its amino terminus. Despite the major differences in the properties of these proteins, their kinetic parameters are similar (kcat = 30-70 s-1, Km of alphaKG = 31-50 muM, Km of xanthine approximately 45 muM, and pH optima at 7.0-7.4). The enzyme exhibits no significant isotope effect when [8-2H]xanthine is used; however, it demonstrates a 2-fold solvent deuterium isotope effect. CuII and ZnII potently inhibit the FeII-specific enzyme, whereas CoII, MnII, and NiII are weaker inhibitors. NaCl decreases the kcat and increases the Km of both alphaKG and xanthine. The alphaKG cosubstrate can be substituted with alpha-ketoadipate (9-fold decrease in kcat and 5-fold increase in the Km compared to those of the normal alpha-keto acid), while the alphaKG analogue N-oxalylglycine is a competitive inhibitor (Ki = 0.12 muM). No alternative purines effectively substitute for xanthine as a substrate, and only one purine analogue (6,8-dihydroxypurine) results in significant inhibition. Quenching of the endogenous fluorescence of the two enzyme forms by xanthine, alphaKG, and DHP was used to characterize their binding properties. A XanA homology model was generated on the basis of the structure of the related enzyme TauD (PDB entry 1OS7) and provided insights into the sites of posttranslational modification and substrate binding. These studies represent the first biochemical characterization of purified xanthine/alphaKG dioxygenase.     

Hypohidrotic ectodermal dysplasia

Summary A family carrying the X-linked gene for hypohidrotic ectodermal dysplasia (hereditary ectodermal polydysplasia or Christ-Siemens-Touraine syndrome) over three generations was monitored for more than 15 years. Two prenatal diagnoses were carried out by fetoscopy on skin biopsies. Polymorphic probes were used in the segregation analysis of the Xq11–21 region carried out on 30 members of the family. Current screening possiblitities for the carriers and prenatal diagnosis are discussed.     

Tandemly duplicated Arabidopsis genes that encode polygalacturonase-inhibiting proteins are regulated coordinately by different signal transduction pathways in response to fungal infection

Polygalacturonase-inhibiting proteins (PGIPs) are plant proteins that counteract fungal polygalacturonases, which are important virulence factors. Like many other plant defense proteins, PGIPs are encoded by gene families, but the roles of individual genes in these families are poorly understood. Here, we show that in Arabidopsis, two tandemly duplicated PGIP genes are upregulated coordinately in response to Botrytis cinerea infection, but through separate signal transduction pathways. AtPGIP2 expression is mediated by jasmonate and requires COI1 and JAR1, whereas AtPGIP1 expression is upregulated strongly by oligogalacturonides but is unaffected by salicylic acid, jasmonate, or ethylene. Both AtPGIP1 and AtPGIP2 encode functional inhibitors of polygalacturonase from Botrytis, and their overexpression in Arabidopsis significantly reduces Botrytis disease symptoms. Therefore, gene duplication followed by the divergence of promoter regions may result in different modes of regulation of similar defensive proteins, thereby enhancing the likelihood of defense gene activation during pathogen infection.     

Phylogenetic Properties of RNA Viruses

A new word, phylodynamics, was coined to emphasize the interconnection between phylogenetic properties, as observed for instance in a phylogenetic tree, and the epidemic dynamics of viruses, where selection, mediated by the host immune response, and transmission play a crucial role. The challenges faced when investigating the evolution of RNA viruses call for a virtuous loop of data collection, data analysis and modeling. This already resulted both in the collection of massive sequences databases and in the formulation of hypotheses on the main mechanisms driving qualitative differences observed in the (reconstructed) evolutionary patterns of different RNA viruses. Qualitatively, it has been observed that selection driven by the host immune response induces an uneven survival ability among co-existing strains. As a consequence, the imbalance level of the phylogenetic tree is manifestly more pronounced if compared to the case when the interaction with the host immune system does not play a central role in the evolutive dynamics. While many imbalance metrics have been introduced, reliable methods to discriminate in a quantitative way different level of imbalance are still lacking. In our work, we reconstruct and analyze the phylogenetic trees of six RNA viruses, with a special emphasis on the human Influenza A virus, due to its relevance for vaccine preparation as well as for the theoretical challenges it poses due to its peculiar evolutionary dynamics. We focus in particular on topological properties. We point out the limitation featured by standard imbalance metrics, and we introduce a new methodology with which we assign the correct imbalance level of the phylogenetic trees, in agreement with the phylodynamics of the viruses. Our thorough quantitative analysis allows for a deeper understanding of the evolutionary dynamics of the considered RNA viruses, which is crucial in order to provide a valuable framework for a quantitative assessment of theoretical predictions.     

Morphological changes induced in erythrocyte by amyloid beta peptide and glucose depletion: A combined atomic force microscopy and biochemical study

Circulating red blood cells (RBCs) undergo aging, a fundamental physiological phenomenon that regulates their turnover. We show that treatment with beta amyloid peptide 1–42 (Aβ) accelerates the occurrence of morphological and biochemical aging markers in human RBCs and influences the cell metabolism leading to intracellular ATP depletion. The morphological pattern has been monitored using Atomic Force Microscopy (AFM) imaging and measuring the RBCs' plasma membrane roughness employed as a morphological parameter capable to provide information on the structure and integrity of the membrane-skeleton. Results evidence that Aβ boosts the development of crenatures and proto-spicules simultaneously to acceleration in the weakening of the cell-cytoskeleton contacts and to the induction of peculiar nanoscale features on the cell membrane. Incubation in the presence of glucose can remove all but the latter Aβ-induced effects.Biochemical data demonstrate that contemporaneously to morphological and structural alterations, Aβ and glucose depletion trigger a complex signaling pathway involving caspase 3, protein kinase C (PKC) and nitric oxide derived metabolites.As a whole, the collected data revealed that, the damaging path induced by Aβ in RBC provide a sequence of morphological and functional intermediates following one another along RBC life span, including: (i) an acceleration in the development of shape alteration typically observed along the RBC's aging; (ii) the development of characteristic membrane features on the plasma membrane and (iii) triggering a complex signaling pathway involving caspase 3, PKC and nitric oxide derived metabolites.     

Mobile Communication in the Public Mind: Insights from Free Associations Related to Mobile Phone Base Stations

The number of people using mobile phones has dramatically increased. At the same time, many people are unsettled about the potential health effects from the electromagnetic fields generated by mobile phone base stations. Research indicates that the risks associated with base stations are perceived differently by experts, laypeople, and base station opponents. Using a free association method, we analyzed these differences in more detail. In our first study, we found no difference between experts and laypeople but a marked distinction in the associations of opponents as opposed to the first two groups. The prevalence of free associations in a large random sample from the general population was explored via correspondence analysis in the second study. People who assign high risks to mobile communication had different, more negative associations in mind (e.g., “senselessness,” “hazard”) compared to people with low risk-perception (e.g., “mast,” “acceptance”). Our research is in line with the assumption that the affect heuristic guides risk and benefit assessments, and highlights the role of affect in risk perception and communication.     

Exome Sequencing Identifies Rare Deleterious Mutations in DNA Repair Genes FANCC and BLM as Potential Breast Cancer Susceptibility Alleles

Despite intensive efforts using linkage and candidate gene approaches, the genetic etiology for the majority of families with a multi-generational breast cancer predisposition is unknown. In this study, we used whole-exome sequencing of thirty-three individuals from 15 breast cancer families to identify potential predisposing genes. Our analysis identified families with heterozygous, deleterious mutations in the DNA repair genes FANCC and BLM, which are responsible for the autosomal recessive disorders Fanconi Anemia and Bloom syndrome. In total, screening of all exons in these genes in 438 breast cancer families identified three with truncating mutations in FANCC and two with truncating mutations in BLM. Additional screening of FANCC mutation hotspot exons identified one pathogenic mutation among an additional 957 breast cancer families. Importantly, none of the deleterious mutations were identified among 464 healthy controls and are not reported in the 1,000 Genomes data. Given the rarity of Fanconi Anemia and Bloom syndrome disorders among Caucasian populations, the finding of multiple deleterious mutations in these critical DNA repair genes among high-risk breast cancer families is intriguing and suggestive of a predisposing role. Our data demonstrate the utility of intra-family exome-sequencing approaches to uncover cancer predisposition genes, but highlight the major challenge of definitively validating candidates where the incidence of sporadic disease is high, germline mutations are not fully penetrant, and individual predisposition genes may only account for a tiny proportion of breast cancer families.     

Association Between the Rat Homologue of CO-029, a Metastasis-associated Tetraspanin Molecule and Consumption Coagulopathy

Recently, we have described a panel of metastasis-associated antigens in the rat, i.e., of molecules expressed on metastasizing, but not on nonmetastasizing tumor lines. One of these molecules, recognized by the monoclonal antibody D6.1 and named accordingly D6.1A, was found to be abundantly expressed predominantly on mesenchyme-derived cells. The DNA of the antigen has been isolated and cloned. Surprisingly, the gene product proved to interfere strongly with coagulation.

The 1.182-kb cDNA codes for a 235–amino acid long molecule with a 74.2% homology in the nucleotide and a 70% homology in the amino acid sequence to CO-029, a human tumor-associated molecule. According to the distribution of hydrophobic and hydrophilic amino acids, D6.1A belongs to the tetraspanin superfamily. Western blotting of D6.1A-positive metastasizing tumor lines revealed that the D6.1A, like many tetraspanin molecules, is linked to further membrane molecules, one of which could be identified as α6β1 integrin. Transfection of a low-metastasizing tumor cell line with D6.1A cDNA resulted in increased metastatic potential and provided a clue as to the functional role of D6.1A. We noted massive bleeding around the metastases and, possibly as a consequence, local infarctions predominantly in the mesenteric region and all signs of a consumption coagulopathy. By application of the D6.1 antibody the coagulopathy was counterregulated, though not prevented.

It has been known for many years that tumor growth and progression is frequently accompanied by thrombotic disorders. Our data suggest that the phenomenon could well be associated with the expression of tetraspanin molecules.

     

Spatial and temporal scale of density-dependent body growth and its implications for recruitment, population dynamics and management of stream-dwelling salmonid populations

Density-dependent variations in body growth and size have important consequences for the population dynamics of stream-dwelling salmonid populations, since body size is related to a variety of ecologically relevant characteristics. These include survival and fecundity, competitive and predatory abilities, and foraging behavior. However, little work has been done to understand how density-dependent body growth varies across temporal and spatial scales and when this compensatory process is relevant for recruitment and population dynamics of stream-dwelling salmonids. Increased intra- or inter-cohort competition reduces growth rates of juveniles. Both within- and among-cohort differences at the juvenile stage are likely to be maintained through the lifetime. Limited movement or dispersal can lead to subdivision of a population into several local populations with independent dynamics. The spatial and temporal variation in movement and the patchy distribution of resources make fish likely to experience density-dependence across location, life-stage, and season. The relaxation of density-dependent suppression of body growth at low densities constitutes a potential mechanism for salmonids to persist in the face of environmental perturbation and may contribute to explaining the peculiar resilience to population collapses often showed by salmonids. The inclusion of density-dependent growth in population models may increase the usefulness of model predictions in management contexts. Models not accounting for density-dependent growth may underestimate the recovery potential of resident salmonid populations when they collapse to low densities.     

Hox clusters as models for vertebrate genome evolution

The surprising variation in the number of Hox clusters and the genomic architecture within vertebrate lineages, especially within the ray-finned fish, reflects a history of duplications and subsequent lineage-specific gene loss. Recent research on the evolution of conserved non-coding sequences (CNS) in Hox clusters promises to reveal interesting results for functional and phenotypic diversification.