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.     

Purification, characterization, and cloning of a serine proteinase inhibitor from the ectoparasite Haematobia irritans irritans (Diptera: Muscidae)

The fly Haematobia irritans irritans is one of the most important ectoparasites in cattle production, due to its ability to suck large amounts of blood. This report describes the purification and characterization of a serine proteinase inhibitor (HiTI) present in H. i. irritans head and thorax extracts. The HiTI purified by affinity chromatography on trypsin-Sepharose has a molecular mass of 7029Da by MALDI-TOF mass spectrometry. HiTI inhibited bovine trypsin, human neutrophil elastase, and a trypsin-like enzyme purified from H. i. irritans abdomen with dissociation constants of 0.57, 1.30, and 0.20nM, respectively. The HiTI partial amino acid sequence allowed its classification into the BPTI-Kunitz-type family. An HiTI cDNA fragment was cloned in the pGEMT vector using RT-PCR. The translated amino acid sequence of HiTI cDNA confirmed a unique Kunitz-type-domain protein. Our results suggest that HiTI could control some endogenous enzyme, e.g., the H. i. irritans trypsin-like protein.     

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.     

Chlamydia pneumoniae infection acts as an endothelial stressor with the potential to initiate the earliest heat shock protein 60-dependent inflammatory stage of atherosclerosis

We identified increased expression and redistribution of the intracellular protein 60-kDa human heat shock protein (hHSP60) (HSPD1) to the cell surface in human endothelial cells subjected to classical atherosclerosis risk factors and subsequent immunologic cross-reactivity against this highly conserved molecule, as key events occurring early in the process of atherosclerosis. The present study aimed at investigating the role of infectious pathogens as stress factors for vascular endothelial cells and, as such, contributors to early atherosclerotic lesion formation. Using primary donor-matched arterial and venous human endothelial cells, we show that infection with Chlamydia pneumoniae leads to marked upregulation and surface expression of hHSP60 and adhesion molecules. Moreover, we provide evidence for an increased susceptibility of arterial endothelial cells for redistribution of hHSP60 to the cellular membrane in response to C. pneumoniae infection as compared to autologous venous endothelial cells. We also show that oxidative stress has a central role to play in endothelial cell activation in response to chlamydial infection. These data provide evidence for a role of C. pneumoniae as a potent primary endothelial stressor for arterial endothelial cells leading to enrichment of hHSP60 on the cellular membrane and, as such, a potential initiator of atherosclerosis.     

Biosynthesis of riboflavin in archaea. 6,7-dimethyl-8-ribityllumazine synthase of Methanococcus jannaschii.

Heterologous expression of the putative open reading frame MJ0303 of Methanococcus jannaschii provided a recombinant protein catalysing the formation of the riboflavin precursor, 6,7-dimethyl-8-ribityllumazine, by condensation of 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione and 3,4-dihydroxy-2-butanone 4-phosphate. Steady state kinetic analysis at 37 degrees C and pH 7.0 indicated a catalytic rate of 11 nmol.mg-1.min-1; Km values for 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione and 3,4-dihydroxybutanone 4-phosphate were 12.5 and 52 micro m, respectively. The enzyme sediments at an apparent velocity of about 12 S. Sedimentation equilibrium analysis indicated a molecular mass around 1 MDa but was hampered by nonideal solute behaviour. Negative-stained electron micrographs showed predominantly spherical particles with a diameter of about 150 A. The data suggest that the enzyme from M. jannaschii can form capsids with icosahedral 532 symmetry consisting of 60 subunits.     

Genomic organisation and alternative splicing of mouse and human thioredoxin reductase 1 genes

Background  

Thioredoxin reductase (TR) is a redox active protein involved in many cellular processes as part of the thioredoxin system. Presently there are three recognised forms of mammalian thioredoxin reductase designated as TR1, TR3 and TGR, that represent the cytosolic, mitochondrial and novel forms respectively. In this study we elucidated the genomic organisation of the mouse (Txnrd1) and human thioredoxin reductase 1 genes (TXNRD1) through library screening, restriction mapping and database mining.     

Alteration in the endogenous intestinal flora of Swiss Webster mice by experimental Angiostrongylus costaricensis infection

The association between worm infections and bacterial diseases has only recently been emphasized. This study examined the effect of experimental Angiostrongylus costaricensis infection on endogenous intestinal flora of Swiss Webster mice. Eight mice aging six weeks were selected for this experiment. Four were infected with A. costaricensis and the other four were used as controls. Twenty eight days after the worm infection, all mice in both groups were sacrificed and samples of the contents of the ileum and colon were obtained and cultured for aerobic and anaerobic bacteria. In the mice infected with A. costaricensis there was a significant increase in the number of bacteria of the endogenous intestinal flora, accompanied by a decrease in the number of Peptostreptococcus spp. This alteration in the intestinal flora of mice infected by the nematode may help to understand some bacterial infections described in humans.     

Polythiophenes inhibit prion propagation by stabilizing prion protein (PrP) aggregates

Luminescent conjugated polymers (LCPs) interact with ordered protein aggregates and sensitively detect amyloids of many different proteins, suggesting that they may possess antiprion properties. Here, we show that a variety of anionic, cationic, and zwitterionic LCPs reduced the infectivity of prion-containing brain homogenates and of prion-infected cerebellar organotypic cultured slices and decreased the amount of scrapie isoform of PrP(C) (PrP(Sc)) oligomers that could be captured in an avidity assay. Paradoxically, treatment enhanced the resistance of PrP(Sc) to proteolysis, triggered the compaction, and enhanced the resistance to proteolysis of recombinant mouse PrP(23-231) fibers. These results suggest that LCPs act as antiprion agents by transitioning PrP aggregates into structures with reduced frangibility. Moreover, ELISA on cerebellar organotypic cultured slices and in vitro conversion assays with mouse PrP(23-231) indicated that poly(thiophene-3-acetic acid) may additionally interfere with the generation of PrP(Sc) by stabilizing the conformation of PrP(C) or of a transition intermediate. Therefore, LCPs represent a novel class of antiprion agents whose mode of action appears to rely on hyperstabilization, rather than destabilization, of PrP(Sc) deposits.