Phylogenetic analysis of Myobia musculi (Schranck, 1781) by using the 18S small ribosomal subunit sequence

We used high-fidelity PCR to amplify 2 overlapping regions of the ribosomal gene complex from the rodent fur mite Myobia musculi. The amplicons encompassed a large portion of the mite's ribosomal gene complex spanning 3128 nucleotides containing the entire 18S rRNA, internal transcribed spacer (ITS) 1,5.8S rRNA, ITS2, and a portion of the 5'-end of the 28S rRNA. M. musculi's 179-nucleotide 5.8S rRNA nucleotide sequence was not conserved, so this region was identified by conservation of rRNA secondary structure. Maximum likelihood and Bayesian inference phylogenetic analyses were performed by using multiple sequence alignment consisting of 1524 nucleotides of M. musculi 18S rRNA and homologous sequences from 42 prostigmatid mites and the tick Dermacentor andersoni. The phylograms produced by both methods were in agreement regarding terminal, secondary, and some tertiary phylogenetic relationships among mites. Bayesian inference discriminated most infraordinal relationships between Eleutherengona and Parasitengona mites in the suborder Anystina. Basal relationships between suborders Anystina and Eupodina historically determined by comparing differences in anatomic characteristics were less well-supported by our molecular analysis. Our results recapitulated similar 18S rRNA sequence analyses recently reported. Our study supports M. musculi as belonging to the suborder Anystina, infraorder Eleutherenona, and superfamily Cheyletoidea.     

KIAA0101 interacts with BRCA1 and regulates centrosome number

To find genes and proteins that collaborate with BRCA1 or BRCA2 in the pathogenesis of breast cancer, we used an informatics approach and found a candidate BRCA interactor, KIAA0101, to function like BRCA1 in exerting a powerful control over centrosome number. The effect of KIAA0101 on centrosomes is likely direct, as its depletion does not affect the cell cycle, KIAA0101 localizes to regions coincident with the centrosomes, and KIAA0101 binds to BRCA1. We analyzed whether KIAA0101 protein is overexpressed in breast cancer tumor samples in tissue microarrays, and we found that overexpression of KIAA0101 correlated with positive Ki67 staining, a biomarker associated with increased disease severity. Furthermore, overexpression of the KIAA0101 gene in breast tumors was found to be associated with significantly decreased survival time. This study identifies KIAA0101 as a protein important for breast tumorigenesis, and as this factor has been reported as a UV repair factor, it may link the UV damage response to centrosome control.     

The effects of low-impact mutations in digital organisms

Background  

Avida is a computer program that performs evolution experiments with digital organisms. Previous work has used the program to study the evolutionary origin of complex features, namely logic operations, but has consistently used extremely large mutational fitness effects. The present study uses Avida to better understand the role of low-impact mutations in evolution.     

Extremely high Tp53 mutation load in esophageal squamous cell carcinoma in Golestan Province, Iran

Background

Golestan Province in northeastern Iran has one of the highest incidences of esophageal squamous cell carcinoma (ESCC) in the world with rates over 50 per 100,000 person-years in both sexes. We have analyzed TP53 mutation patterns in tumors from this high-risk geographic area in search of clues to the mutagenic processes involved in causing ESCC.

Methodology/Principal Findings

Biopsies of 119 confirmed ESCC tumor tissue from subjects enrolled in a case-control study conducted in Golestan Province were analyzed by direct sequencing of TP53 exons 2 through 11. Immunohistochemical staining for p53 was carried out using two monoclonal antibodies, DO7 and 1801. A total of 120 TP53 mutations were detected in 107/119 cases (89.9%), including 11 patients with double or triple mutations. The mutation pattern was heterogeneous with infrequent mutations at common TP53 “hotspots” but frequent transversions potentially attributable to environmental carcinogens forming bulky DNA adducts, including 40% at bases known as site of mutagenesis by polycyclic aromatic hydrocarbons (PAHs). Mutations showed different patterns according to the reported temperature of tea consumption, but no variation was observed in relation to ethnicity, tobacco or opium use, and alcoholic beverage consumption or urban versus rural residence.

Conclusion/Significance

ESCC tumors in people from Golestan Province show the highest rate of TP53 mutations ever reported in any cancer anywhere. The heterogeneous mutation pattern is highly suggestive of a causative role for multiple environmental carcinogens, including PAHs. The temperature and composition of tea may also influence mutagenesis.     

Dehalogenation of the herbicides bromoxynil (3,5-dibromo-4-hydroxybenzonitrile) and ioxynil (3,5-diiodino-4-hydroxybenzonitrile) by Desulfitobacterium chlororespirans

Desulfitobacterium chlororespirans has been shown to grow by coupling the oxidation of lactate to the metabolic reductive dehalogenation of ortho chlorines on polysubstituted phenols. Here, we examine the ability of D. chlororespirans to debrominate and deiodinate the polysubstituted herbicides bromoxynil (3,5-dibromo-4-hydroxybenzonitrile), ioxynil (3,5-diiodo-4-hydroxybenzonitrile), and the bromoxynil metabolite 3,5-dibromo-4-hydroxybenzoate (DBHB). Stoichiometric debromination of bromoxynil to 4-cyanophenol and DBHB to 4-hydroxybenzoate occurred. Further, bromoxynil (35 to 75 microM) and DBHB (250 to 260 microM) were used as electron acceptors for growth. Doubling times for growth (means +/- standard deviations for triplicate cultures) on bromoxynil (18.4 +/- 5.2 h) and DBHB (11.9 +/- 1.4 h), determined by rate of [14C]lactate uptake into biomass, were similar to those previously reported for this microorganism during growth on pyruvate (15.4 h). In contrast, ioxynil was not deiodinated when added alone or when added with bromoxynil; however, ioxynil dehalogenation, with stoichiometric conversion to 4-cyanophenol, was observed when the culture was amended with 3-chloro-4-hydroxybenzoate (a previously reported electron acceptor). To our knowledge, this is the first direct report of deiodination by a bacterium in the Desulfitobacterium genus and the first report of an anaerobic pure culture with the ability to transform bromoxynil or ioxynil. This research provides valuable insights into the substrate range of D. chlororespirans.     

A rapid and efficient protocol to purify biologically active recombinant proteins from mammalian cells

Here, we describe a simple and efficient method for the expression and purification of active recombinant proteins in mammalian cells. This method uses the expression of T7 epitope-tagged proteins in transiently transfected 293T cells grown in monolayer, followed by anti-T7-agarose affinity chromatography. This procedure yields approximately between 75 and 100 microg of biologically active protein/150 cm(2) flask that can be used for biochemical studies. We have tested this protocol for the expression of the prototype SR protein, SF2/ASF, which is a member of the SR protein family with a role in constitutive and alternative splicing. We show that SF2/ASF purified using this protocol is able to complement an S100 HeLa extract, demonstrating that is biologically active. Moreover, expression of a novel SR-related protein that it is required for the second step of pre-mRNA splicing also rendered an active protein. In summary, we present a protocol based on transient transfection of mammalian cells that results in easy purification of significant amounts of biologically active proteins.     

Integrin regulation

Integrin signaling is bidirectional. 'Inside-out' signals regulate integrin affinity for adhesive ligands, and ligand-dependent 'outside-in' signals regulate cellular responses to adhesion. Integrin extracellular domains are yielding to high-resolution structural analyses, and intracellular proteins involved in integrin signaling are being identified. However, a key unresolved question is how integrins propagate signals across the plasma membrane.     

Integrins and Src: dynamic duo of adhesion signaling

Src family protein tyrosine kinases (SFKs) play important roles downstream of integrin adhesion receptors, and they are necessary for the generation of "outside-in signals" that regulate cytoskeletal organization, cell motility and gene expression in response to cell adhesion. One relatively under-explored facet of this relationship is the possible physical interaction of integrins with SFKs. Recently, it has been established that beta3 integrins and c-Src can interact directly, and this pool of c-Src is activated by cell adhesion to initiate outside-in signaling in platelets, osteoclasts and cells of the vasculature. Here, the biochemical basis for and biological significance of this integrin-SFK interaction is summarized, and I propose a general mechanism for initiation of outside-in integrin signaling.