Copy number variation across European populations

Genome analysis provides a powerful approach to test for evidence of genetic variation within and between geographical regions and local populations. Copy number variants which comprise insertions, deletions and duplications of genomic sequence provide one such convenient and informative source. Here, we investigate copy number variants from genome wide scans of single nucleotide polymorphisms in three European population isolates, the island of Vis in Croatia, the islands of Orkney in Scotland and the South Tyrol in Italy. We show that whereas the overall copy number variant frequencies are similar between populations, their distribution is highly specific to the population of origin, a finding which is supported by evidence for increased kinship correlation for specific copy number variants within populations.     

Unraveling the mechanism of radiosensitization by gemcitabine: the role of TP53

Gemcitabine has excellent radiosensitizing properties, as shown in both preclinical and clinical studies. Radiosensitization correlated with the early S-phase block of gemcitabine. In the present study, we investigated the role of TP53 in the radiosensitizing effect of gemcitabine. Isogenic A549 cells differing in TP53 status were treated with gemcitabine during the 24 h prior to irradiation. Cell survival was determined 7 days after irradiation by the sulforhodamine B test. In addition, cell cycle perturbation was determined by flow cytometry and TP53 expression by Western blot analysis. Gemcitabine caused a concentration-dependent radiosensitizing effect in all cell lines. Transformed A549 cells were less sensitive to the cytotoxic effect of gemcitabine. The cell cycle arrest early in the S phase was dependent on the drug dose but was comparable in the different cell lines and was not related to functional TP53. Using isogenic cell lines, we have shown that neither TP53 status nor the transfection procedure influenced the radiosensitizing effect of gemcitabine. Since both the radiosensitizing effect at equitoxic concentrations and the cell cycle effect of gemcitabine were independent of TP53 expression, it is likely that TP53 protein does not play a crucial role in the radiosensitizing mechanism of gemcitabine.     

The delta e13 isoform of the calcitonin receptor forms a six-transmembrane domain receptor with dominant-negative effects on receptor surface expression and signaling

The CTRdelta e13 splice variant of the rabbit calcitonin receptor, which lacks the 14 amino acids of the seventh transmembrane domain (TMD) that are encoded by exon 13, is poorly expressed on the cell surface, fails to mobilize intracellular calcium or activate Erk, and inhibits the cell surface expression of the full-length C1a isoform. Nuclear magnetic resonance- and fluorescence-activated cell sorter-based experiments showed that the residual seventh TMD of CTRdelta e13 fails to partition into the lipid bilayer, resulting in an extracellular C terminus. Truncating the receptor after residue 397 to delete the cytoplasmic tail resulted in reduced cell surface expression and an inability to mobilize intracellular calcium or activate Erk, but the truncated receptor did not inhibit C1a cell surface expression. In contrast, when the receptor was truncated after residue 374 to eliminate the entire seventh TMD domain and the C-terminal domain, the resulting receptor reduced the cell surface expression of C1a in a manner similar to that of CTRdelta e13. Thus, normal cell surface expression, mobilization of intracellular calcium, and Erk activation requires the cytoplasmic C-terminal tail of the CTR, whereas the absence of the seventh TMD in the transmembrane helical bundle causes the dominant-negative effect on the surface expression of C1a.     

Protein disorder: conformational distribution of the flexible linker in a chimeric double cellulase

The structural properties of the linker peptide connecting the cellulose-binding module to the catalytic module in bimodular cellulases have been investigated by small-angle x-ray scattering. Since the linker and the cellulose-binding module are relatively small and cannot be readily detected separately, the conformation of the linker was studied by means of an artificial fusion protein, Cel6BA, in which an 88-residue linker connects the large catalytic modules of the cellulases Cel6A and Cel6B from Humicola insolens. Our data showed that Cel6BA is very elongated with a maximum dimension of 178 A, but could not be described by a single conformation. Modeling of a series of Cel6BA conformers with interdomain separations ranging between 10 A and 130 A showed that good Guinier and P(r) profile fits were obtained by a weighted average of the scattering curves of all the models where the linker follows a nonrandom distribution, with a preference for the more compact conformers. These structural properties are likely to be essential for the function of the linker as a molecular spring between the two functional modules. Small-angle x-ray scattering therefore provides a unique tool to quantitatively analyze the conformational disorder typical of proteins described as natively unfolded.     

Hot fusion

A team from Harvard University has demonstrated that human embryonic stem cells have the capacity to reprogram an adult cell nucleus, leading to a new way of deriving human embryonic stem cell lines without using oocytes.     

Characterization of a TET-like aminopeptidase complex from the hyperthermophilic archaeon Pyrococcus horikoshii

Pyrococcus horikoshii open reading frame PH1527 encodes a 39014 Da protein that shares about 30% identity with endoglucanases and members of the M42 peptidase family. Analytical ultracentrifugation and electron microscopy studies showed that the purified recombinant protein forms stable, large dodecameric complexes with a tetrahedral shape similar to the one described for DAP, a deblocking aminopeptidase that was characterized in the same organism. The two related proteins were named PhTET1 (for DAP) and PhTET2 (for PH1527). The substrate specificity and the mode of action of the PhTET2 complex were studied in detail and compared to those of PhTET1 and other assigned M42 peptidases. When assayed with short chromogenic peptides, PhTET2 was found to be an aminopeptidase, with a clear preference for leucine as the N-terminal amino acid. However, the enzyme can cleave moderately long polypeptide substrates of various compositions in a fairly unspecific manner. The hydrolytic mechanism was found to be nonprocessive. The enzyme has neither carboxypeptidase nor endoproteolytic activities, and it is devoid of N-terminal deblocking activity. PhTET2 was inhibited in the presence of EDTA and bestatin, and cobalt was found to be an activating metal. The PhTET2 protein is a highly thermostable enzyme that displays optimal activity around 100 degrees C over a broad pH array.     

Conservation of epidermal growth factor receptor function in the human parasitic helminth Schistosoma mansoni

The epidermal growth factor receptor (EGF-R) plays an important role in development and cell differentiation, and homologues of EGF-R have been identified in a broad range of vertebrate and invertebrate organisms. This work concerns the functional characterization of SER, the EGF-R-like molecule previously identified in the helminth parasite Schistosoma mansoni. Transactivation assays performed in epithelial Madin-Darby canine kidney cells co-transfected with SER and a Ras-responsive reporter vector indicated that SER was able to trigger a Ras/ERK pathway in response to human epidermal growth factor (EGF). These results were confirmed in Xenopus oocytes showing that human EGF induced meiosis reinitiation characterized by germinal vesicle breakdown in SER-expressing oocytes. Germinal vesicle breakdown induced by EGF was dependent on receptor kinase activity and shown to be associated with phosphorylation of SER and of downstream ERK proteins. (125)I-EGF binding experiments performed on SER-expressing oocytes revealed high affinity (2.9 x 10(-9) M) of the schistosome receptor for human EGF. Phosphorylation of the native SER protein present in S. mansoni membranes was also shown to occur upon binding of human EGF. These data demonstrate the ability of the SER schistosome receptor to be activated by vertebrate EGF ligands as well as to activate the classical ERK pathway downstream, indicating the conservation of EGF-R function in S. mansoni. Moreover, human EGF was shown to increase protein and DNA synthesis as well as protein phosphorylation in parasites, supporting the hypothesis that host EGF could regulate schistosome development. The possible role of SER as a receptor for host EGF peptides and its implication in host-parasite signaling and parasite development are discussed.     

SFTG international collaborative study on in vitro micronucleus test III. Using CHO cells

In this report, results are presented from an international study of the in vitro micronucleus assay using Chinese hamster ovary cells. This study was coordinated by an organizing committee supported by the SFTG (the French branch of the European Environmental Mutagen Society). Test chemicals included mannitol, bleomycin, cytosine arabinoside, urethane and diethylstilboestrol. Mitomycin C was used as a positive control. Each chemical was evaluated in at least two laboratories following a variety of different protocols (short and long exposures, varying recovery times, with and without cytochalasin B) in order to help determine a standard protocol for routine testing in Chinese hamster ovary cells. Mannitol and urethane were negative, while bleomycin, cytosine arabinoside and diethylstilboestrol induced a dose dependent increase in micronucleated cells. In the presence of cytochalasin B, increases in micronuclei were observed in binucleated as well as mononucleated cells in cultures treated with bleomycin, cytosine arabinoside or diethylstilboestrol. Importantly, all three of these chemicals were detected in each of the different treatment/recovery regimens. No differences were seen in the sensitivity or accuracy of the responses in the presence of absence of cytochalasin B. Overall, these results demonstrate the suitability of Chinese hamster ovary cells for the in vitro micronucleus assay.     

Newbouldiaquinone A: A naphthoquinone-anthraquinone ether coupled pigment, as a potential antimicrobial and antimalarial agent from Newbouldia laevis

The study of the chemical constituents of the roots of Newbouldia laevis (Bignoniaceae) has resulted in the isolation and characterization of a naphthoquinone-anthraquinone coupled pigment named newbouldiaquinone A (1) together with 14 known compounds: apigenin, chrysoeriol, newbouldiaquinone, lapachol, 2-methylanthraquinone, 2-acetylfuro-1,4-naphthoquinone, 2,3-dimethoxy-1,4-benzoquinone, oleanolic acid, canthic acid, 2-(4-hydroxyphenyl)ethyl triacontanoate, newbouldiamide, 5,7-dihydroxydehydroiso-alpha-lapachone, beta-sitosterol, and beta-sitosterol glucopyranoside. The structure elucidation of the isolated compounds was established based on spectroscopic studies, notably of the 2D NMR spectra. The antimalarial activity of compound (1) against Plasmodium falciparum in vitro shows moderate chemo suppression of parasitic growth. Its antimicrobial activity against a wide range of microorganisms was 13- and 24-fold more active against Candida gabrata and Enterobacter aerogens than the reference antibiotics nystatin and gentamycin.