A Sequence-Tagged Site Map of Human Chromosome 11

We report the construction of 370 sequence-tagged sites (STSs) that are detectable by PCR amplification under sets of standardized conditions and that have been regionally mapped to human chromosome 11. DNA sequences were determined by sequencing directly from cosmid templates using primers complementary to T3 and T7 promoters present in the cloning vector. Oligonucleotide PCR primers were predicted by computer and tested using a battery of genomic DNAs. Cosmids were regionally localized on chromosome 11 by using fluorescence in situ hybridization or by analyzing a somatic cell hybrid panel. Additional STSs corresponding to known genes and markers on chromosome 11 were also produced under the same series of standardized conditions. The resulting STSs provide uniform coverage of chromosome 11 with an average spacing of 340 kb. The DNA sequence determined for use in STS production corresponds to about 0.1% (116 kb) of chromosome 11 and has been analyzed for the presence of repetitive sequences, similarities to known genes and motifs, and possible exons. Computer analysis of this sequence has identified and therefore mapped at least eight new genes on chromosome 11.     

Multiple Enzymatic Activities Associated with Recombinant NS3 Protein of Hepatitis C Virus

The hepatitis C virus (HCV) nonstructural 3 protein (NS3) contains at least two domains associated with multiple enzymatic activities; a serine protease activity resides in the N-terminal one-third of the protein, whereas RNA helicase activity and RNA-stimulated nucleoside triphosphatase activity are associated with the C-terminal portion. To study the possible mutual influence of these enzymatic activities, a full-length NS3 polypeptide of 67 kDa was expressed as a nonfusion protein in Escherichia coli, purified to homogeneity, and shown to retain all three enzymatic activities. The protease activity of the full-length NS3 was strongly dependent on the activation by a synthetic peptide spanning the central hydrophobic core of the NS4A cofactor. Once complexed with the NS4A-derived peptide, the full-length NS3 protein and the isolated N-terminal protease domain cleaved synthetic peptide substrates with comparable efficiency. We show that, as in the case of the isolated protease domain, the protease activity of full-length NS3 undergoes inhibition by the N-terminal cleavage products of substrate peptides corresponding to the NS4A-NS4B and NS5A-NS5B. We have also characterized and quantified the NS3 ATPase, RNA helicase, and RNA-binding activities under optimized reaction conditions. Compared with the isolated N-terminal and C-terminal domains, recombinant full-length NS3 did not show significant differences in the three enzymatic activities analyzed in independent in vitro assays. We have further explored the possible interdependence of the NS3 N-terminal and C-terminal domains by analyzing the effect of polynucleotides on the modulation of all NS3 enzymatic functions. Our results demonstrated that the observed inhibition of the NS3 proteolytic activity by single-stranded RNA is mediated by direct interaction with the protease domain rather than with the helicase RNA-binding domain.     

Toxicity and genotoxicity of surface water before and after various potabilization steps

Many studies have revealed the presence of compounds with genotoxic activity in drinking water by means of short-term mutagenicity tests. In this study, the influence of the different steps of surface water treatment on the mutagenicity of drinking water was evaluated. Four different types of samples were collected: raw lake water, water after pre-disinfection with chlorine dioxide, water after filtration on granular activated carbon, and tap water. Water extracts underwent a bacterial toxicity test (Microtox test) and different in vitro genotoxicity tests: a test with Salmonella typhimurium strains, a Saccharomyces cerevisiae test, the SOS Chromotest with Escherichia coli and the Mutatox test with Vibrio fischeri. The Microtox test revealed high toxicity in the treated water samples. The disinfection steps increased the toxicity: the Mutatox test confirmed these results and the Salmonella/microsome test at the highest doses showed toxicity that could conceal mutagenicity. The SOS Chromotest was positive in all treated water samples without metabolic activation. In the test with S. cerevisiae both toxicity and genotoxicity generally increased during the water treatment steps, especially in cells without induction of cytochrome P450.     

Role of cross-talk between IFN-alpha-induced monocyte-derived dendritic cells and NK cells in priming CD8+ T cell responses against human tumor antigens

In the present study we evaluated the role of IFN-alpha in the generation of dendritic cells (IFN-DCs) with priming activity on CD8(+) T lymphocytes directed against human tumor Ags. A 3-day treatment of monocytes, obtained as adherent PBMCs from HLA-A*0201(+) healthy donors, with IFN-alpha and GM-CSF led to the differentiation of DCs displaying a semimature phenotype, but promptly inducing CD8(+) T cell responses after one in vitro sensitization with peptides derived from melanoma (gp100(209-217) and MART-1/Melan-A(27-35)) and adenocarcinoma (CEA(605-613)) Ags. However, these features were lost when IFN-DCs were generated from immunosorted CD14(+) monocytes. The ability of adherent PBMCs to differentiate into IFN-DCs expressing higher levels of costimulatory molecules and exerting efficient T cell priming capacity was associated with the presence of contaminating NK cells, which underwent phenotypic and functional activation upon IFN-alpha treatment. NK cell boost appeared to be mediated by both direct and indirect (i.e., mediated by IFN-DCs) mechanisms. Experiments performed to prove the role of contaminating NK cells in DC differentiation showed that IFN-DCs generated in the absence of NK were phenotypically less mature and could not efficiently prime antitumor CD8(+) lymphocytes. Reciprocally, IFN-DCs raised from immunosorted CD14(+) monocytes regained their T cell priming activity when NK cells were added to the culture before IFN-alpha and GM-CSF treatment. Together, our data suggest that the ability of IFN-DCs to efficiently prime anti-tumor CD8(+) T lymphocytes relied mostly on the positive cross-talk occurring between DCs and NK cells upon stimulation with IFN-alpha.     

Determination of urinary ortho- and meta-cresol in humans by headspace SPME gas chromatography/mass spectrometry

ortho-Cresol (o-C) and meta-cresol (m-C) are minor urinary metabolites of toluene, a widely used chemical with neurotoxicological properties. A new assay for their determination in human urine is here proposed. Urinary cresol sulphates and glucuronates are submitted to acid hydrolysis, urine is neutralized, added with o-cresols-d8, and analytes are sampled in the headspace of urine by SPME using a polydimethylsiloxane fiber. Analysis is performed by GC/MS using, for separation, either a SupelcoWax10 (for o-C) or a chiral CP Cresol (for o-C and m-C) column. The method is very specific, with a range of linearity 0-5.0 mg/l, within- and between-run precision, as coefficient of variation, <15% and <19%, limit of detection of 0.006 mg/l for o-C and 0.007 mg/l for m-C. The procedure is applied to the quantification of cresols in urine from workers exposed to toluene and from subjects belonging to the general population.     

A Rapid lmmunostaining Method for Frozen Sections

A simple and rapid one-step method for demonstrating immunohistochemical markers (leukocyte common antigen, cytokeratin, etc.) is described, which can help define the nature of poorly differentiated neoplasms for diagnosis using frozen section. Microwave irradiation was used to speed immunohistochemical analysis using “Enhanced Polymer One-step Staining” (EPOS) reagents on cryostat sections from a variety of pathologic samples. Reproducible results were obtained using EPOS reagents for leukocyte common antigen and cytokeratin. The overall procedure takes less than 10 min and can be completed during surgery.     

IFNL4 and IFNL3 Associated Polymorphisms Strongly Influence the Spontaneous IFN-Alpha Receptor-1 Expression in HCV-Infected Patients

Single-nucleotide polymorphism in IFNL3 gene (rs12979860) predicts spontaneous and therapy-induced HCV clearance. In a previous study from our group PBMC from patients with favourable rs12979860 genotype showed higher levels of IFNAR-1 mRNA. Recently, a dinucleotide polymorphism, ss469415590 (TT or ΔG), has been discovered in the region upstream IFNL3 gene, which is in high linkage disequilibrium with rs12979860. ss469415590[ΔG] is a frameshift variant that creates a novel gene, designed IFNL4, encoding the interferon-lambda 4 protein (IFNL4). The aim of the present study was to extend the analysis of IFNAR-1 mRNA levels to the ss469415590 variants. Our results highlight that the difference of IFNAR-1 mRNA levels between favourable and unfavourable genotype combinations, at both rs12979860 and ss469415590 loci, is stronger than that observed for single polymorphisms at each locus. These findings suggest may represent the biological basis for the observed association between IFNL3 CC and IFNL4 TT/TT genotypes and favourable outcome of either natural HCV infection (clearance vs chronic evolution) or IFN-based therapy.     

Optimization of in vivo activity of a bifunctional homing endonuclease and maturase reverses evolutionary degradation

The LAGLIDADG homing endonuclease (LHE) I-AniI has adopted an extremely efficient secondary RNA splicing activity that is beneficial to its host, balanced against inefficient DNA cleavage. A selection experiment identified point mutations in the enzyme that act synergistically to improve endonuclease activity. The amino-acid substitutions increase target affinity, alter the thermal cleavage profile and significantly increase targeted recombination in transfected cells. The RNA splicing activity is not affected by these mutations. The improvement in DNA cleavage activity is largely focused on one of the enzyme's two active sites, corresponding to a rearrangement of a lysine residue hypothesized to act as a general base. Most of the constructs isolated in the screen contain one or more mutations that revert an amino-acid identity to a residue found in one or more close homologues of I-AniI. This implies that mutations that have previously reduced the endonuclease activity of I-AniI are identified and reversed, sometimes in combination with additional ‘artificial’ mutations, to optimize its in vivo activity.     

Effects of species traits on the genetic diversity of high-mountain plants: a multi-species study across the Alps and the Carpathians

Aim  To test the influence of various species traits, elevation and phylogeographical history on the genetic diversity of high-mountain plants in the Alps and Carpathians.
Location  The regular sampling grid comprised the whole range of the European Alps and the Carpathians.
Methods  Twenty-two high-mountain plant species were exhaustively sampled and their genetic diversity was assessed with amplified fragment length polymorphisms (AFLPs). ANOVAs were used to check for relationships between species traits and species genetic diversity, and to test whether genetic diversity was influenced by altitude and phylogeographical history (i.e. Alps versus Carpathians).
Results  In both mountain systems, species dispersed and pollinated by wind showed higher genetic diversity than species with self or insect pollination, and with animal- or gravity-dispersed seeds. Only in the Alps did altitudinal range size affect species genetic diversity significantly: species with narrow altitudinal ranges in the highest vegetation belts had significantly higher genetic diversity than those expanding over wide altitudinal ranges. Genetic diversity was species specific and significantly higher in the Alps than in the Carpathians, but it was not influenced by elevation.
Main conclusions  Wind pollination and wind dispersal seem to foster high genetic diversity. However, species traits are often associated and their effects on genetic diversity cannot be clearly disentangled. As genetic diversity is species specific, comparisons across species need to be interpreted with care. Genetic diversity was generally lower in the Carpathians than in the Alps, due to higher topographical isolation of alpine habitats in the Carpathians and this mountain massif's divergent phylogeographical history. Elevation did not influence genetic diversity, challenging the long-held view of decreasing genetic diversity with increasing elevation in mountain plants.