Structure and Oligonucleotide Binding Efficiency of Differently Prepared Click Chemistry-Type DNA Microarray Slides Based on 3-Azidopropyltrimethoxysilane.

The structural characterization of glass slides surface-modified with 3-azidopropyltrimethoxysilane and used for anchoring nucleic acids, resulting in the so-called DNA microarrays, is presented. Depending on the silanization conditions, the slides were found to show different oligonucleotide binding efficiency, thus, an attempt was made to correlate this efficiency with the structural characteristics of the silane layers. Atomic force microscopy (AFM), X-ray photoelectron spectroscopy (XPS) and X-ray reflectometry (XRR) measurements provided information on the surface topography, chemical composition and thickness of the silane films, respectively. The surface for which the best oligonucleotides binding efficiency is observed, has been found to consist of a densely-packed silane layer, decorated with a high-number of additional clusters that are believed to host exposed azide groups.

Polymorphisms in the P1 promoter of the IGF-1 gene in children with growth disorders.

BACKGROUND/AIMS: The aim of this study was to associate children's growth disorders with polymorphisms detected in the P1 promoter region of IGF1 (including SNP and (CA) n microsatellite repeat polymorphism) and IGF1 and IGFPB3 levels. METHODS: IGF-1 gene P1 promoter polymorphism was analyzed in DNA obtained from the blood of 51 children with growth disorders and 50 healthy children without growth disorders by means of PCR-SSCP and sequencing. RESULTS: Among children with growth disorders and the control group we found previously described polymorphisms in the P1 promoter of the IGF-1 gene (rs35767, rs5742612) and different genotypes. The frequency of both detected polymorphisms was no significantly different in the study and the control groups. The CA repeat sequence within the group of children in the study ranged from 11 to 21. The most common were homozygote 19/19 (49.02%) and heterozygote 19/20 (27.45%). Our results did not show any association between polymorphisms in the P1 promoter and IGF-1 levels in the serum of children with growth disorders. CONCLUSIONS: This study demonstrated that SNP and (CA) n microsatellite repeat polymorphisms by themselves are not the primary regulatory elements of IGF-1 expression. However, our bioinformatics analysis has shown that the (CA) n microsatellite region in the P1 promoter of IGF-1 is able to form DNA loop structures which can modulate transcription.

Pathogenicity of different PR8 influenza A virus variants in mice is determined by both viral and host factors.

Experimental mouse models were used to compare virulence and reproduction rate of three mouse-adapted variants of the PR8 influenza A virus strain. We observed large differences in pathogenicity in two mouse strains. The PR8M variant was lethal in DBA/2J mice but not in C57BL/6J mice, whereas PR8F and hvPR8 variants were lethal in both mouse strains. High lethality of PR8M in DBA/2J correlated with high viral load at early time points after infection and spread of the virus into alveolar regions. Also, higher viral loads and mortality in mice infected with PR8F resulted in a higher number of infiltrating leukocytes. 3D-protein structure predictions of the HA indicated amino acid sequence alterations which may render the HA cleavage site in PR8F more accessible to host proteases. Infection of C57BL/6J mice with a re-assorted PR8 virus revealed that the HA gene is the main determinant of virulence of the PR8F variant.

Structural basis for the methylation of A1408 in 16S rRNA by a panaminoglycoside resistance methyltransferase NpmA from a clinical isolate and analysis of the NpmA interactions with the 30S ribosomal subunit.

NpmA, a methyltransferase that confers resistance to aminoglycosides was identified in an Escherichia coli clinical isolate. It belongs to the kanamycin-apramycin methyltransferase (Kam) family and specifically methylates the 16S rRNA at the N1 position of A1408. We determined the structures of apo-NpmA and its complexes with S-adenosylmethionine (AdoMet) and S-adenosylhomocysteine (AdoHcy) at 2.4, 2.7 and 1.68 Å, respectively. We generated a number of NpmA variants with alanine substitutions and studied their ability to bind the cofactor, to methylate A1408 in the 30S subunit, and to confer resistance to kanamycin in vivo. Residues D30, W107 and W197 were found to be essential. We have also analyzed the interactions between NpmA and the 30S subunit by footprinting experiments and computational docking. Helices 24, 42 and 44 were found to be the main NpmA-binding site. Both experimental and theoretical analyses suggest that NpmA flips out the target nucleotide A1408 to carry out the methylation. NpmA is plasmid-encoded and can be transferred between pathogenic bacteria; therefore it poses a threat to the successful use of aminoglycosides in clinical practice. The results presented here will assist in the development of specific NpmA inhibitors that could restore the potential of aminoglycoside antibiotics.

Virtual screening and experimental verification to identify potential inhibitors of the ErmC methyltransferase responsible for bacterial resistance against macrolide antibiotics.

Methyltransferases from the Erm family catalyze S-adenosyl-L-methionine-dependent modification of a specific adenine residue in bacterial 23S rRNA, thereby conferring resistance to clinically important macrolide, lincosamide, and streptogramin B antibiotics. Thus far, no inhibitors of these enzymes have been identified or designed that would effectively abolish the resistance in vivo. We used the crystal structure of ErmC' methyltransferase as a target for structure-based virtual screening of a database composed of 58,679 lead-like compounds. Among 77 compounds selected for experimental validation (63 predicted to bind to the catalytic pocket and 14 compounds predicted to bind to the putative RNA binding site), we found several novel inhibitors that decrease the minimal inhibitory concentration of a macrolide antibiotic erythromycin toward an Escherichia coli strain that constitutively expresses ErmC'. Eight of them have IC(50) values in the micromolar range. Analysis of docking models of the identified inhibitors suggests a novel strategy to develop potent and clinically useful inhibitors.