Enterocin Cross-Resistance Mediated by ABC Transport Systems.

In their struggle for life, bacteria frequently produce antagonistic substances against competitors. Antimicrobial peptides produced by bacteria (known as bacteriocins) are active against other bacteria, but harmless to their producer due to an associated immunity gene that prevents self-inhibition. However, knowledge of cross-resistance between different types of bacteriocin producer remains very limited. The immune function of certain bacteriocins produced by the Enterococcus genus (known as enterocins) is mediated by an ABC transporter. This is the case for enterocin AS-48, a gene cluster that includes two ABC transporter-like systems (Transporter-1 and 2) and an immunity protein. Transporter-2 in this cluster shows a high similarity to the ABC transporter-like system in MR10A and MR10B enterocin gene clusters. The aim of our study was to determine the possible role of this ABC transporter in cross-resistance between these two different types of enterocin. To this end, we designed different mutants (Tn5 derivative and deletion mutants) of the as-48 gene cluster in Enterococcus faecalis and cloned them into the pAM401 shuttle vector. Antimicrobial activity assays showed that enterocin AS-48 Transporter-2 is responsible for cross-resistance between AS-48 and MR10A/B enterocin producers and allowed identification of the MR10A/B immunity gene system. These findings open the way to the investigation of resistance beyond homologous bacteriocins.

Gene expression profiling reveals novel biomarkers in nonsmall cell lung cancer.

The development of reliable gene expression profiling technology is having an increasing impact on our understanding of lung cancer biology. Our study aimed to determine any correlation between the phenotypic heterogeneity and genetic diversity of lung cancer. Microarray analysis was performed on a set of 46 tumor samples and 45 paired nontumor samples of nonsmall cell lung cancer (NSCLC) samples to establish gene signatures in primary adenocarcinomas and squamous-cell carcinomas, determine differentially expressed gene sequences at different stages of the disease and identify sequences with biological significance for tumor progression. After the microarray analysis, the expression level of 92 selected genes was validated by qPCR and the robust Bonferroni test in an independent set of 70 samples composed of 48 tumor samples and 22 nontumor samples. Gene sequences were differentially expressed as a function of tumor type, stage and differentiation grade. High upregulation was observed for KRT15 and PKP1, which may be good markers to distinguish squamous-cell carcinoma samples. High downregulation was observed for DSG3 in stage IA adenocarcinomas.

Gene expression signatures in breast cancer distinguish phenotype characteristics, histologic subtypes, and tumor invasiveness.

BACKGROUND: The development of reliable gene expression profiling technology is having an increasing impact on the understanding of breast cancer biology. METHODS: In this study, microarray analysis was performed to establish gene signatures for different breast cancer phenotypes, to determine differentially expressed gene sequences at different stages of the disease, and to identify sequences with biologic significance for tumor progression. Samples were taken from patients before their treatment. After microarray analysis, the expression level of 153 selected genes was studied by real-time quantitative polymerase chain reaction analysis. RESULTS: Several gene sequences were expressed differentially in tumor samples versus control samples and also were associated with different breast cancer phenotypes, estrogen receptor status, tumor histology, and grade of tumor differentiation. In lymph node-negative tumors were identified a set of genes related to tumor differentiation grade. CONCLUSIONS: Several differentially expressed gene sequences were identified at different stages of breast cancer.

Use of capillary electrophoresis for accurate determination of CAG repeats causing Huntington disease. An oligonucleotide design avoiding shadow bands.

Huntington disease (HD) is a neurodegenerative disorder associated with the expansion of a polymorphic trinucleotide CAG repeat in the HD gene. We have developed an assay to accurately determine CAG repeats that combines a novel oligonucleotide design and the resolution of capillary electrophoresis. A mismatch in the second nucleotide from the 3' end enhanced specificity by avoiding mispriming and diminishing shadow bands and artifactual PCR products. The coupling of capillary electrophoresis analysis with the assay added the advantages of accuracy, high resolution, semi-automation, rapid analysis and low sample consumption. Analysis of 200 chromosomes in the Spanish population sample studied (control group) gave a peak frequency for 16 CAG repeats and of 7 triplets for CCG repeats. Diagnosis of HD was confirmed in 22 of 34 individuals with a range of CAG repeats from 39 to 52. Predictive testing was also carried out for 19 relatives of the HD families diagnosed at our laboratory. The method proposed in this article provides an accurate sizing of DNA repeats that can be applied to the analysis of DNA size-related disorders.

A family with atypical cystic fibrosis: brother and sister with heterozygosity for both G542X and R117H.

Clinical manifestations of cystic fibrosis (CF) are variable. Genetic and environmental factors that determine whether an individual will develop associated complications are still under investigation. The present study reports the genetic analysis of a family with different clinical forms of CF and addresses the difficulty of CF diagnosis in an individual with mutant alleles G542X and R117H because of the variable phenotype associated with R117H mutation. Both children in this family were heterozygous for G542X/R117H with the same thymine sequence (7T/9T) in intron 8 of CF transmembrane conductance regulator. The girl was diagnosed with CF, whereas the boy was diagnosed with azoospermia as the sole clinical manifestation. The possible implication of the hemochromatosis gene as a CF modifier locus was analyzed because the 2 children had the same genotype. No genetic differences were detected between brother and sister that explained the different clinical manifestations of CF.

Frequency and clinical expression of HFE gene mutations in a Spanish population of subjects with abnormal iron metabolism.

Three HFE gene mutations (HFE 845 G-->A, 187 C-->G and 193 A-->T) are the most common mutations related to hereditary haemochromatosis (HH). The genotype for these mutations was analysed in 359 Spanish individuals with altered iron metabolism and iron overload. Various biochemical parameters were measured in serum samples from 96 of these individuals, and the effect of the genotype on these parameters was studied. Allele frequencies were 12.95% for the HFE C282Y variant, 28.97% for the HFE H63D variant and 0.69% for the HFE S65C variant, calculated in a total of 718 chromosomes. Multiple comparisons analysis showed very significant differences (p=0.001) in transferrin saturation index (TSI) between the HFE C282Y variant homozygous and control (ten healthy volunteers) groups. Highly significant (p=0.0001) and significant (p=0.005) differences in serum ferritin values were found between the HFE C282Y variant homozygous and control groups and between compound (HFE C282Y/H63D variant) heterozygous and control groups, respectively. Very significant differences (p=0.001) in serum iron values were observed between the HFE C282Y variant homozygous and control groups. TSI and serum ferritin values detected most HFE C282Y variant homozygotes and are recommended to facilitate the clinical diagnosis of HH.

Trypanosoma cruzi contains a single detectable uracil-DNA glycosylase and repairs uracil exclusively via short patch base excision repair.

Enzymes involved in genomic maintenance of human parasites are attractive targets for parasite-specific drugs. The parasitic protozoan Trypanosoma cruzi contains at least two enzymes involved in the protection against potentially mutagenic uracil, a deoxyuridine triphosphate nucleotidohydrolase (dUTPase) and a uracil-DNA glycosylase belonging to the highly conserved UNG-family. Uracil-DNA glycosylase activities excise uracil from DNA and initiate a multistep base-excision repair (BER) pathway to restore the correct nucleotide sequence. Here we report the biochemical characterisation of T.cruzi UNG (TcUNG) and its contribution to the total uracil repair activity in T.cruzi. TcUNG is shown to be the major uracil-DNA glycosylase in T.cruzi. The purified recombinant TcUNG exhibits substrate preference for removal of uracil in the order ssU>U:G>U:A, and has no associated thymine-DNA glycosylase activity. T.cruzi apparently repairs U:G DNA substrate exclusively via short-patch BER, but the DNA polymerase involved surprisingly displays a vertebrate POLdelta-like pattern of inhibition. Back-up UDG activities such as SMUG, TDG and MBD4 were not found, underlying the importance of the TcUNG enzyme in protection against uracil in DNA and as a potential target for drug therapy.

Multiplex analysis of the most common mutations related to hereditary haemochromatosis: two methods combining specific amplification with capillary electrophoresis.

We present the first application of a multiplex multicolour assay for the simultaneous detection of three of the most frequent mutations related to hereditary haemochromatosis (C282Y, H63D and S65C), using fluorescent detection and capillary electrophoresis. We describe two methods: the first is based on a single base extension assay, resulting in a single base difference of the extended products; and the second is a competitive allele-specific polymerase chain reaction (PCR), based on competition between allele-specific primers. Specificity of the latter primers is enhanced with a mismatch at the antepenultimate nucleotide. Primers are designed to amplify products of different sizes and with different fluorescent dyes in order to accurately distinguish all possible combinations of genotypes (homozygous and heterozygous for each mutation) in a multiplex PCR analysis. An advantage of the present approach is that capillary electrophoresis analysis of the amplified products enables easy, rapid, unambiguous and high resolution discrimination between wild-type and mutant alleles, although different mutations may be present in the multiplex analysis. This will facilitate automated genotyping for routine molecular diagnostics and large-scale genetic studies.