La inestabilidad microsatélite y la hipermetilación del promotor mlh1 se asocian con la aparición tardía del cáncer de endometrio

[ES] El silenciamiento del gen reparador de mutaciones MLH1 por hipermetilación de su promotor es la principal causa de inestabilidad microsatélite (MSI) en tumores esporádicos tanto de origen colorectal (CC) como endometrial (CE). Los CC con MSI y MLH1 metilado ocurren en pacientes con más edad que los CC sin estas alteraciones moleculares. Sin embargo, en CE esta relación es controvertida. En este trabajo hemos estudiado la relación entre la edad y el estado MSI y de metilación de MLH1, en una serie amplia de pacientes con CE. El MSI se estableció usando tres marcadores de inestabilidad (BAT-26, BAT-25 y APA3). Para determinar la hipermetilación del promotor MLH1 se usó el MS-SSCA (methylation-sensitive single-strand conformation analysis). El 20% de las muestras de CE eran MSI, y el 80% de estas tenían MLH1 metilado. La edad en el diagnóstico de las pacientes con tumores MSI fue significativamente más elevada que la de las pacientes con tumores que presentaban estabilidad de microsatélites (MSS). (edad±SD: 67.1±9.6 años vs 63.4±10.0 años, p=0,034, test de Fisher). Los tumores MSI con MLH1 metilado ocurrieron en pacientes de más edad que los que presentan MLH1 no metilado o MSS. (68.5±9.5 años vs 61.4±8.0 años vs 63.4 ± 10.0 años, respectivamente, p=0,008, test de Chi-cuadrado). Estos resultados sugieren la existencia de diferencias epidemiológicas en pacientes con CE con diferentes estados de inestabilidad de microsatélites y de metilación del promotor MLH1.

Study of tumor suppressor genes p53 and Rb in acute myelogenous leukemia

Loss of antiproliferative function of p53 by point mutation occurred frequently in various solid tumors. However, the genetic change of p53 by deletion or point mutation was a rare event (6%) in the cells of 49 AML patients analyzed by single-stranded conformation polymorphism and sequencing. Despite infrequent point mutation, abundant levels of p53 protein were detected in 75% of AML patients studied by immunoprecipitation with p53 specific antibodies. Furthermore, p53 protein in most cases had an altered conformation as analyzed by the reactivity to PAb240 which recognizes mutant p53; p53 protein in mitogen stimulated normal lymphocytes also had similar altered conformation. This altered conformation may be another mechanism for inactivation of p53 function in the growth stimulated environment. Some evidence indicated that posttranslational modification by phosphorylation may contribute to the conformational change of p53.^ Retinoblastoma (Rb) gene inactivation by deletion, rearrangement or mutation has also been implicated in many types of solid tumors. Our studies showed that absence or low levels of Rb protein were observed in more than 20% of AML patients at diagnosis, and the low levels of Rb correlated with shorter survival of patients. The absence of Rb protein was due to gene inactivation in some cases and to abnormal regulation of Rb expression in others. ^

Phylogenetic assessment of length variation at a microsatellite locus

Sixty-six haplotypes at a locus containing a simple dinucleotide (CA)n microsatellite repeat were isolated by PCR–single-strand conformational polymorphism from populations of the horseshoe crab Limulus polyphemus. These haplotypes were sequenced to assess nucleotide variation directly. Thirty-four distinct sequences (alleles) were identified in a region 570 bp long that included the microsatellite motif. In the repeat region itself, CA-number varied in integer values from 5 to 11 across alleles, except that a (CA)8 class was not observed. Differences among alleles were due also to polymorphisms at 22 sites in regions immediately flanking the microsatellite repeats. Nucleotide substitutions in these regions were used to estimate phylogenetic relationships among alleles, and the gene phylogeny was used to trace the evolution of length variation and CA repeat numbers. A low correlation between size variation and genealogical relationships among alleles suggests that absolute fragment size (as normally scored in microsatellite assays) is an unreliable indicator of historical affinities among alleles. This finding on the molecular fine structure of microsatellite variation suggests the need for caution in the use of repeat counts at microsatellite loci as secure indicators of allelic relationships.

Sequence variation in the Fanconi anemia gene FAA

Fanconi anemia (FA) is a genetically heterogeneous autosomal recessive syndrome associated with chromosomal instability, hypersensitivity to DNA crosslinking agents, and predisposition to malignancy. The gene for FA complementation group A (FAA) recently has been cloned. The cDNA is predicted to encode a polypeptide of 1,455 amino acids, with no homologies to any known protein that might suggest a function for FAA. We have used single-strand conformational polymorphism analysis to screen genomic DNA from a panel of 97 racially and ethnically diverse FA patients from the International Fanconi Anemia Registry for mutations in the FAA gene. A total of 85 variant bands were detected. Forty-five of the variants are probably benign polymorphisms, of which nine are common and can be used for various applications, including mapping studies for other genes in this region of chromosome 16q. Amplification refractory mutation system assays were developed to simplify their detection. Forty variants are likely to be pathogenic mutations. Seventeen of these are microdeletions/microinsertions associated with short direct repeats or homonucleotide tracts, a type of mutation thought to be generated by a mechanism of slipped-strand mispairing during DNA replication. A screening of 350 FA probands from the International Fanconi Anemia Registry for two of these deletions (1115–1118del and 3788–3790del) revealed that they are carried on about 2% and 5% of the FA alleles, respectively. 3788–3790del appears in a variety of ethnic groups and is found on at least two different haplotypes. We suggest that FAA is hypermutable, and that slipped-strand mispairing, a mutational mechanism recognized as important for the generation of germ-line and somatic mutations in a variety of cancer-related genes, including p53, APC, RB1, WT1, and BRCA1, may be a major mechanism for FAA mutagenesis.

Energetics of the interaction between water and the helical peptide group and its role in determining helix propensities

The alanine helix provides a model system for studying the energetics of interaction between water and the helical peptide group, a possible major factor in the energetics of protein folding. Helix formation is enthalpy-driven (−1.0 kcal/mol per residue). Experimental transfer data (vapor phase to aqueous) for amides give the enthalpy of interaction with water of the amide group as ≈−11.5 kcal/mol. The enthalpy of the helical peptide hydrogen bond, computed for the gas phase by quantum mechanics, is −4.9 kcal/mol. These numbers give an enthalpy deficit for helix formation of −7.6 kcal/mol. To study this problem, we calculate the electrostatic solvation free energy (ESF) of the peptide groups in the helical and β-strand conformations, by using the delphi program and parse parameter set. Experimental data show that the ESF values of amides are almost entirely enthalpic. Two key results are: in the β-strand conformation, the ESF value of an interior alanine peptide group is −7.9 kcal/mol, substantially less than that of N-methylacetamide (−12.2 kcal/mol), and the helical peptide group is solvated with an ESF of −2.5 kcal/mol. These results reduce the enthalpy deficit to −1.5 kcal/mol, and desolvation of peptide groups through partial burial in the random coil may account for the remainder. Mutant peptides in the helical conformation show ESF differences among nonpolar amino acids that are comparable to observed helix propensity differences, but the ESF differences in the random coil conformation still must be subtracted.

DHPLC analysis of patients with nevoid basal cell carcinoma syndrome reveals novel PTCH missense mutations in the serol-sensing domain

Nevoid Basal Cell Carcinoma Syndrome (NBCCS) is an autosomal dominant disorder characterised by multiple basal cell carcinomas, palmar and plantar pitting, odontogenic keratocysts of the jaws and bilamellar calcification of the falx. Mutations in the PTCH gene are responsible for NBCCS but most studies have found mutations in less than half of the cases tested. We used denaturing high performance liquid chromatography (DHPLC) to screen for PTCH mutations in 28 NBCCS cases, most of whom had been previously evaluated by single stranded conformation polymorphism analysis but found to be negative. Protein truncating (n = 10) and missense or indel (n = 4) mutations were found in 14/28 (50%) cases and one additional case carried an unclassified variant, c.2777G>C. Thirteen of the variants were novel. The mutation frequency was similar in inherited and de novo cases. Three of the missense and indel mutations were in the sterol-sensing domain, and one was in the sixth transmembrane domain.

Mitochondrial inheritance during a parasexual cycle in {\it Fusarium oxysporum\/} forma specialis {\it cubense\/}

Fusarium oxysporum is a diverse, asexual fungal species composed of both saprophytic and pathogenic members. The destructive phytopathogens are classified into formae speciales based on the host species and into vegetative compatibility groups (VCGs) based on the ability of two individuals to form heterokaryons. Parasexuality, a non-sexual mode of genetic exchange unique to some fungi has been demonstrated in the laboratory in Fusarium oxysporum f. sp. cubense (FOC). The goals of this dissertation were threefold: to ascertain whether mitochondrial (mt) markers can distinguish race differences in FOC; to determine genetic relatedness of VCGs in FOC based on a mt marker; and to discover the mode of mt inheritance during a parasexual cycle.^ Band patterns produced by electrophoresis of Hae III digested genomic DNA indicated that VCG differences, not race, could be discerned by mtDNA analysis. Primers were designed to amplify a mt intergenic locus which served as a molecular marker to screen 55 strains of FOC in 16 VCGs using both single strand conformational polymorphism and DNA sequencing. Based on homogeneity of the locus, strains were assigned to seven mitotypes, a classification unit which I introduced and found informative for grouping related VCGs.^ To determine the mode of mt inheritance during a parasexual cycle, strains in different mitotypes were paired. Mitochondrial inheritance in all hybrid progeny was found to be uniparental. I speculated that if a parasexual cycle occurs in nature there would be greater variation in the nuclear genome than the mt. This could produce multiple VCGs within a mitotype, a phenomenon observed in FOC. Based on these data, I concluded that parasexuality in nature may contribute to the diversity observed in Fusarium oxysporum. ^

The isolation, characterization, and application of WRKY genes as useful molecular markers in tropical trees

The improvement of tropical tree crops using conventional breeding methods faces challenges due to the length of time involved. Thus, like most crops, there is an effort to utilize molecular genetic markers in breeding programs to select for desirable agronomic traits. Known as marker assisted breeding or marker assisted selection, genetic markers associated with a phenotype of interest are used to screen and select material reducing the time necessary to evaluate candidates. As the focus of this research was improving disease resistance in tropical trees, the usefulness of the WRKY gene superfamily was investigated as candidates for generating useful molecular genetic markers. WRKY genes encode plant-specific transcriptional factors associated with regulating plants' responses to both biotic and abiotic stress. ^ One pair of degenerate primers amplified 48 WRKY gene fragments from three taxonomically distinct, economically important, tropical tree crop species: 18 from Theobroma cacao L., 21 from Cocos nucifera L. and 9 from Persea americana Mill. Several loci from each species were polymorphic because of single nucleotide substitutions present within a putative non-coding region of the loci. Capillary array electrophoresis-single strand conformational polymorphism (CAE-SSCP) mapped four WRKY loci onto a genetic linkage map of a T. cacao F2 population segregating for resistance to witches' broom disease. Additionally, PCR primers specific for four T. cacao loci successfully amplified WRKY loci from 15 members of the Byttneriae tribe. A method was devised to allow the reliable discrimination of alleles by CAE-SSCP using only the mobility assigned to the sample peaks. Once this method was validated, the diversity of three WRKY loci was evaluated in a germplasm collection of T. cacao . One locus displayed high diversity in the collection, with at least 18 alleles detected from mobility differences of the product peaks. The number of WRKY loci available within the genome, ease of isolation by degenerate PCR, codominant segregation demonstrated in the F2 population, and usefulness for screening germplasm collections and closely related wild species demonstrates that the WRKY superfamily of genes are excellent candidates for developing a number of genetic molecular markers for breeding purposes in tropical trees. ^

Focused molecular analysis of small cell lung cancer: feasibility in routine clinical practice.

Background: There is an urgent need to identify molecular signatures in small cell lung cancer (SCLC) that may select patients who are likely to respond to molecularly targeted therapies. In this study, we investigate the feasibility of undertaking focused molecular analyses on routine diagnostic biopsies in patients with SCLC.

Methods: A series of histopathologically confirmed formalin-fixed, paraffin-embedded SCLC specimens were analysed for epidermal growth factor receptors (EGFR), KRAS, NRAS and BRAF mutations, ALK gene rearrangements and MET amplification. EGFR and KRAS mutation testing was evaluated using real time polymerase chain reaction (RT-PCR cobas®), BRAF and NRAS mutations using multiplex PCR and capillary electrophoresis-single strand conformation analysis, and ALK and MET aberrations with fluorescent in situ hybridization. All genetic aberrations detected were validated independently.

Results: A total of 105 patients diagnosed with SCLC between July 1990 and September 2006 were included. 60 (57 %) patients had suitable tumour tissue for molecular testing. 25 patients were successfully evaluated for all six pre-defined molecular aberrations. Eleven patients failed all molecular analysis. No mutations in EGFR, KRAS and NRAS were detected, and no ALK gene rearrangements or MET gene amplifications were identified. A V600E substitution in BRAF was detected in a Caucasian male smoker diagnosed with SCLC with squamoid and glandular features.

Conclusion: The paucity of patients with sufficient tumour tissue, quality of DNA extracted and low frequency of aberrations detected indicate that alternative molecular characterisation approaches are necessary, such as the use of circulating plasma DNA in patients with SCLC.

Concomitant polymorphism of an antiferromagnetically coupled dicopper(II,II) complex with single strand helical assembly: synthesis, structure, DSC, magnetic and heterogeneous catalytic studies

Two concomitant polymorphic coordination complexes (dark blue - I and black - II) with the formula (Cu2C44H60N4O4) have been synthesized and characterized crystallographically. Magnetic measurements show the presence of a strong antiferromagnetic interaction and the 2J value corresponds extremely well to the theoretically calculated one, indicating the fact that it follows nicely the magneto-structural relationship. Immobilization of the copper(II) complex I on a 2D-hexagonal mesoporous silica showed good catalytic efficiency in the liquid phase partial oxidation of olefins in the presence of TBHP as an oxidant. (C) 2009 Elsevier Ltd. All rights reserved.

Macrocycles mimic the extended peptide conformation recognized by aspartic, serine, cysteine and metallo proteases

It has been previously demonstrated that aspartic, serine, metallo and cysteine proteases bind to their inhibitors and substrate analogues in a single conformation, the saw-tooth or extended beta-strand. Consequently a generic approach to the development of protease inhibitors is the use of constraints that conformationally restrict putative inhibitor molecules to an extended form. In this way the inhibitor is pre-organized for binding to a protease and does not need to rearrange its structure. One constraining device that has proven to be effective for such pre-organization is macrocyclization. This article illustrates the general principle that macrocycles, especially those composed of 3-4 amino acids and usually 13-17 ring atoms, can effectively mimic the extended conformation of short peptide sequences. Such structure-stabilising macrocycles are stable to degradation by proteases, valuable components of potent protease inhibitors, and in many cases they are also bioavailable.

XRCC3241 Polymorphism influence on genotoxicity biomarkers frequency in workers occupationaly exposed to formaldehyde

Formaldehyde (FA) is ubiquitous in the environment and is a chemical agent that possesses high reactivity. Occupational exposure to FA has been shown to induce nasopharyngeal cancer and has been classified as carcinogenic to humans (group 1) on the basis of sufficient evidence in humans and sufficient evidence in experimental animals. The exposure to this substance is epidemiologically linked to cancer and nuclear changes detected by the cytokinesis-block micronucleus test (CBMN). This method is extensively used in molecular epidemiology, since it determines several biomarkers of genotoxicity, such as micronucleus (biomarkers of chromosomes breakage or loss), nucleoplasmic bridges (biomarker of chromosome rearrangement, poor repair and / or telomeres fusion) and nuclear buds (biomarker of elimination of amplified DNA). The gene X-ray repair cross-complementing group 3 (XRCC3) is involved in homologous recombination repair of cross-links and chromosomal double-strand breaks and at least one polymorphism has been reported in codon 241, a substitution of a methionine for a threonine.

Interaction of formaldehyde and tobacco smoking in the frequency of micronucleus and the XRCC3 Thr241Met polymorphism

Formaldehyde is classified by IARC as carcinogenic to humans (nasopharyngeal cancer). Tobacco smoke has been epidemiologically associated to a higher risk of development of cancer, especially in the oral cavity, larynx and lungs, as these are places of direct contact with many carcinogenic tobacco’s compounds. XRCC3 is involved in homologous recombination repair of cross-links and chromosomal double-strand breaks (Thr241Met polymorphism). The aim of the study is to determine whether there is an in vivo association between genetic polymorphism of the gene XRCC3 and the frequency of genotoxicity biomarkers in subjects exposed or not to formaldehyde and with or without tobacco consumption.

Effects of physical exercise training in DNA damage and repair - could the difference be in hOGG1 Ser326Cys polymorphism?

Acute physical exercise is associated with increased oxygen consumption, which could result in an increased formation of reactive oxygen species (ROS). ROS can react with several organic structures, namely DNA, causing strand breaks and a variety of modified bases in DNA. Physical exercise training seems to decrease the incidence of oxidative stress-associated diseases, and is considered as a key component of a healthy lifestyle. This is a result of exercise-induced adaptation, which has been associated with the possible increase in antioxidant activity and in oxidative damage repair enzymes, leading to an improved physiological function and enhanced resistance to oxidative stress (Radak et al. 2008). Human 8-oxoguanine DNA glycosylase 1 (hOGG1) is involved in the base excision repair (BER) pathway and encodes an enzyme responsible for removing the most common product of oxidative damage in DNA, 8-hydroxyguanine (8-OH-G). The genetic polymorphism of hOGG1 at codon 326 results in a serine (Ser) to cysteine (Cys) amino acid substitution (Ser326Cys). It has been suggested that the carriers of at least one hOGG1Cys variant allele exhibit lower 8-OH-G excision activity than the wild-type (Wilson et al. 2011). The aim of this study was to investigate the possible influence of hOGG1 Ser326Cys polymorphism on DNA damage and repair activity in response to 16 weeks of combined physical exercise training, in thirty healthy Caucasian men. Comet assay was carried out using peripheral blood lymphocytes and enabled the evaluation of DNA damage, both strand breaks and FPG-sensitive sites, and DNA repair activity. Genotypes were determined by PCR-RFLP analysis. The subjects with Ser/Ser genotype were considered as wild-type group (n=20), Ser/Cys and Cys/Cys genotype were analyzed together as mutant group (n=10). Regarding differences between pre and post-training in the wild-type group, the results showed a significant decrease in DNA strand breaks (DNA SBs) (p=0.002) and also in FPG-sensitive sites (p=0.017). No significant differences were observed in weight (p=0.389) and in lipid peroxidation (MDA) (p=0.102). A significant increase in total antioxidant capacity (evaluated by ABTS) was observed (p=0.010). Regarding mutant group, the results showed a significant decrease in DNA SBs (p=0.008) and in weight (p=0.028). No significant differences were observed in FPG-sensitive sites (p=0.916), in ABTS (p=0.074) and in MDA (p=0.086). No significant changes in DNA repair activity were observed in both genotype groups. This preliminary study suggests the possibility of different responses in DNA damage to physical exercise training, considering the hOGG1 Ser326Cys polymorphism.

Effect of length and location of heterologous sequences on RecA-mediated strand exchange.

We systematically investigated the effect of heterology on RecA-mediated strand exchange between double-stranded linear and single-stranded circular DNA. Strand exchange took place through heterologies of up to 150-200 base pairs when the insertion was at the proximal (initiating) end of the duplex DNA but was completely blocked by an insert of only 22 base pairs placed at the distal end of the duplex. In the case of medial heterology created by insertion either in the duplex or the single-stranded DNA, the ability of RecA to exchange strands decreased as the heterology was shifted toward the distal end of the duplex. These results suggest that two different strand exchange mechanisms operate in the proximal and distal portions of the duplex substrate.