DNA methyltransferases control telomere length and telomere recombination in mammalian cells

Here, we describe a role for mammalian DNA methyltransferases (DNMTs) in telomere length control. Mouse embryonic stem (ES) cells genetically deficient for DNMT1, or both DNMT3a and DNMT3b have dramatically elongated telomeres compared with wild-type controls. Mammalian telomere repeats (TTAGGG) lack the canonical CpG methylation site. However, we demonstrate that mouse subtelomeric regions are heavily methylated, and that this modification is decreased in DNMT-deficient cells. We show that other heterochromatic marks, such as histone 3 Lys 9 (H3K9) and histone 4 Lys 20 (H4K20) trimethylation, remain at both subtelomeric and telomeric regions in these cells. Lack of DNMTs also resulted in increased telomeric recombination as indicated by sister-chromatid exchanges involving telomeric sequences, and by the presence of 'alternative lengthening of telomeres' (ALT)-associated promyelocytic leukaemia (PML) bodies (APBs). This increased telomeric recombination may lead to telomere-length changes, although our results do not exclude a potential involvement of telomerase and telomere-binding proteins in the aberrant telomere elongation observed in DNMT-deficient cells. Together, these results demonstrate a previously unappreciated role for DNA methylation in maintaining telomere integrity.     

Evidence of saxitoxin derivatives as causative agents in the 1997 mass mortality of monk seals in the Cape Blanc Peninsula

Monk seals in Cape Blanc (Western Sahara coast) suffered a mass mortality during May-July 1997 which was attributed to a morbillivirus. High performance liquid chromatography (HPLC) analysis on tissues of seals killed during the outbreak and on related fauna showed peaks with retention times coincident with those of some saxitoxin derivatives but their identity was not proved. Here we present results of further HPLC analyses that unambiguously prove the identity of these toxins by mass spectrometry (MS), supporting the hypothesis that this mortality of monk seals was caused by biotoxins rather than by a morbillivirus.     

Humics as an electron donor for anaerobic respiration

The possibility that microorganisms might use reduced humic substances (humics) as an electron donor for the reduction of electron acceptors with a more positive redox potential was investigated. All of the Fe(III)- and humics-reducing microorganisms evaluated were capable of oxidizing reduced humics and/or the reduced humics analogue anthrahydroquinone-2,6,-disulphonate (AHQDS), with nitrate and/or fumarate as the electron acceptor. These included Geobacter metallireducens , Geobacter sulphurreducens , Geothrix fermentans , Shewanella alga , Wolinella succinogenes and ' S. barnesii '. Several of the humics-oxidizing microorganisms grew in medium with AHQDS as the sole electron donor and fumarate as the electron acceptor. Even though it does not reduce Fe(III) or humics, Paracoccus denitrificans could use AHQDS and reduced humics as electron donors for denitrification. However, another denitrifier, Pseudomonas denitrificans , could not. AHQDS could also serve as an electron donor for selenate and arsenate reduction by W. succinogenes . Electron spin resonance studies demonstrated that humics oxidation was associated with the oxidation of hydroquinone moieties in the humics. Studies with G. metallireducens and W. succinogenes demonstrated that the anthraquinone-2,6-disulphonate (AQDS)/AHQDS redox couple mediated an interspecies electron transfer between the two organisms. These results suggest that, as microbially reduced humics enter less reduced zones of soils and sediments, the reduced humics may serve as electron donors for microbial reduction of several environmentally significant electron acceptors.     

DNA methylation polymorphisms precede any histological sign of atherosclerosis in mice lacking apolipoprotein E

The present work investigates the occurrence and significance of aberrant DNA methylation patterns during early stages of atherosclerosis. To this end, we asked whether the genetically atherosclerosis-prone APOE-null mice show any changes in DNA methylation patterns before the appearance of histologically detectable vascular lesion. We exploited a combination of various techniques: DNA fingerprinting, in vitro methyl-accepting assay, 5-methylcytosine quantitation, histone post-translational modification analysis, Southern blotting, and PCR. Our results show that alterations in DNA methylation profiles, including both hyper- and hypomethylation, were present in aortas and PBMC of 4-week-old mutant mice with no detectable atherosclerotic lesion. Sequencing and expression analysis of 60 leukocytic polymorphisms revealed that epigenetic changes involve transcribed genic sequences, as well as repeated interspersed elements. Furthermore, we showed for the first time that atherogenic lipoproteins promote global DNA hypermethylation in a human monocyte cell line. Taken together, our results unequivocally show that alterations in DNA methylation profiles are early markers of atherosclerosis in a mouse model and may play a causative role in atherogenesis.     

Fasciola hepatica: identification of molecular markers for resistant and susceptible Pseudosuccinea columella snail hosts

Protein electrophoresis, RAPD-PCR and nuclear rDNA ITS sequencing were performed to search for genetic differences between Pseudosuccinea columella snails susceptible and resistant to Fasciola hepatica infection. Of the 21 enzymatic loci analyzed in both populations, none of them exhibited neither within- or between-group variation. Such an absence of enzyme polymorphism support the hypothesis of selfing as the "prevalent" mating system for this hermaphroditic species. Conversely, the RAPD profiles displayed clear differences between susceptible and resistant isolates for 17 of the 26 primers tested while no within-group variation was detected. rDNA ITS sequence analysis from snails of each isolates showed only two bases that differed between groups accounting for a 0.17% of variation confirming that susceptible and resistant snails belong to the same species. This is the first time that a genetic variation using RAPD markers is demonstrated between susceptible and resistant lymnaeid snails vis-a-vis of F. hepatica infection in absence of experimental selection.     

Inhibition of angiotensin converting enzyme (ACE) activity by flavan-3-ols and procyanidins

It was determined that flavan-3-ols and procyanidins have an inhibitory effect on angiotensin I converting enzyme (ACE) activity, and the effect was dependent on the number of epicatechin units forming the procyanidin. The inhibition by flavan-3-ols and procyanidins was competitive with the two substrates assayed: N-hippuryl-L-histidyl-L-leucine (HHL) and N-[3-(2-furyl)acryloyl]-L-phenylalanylglycylglycine (FAPGG). Tetramer and hexamer fractions were the more potent inhibitors, showing Ki of 5.6 and 4.7 microM, respectively. As ACE is a membrane protein, the interaction of flavanols and procyanidins with the enzyme could be related to the number of hydroxyl groups on the procyanidins, which determine their capacity to be adsorbed on the membrane surface.     

The biotransformation of the diterpene 2beta-hydroxy-ent-13-epi-manoyl oxide by Gibberella fujikuroi

Incubation of the diterpene 2beta-hydroxy-ent-13-epi-manoyl oxide with Gibberella fujikuroi afforded in good yield 2beta,6beta-dihydroxy-ent-13-epi-manoyl oxide, 2beta,12beta-dihydroxy-ent-13-epi-manoyl oxide and 2beta,20-dihydroxy-ent-13-epi-manoyl oxide, confirming that although ent-13-epi-manoyl oxide is a final metabolite of a biosynthetic branch in this fungus, more polar derivatives of this compound can be transformed by this micro-organism.     

Capillary electrophoresis-based method to quantitate DNA-protein interactions

A novel, rapid and simple capillary electrophoretic mobility shift assay (CEMSA) with laser-induced fluorescence (LIF) has been developed for the quantitative study of protein-DNA interactions. This method is particularly useful for the study of basic proteins, the most common of the DNA-interacting proteins. To avoid protein stickiness to the capillary walls we have introduced the use of neutral polyacrylamide that requires the use of reverse polarity. Under these conditions, excellent separation of DNA and protein-DNA complexes was obtained without the requirement of a gel matrix, thereby allowing the easy and reliable quantification of protein-DNA affinities. Analysis of the affinities of histones H2B and H4 for a synthetic oligo have been used to demonstrate the reproducibility and accuracy of this method. We have observed that H4 has a higher affinity for DNA than H2B, with half saturation fractions lying in the micromolar range.     

Eradication of Helicobacter pylori by 7-day triple-therapy regimens combining pantoprazole with clarithromycin, metronidazole, or amoxicillin in patients with peptic ulcer disease: results of two double-blind, randomized studies

Aim. To compare the short‐term (7‐day) safety and efficacy of two triple‐therapy regimens using pantoprazole with those of two dual‐therapy regimens (one with pantoprazole and one without), for Helicobacter pylori eradication in patients with peptic ulcer disease. Methods. H. pylori infection was identified by rapid urease (CLOtest), and confirmed by histology and culture. Patients were enrolled into one of two randomized, double‐blind, multicenter, parallel‐group studies. In study A, patients received oral pantoprazole 40 mg, clarithromycin 500 mg, and metronidazole 500 mg (PCM); pantoprazole, clarithromycin and amoxicillin 1000 mg (PCA); or pantoprazole and clarithromycin (PC). In study B, patients received PCM, PCA, PC, or clarithromycin and metronidazole without pantoprazole (CM). Treatments were given twice daily for 7 days. H. pylori status after therapy was assessed by histology and culture at 4 weeks after completing the course of study treatment. Modified intent‐to‐treat (MITT; each study: n = 424, n = 512) and per‐protocol (PP; each study: n = 371, n = 454) populations were analyzed. The MITT population comprised all patients whose positive H. pylori status was confirmed by culture and histology; the PP population comprised patients who also complied with ≥ 85% of study medication doses. Results. A total of 1016 patients were enrolled. Cure rates among patients with clarithromycin‐susceptible H. pylori strains were 82 and 86% for PCM, and 72 and 71% for PCA, in studies A and B, respectively. Cure rates among patients with metronidazole‐susceptible H. pylori strains were 82 and 87% for PCM, and 71 and 69% for PCA, in studies A and B, respectively. The combined eradication rates observed with the PCM regimen were superior to those of all other regimens tested. Side‐effects were infrequent and mild. Conclusions. PCM had the highest overall eradication rate in these two studies examining 7‐day treatment regimens. All regimens were safe and well tolerated.     

Phase-change related epigenetic and physiological changes in <Emphasis Type="Italic">Pinus radiata</Emphasis> D. Don

DNA methylation and polyamine levels were analysed before and after Pinus radiata D. Don. phase change in order to identify possible molecular and physiological phase markers. Juvenile individuals (without reproductive ability) were characterised by a degree of DNA methylation of 30-35% and a ratio of free polyamines to perchloric acid-soluble polyamine conjugates greater than 1, while mature trees (with reproductive ability) had 60% 5-methylcytosine and a ratio of free polyamines to perchloric acid-soluble polyamine conjugates of less than 1. Results obtained with trees that attained reproductive capacity during the experimental period confirmed that changes in the degree of DNA methylation and polyamine concentrations found among juvenile and mature states come about immediately after the phase change. We suggest that both indicators may be associated with the loss of morphogenic ability during ageing, particularly after phase change, through a number of molecular interactions, which are subsequently discussed.