Evaluation of DNA damage in exfoliated tear duct epithelial cells from individuals exposed to air pollution assessed by single cell gel electrophoresis assay

The search for relevant target cells for human monitoring purposes has increased during the last few years. Cells such as sperm, buccal or nasal and gastric epithelium are being used. In this study, we report the use of exfoliated tear duct epithelial cells as a potential material for human biomonitoring studies, since these cells are a target for environmental pollutants. We employed the alkaline single cell gel electrophoresis (SCGE) assay to evaluate for differences in the basal level of DNA damage between young adults from the south (exposed mainly to high levels of ozone) and from the north (exposed principally to hydrocarbons) regions of Mexico City. We found an increase in DNA migration in tear duct epithelial cells from individuals who live in the southern part of the city compared to those living in the northern part. Moreover, young people who live in the southwest part of the city with the highest values of ozone presented the highest values of DNA damage. These results show the feasibility of using exfoliated tear duct epithelial cells in human biomonitoring studies.     

Recombinagenic and mutagenic activities of fluoroquinolones in Drosophila melanogaster

Fluoroquinolones are widely used in human and in veterinary medicine due to their broad-spectrum antibacterial activity. They act by inhibiting type II DNA topoisomerases (gyrase and topoisomerase IV). Because of the sequence homology between prokaryotic and eukaryotic topoisomerases II, fluoroquinolones can pose a hazard to eukaryotic cells. However, published information concerning the genotoxic profiles of these drugs in vivo is sparse and inconsistent. We have assessed the activities of three fluoroquinolones, ciprofloxacin, enrofloxacin and norfloxacin, in the Drosophila melanogaster Somatic Mutation and Recombination Test (SMART) and measured their mutagenic and recombinagenic potentials. Norfloxacin was non-genotoxic. Ciprofloxacin and enrofloxacin induced significant increases in spot frequencies in trans-heterozygous flies. To test the roles of somatic recombination and mutation in the observed genotoxicity, balancer-heterozygous flies were also analyzed. Ciprofloxacin and enrofloxacin were preferential inducers of homologous recombination in proliferative cells, an event linked to loss of heterozygosity.     

Swine and poultry pathogens: the complete genome sequences of two strains of Mycoplasma hyopneumoniae and a strain of Mycoplasma synoviae

This work reports the results of analyses of three complete mycoplasma genomes, a pathogenic (7448) and a nonpathogenic (J) strain of the swine pathogen Mycoplasma hyopneumoniae and a strain of the avian pathogen Mycoplasma synoviae; the genome sizes of the three strains were 920,079 bp, 897,405 bp, and 799,476 bp, respectively. These genomes were compared with other sequenced mycoplasma genomes reported in the literature to examine several aspects of mycoplasma evolution. Strain-specific regions, including integrative and conjugal elements, and genome rearrangements and alterations in adhesin sequences were observed in the M. hyopneumoniae strains, and all of these were potentially related to pathogenicity. Genomic comparisons revealed that reduction in genome size implied loss of redundant metabolic pathways, with maintenance of alternative routes in different species. Horizontal gene transfer was consistently observed between M. synoviae and Mycoplasma gallisepticum. Our analyses indicated a likely transfer event of hemagglutinin-coding DNA sequences from M. gallisepticum to M. synoviae.     

NOPCHAP1 is a PAQosome cofactor that helps loading NOP58 on RUVBL1/2 during box C/D snoRNP biogenesis

The PAQosome is a large complex composed of the HSP90/R2TP chaperone and a prefoldin-like module. It promotes the biogenesis of cellular machineries but it is unclear how it discriminates closely related client proteins. Among the main PAQosome clients are C/D snoRNPs and in particular their core protein NOP58. Using NOP58 mutants and proteomic experiments, we identify different assembly intermediates and show that C12ORF45, which we rename NOPCHAP1, acts as a bridge between NOP58 and PAQosome. NOPCHAP1 makes direct physical interactions with the CC-NOP domain of NOP58 and domain II of RUVBL1/2 AAA+ ATPases. Interestingly, NOPCHAP1 interaction with RUVBL1/2 is disrupted upon ATP binding. Moreover, while it robustly binds both yeast and human NOP58, it makes little interactions with NOP56 and PRPF31, two other closely related CC-NOP proteins. Expression of NOP58, but not NOP56 or PRPF31, is decreased in NOPCHAP1 KO cells. We propose that NOPCHAP1 is a client-loading PAQosome cofactor that selects NOP58 to promote box C/D snoRNP assembly.     

NUFIP and the HSP90/R2TP chaperone bind the SMN complex and facilitate assembly of U4-specific proteins

The Sm proteins are loaded on snRNAs by the SMN complex, but how snRNP-specific proteins are assembled remains poorly characterized. U4 snRNP and box C/D snoRNPs have structural similarities. They both contain the 15.5K and proteins with NOP domains (PRP31 for U4, NOP56/58 for snoRNPs). Biogenesis of box C/D snoRNPs involves NUFIP and the HSP90/R2TP chaperone system and here, we explore the function of this machinery in U4 RNP assembly. We show that yeast Prp31 interacts with several components of the NUFIP/R2TP machinery, and that these interactions are separable from each other. In human cells, PRP31 mutants that fail to stably associate with U4 snRNA still interact with components of the NUFIP/R2TP system, indicating that these interactions precede binding of PRP31 to U4 snRNA. Knock-down of NUFIP leads to mislocalization of PRP31 and decreased association with U4. Moreover, NUFIP is associated with the SMN complex through direct interactions with Gemin3 and Gemin6. Altogether, our data suggest a model in which the NUFIP/R2TP system is connected with the SMN complex and facilitates assembly of U4 snRNP-specific proteins.     

In vitro segmentation induction of Mesocestoides corti (Cestoda) tetrathyridia

Mesocestoides corti is a suitable model for studying cestode development because of its ability to reproduce asexually and segment in vitro. The cultured parasite is also capable of sexual differentiation and, probably, reproduction. To establish conditions that increase the efficiency of in vitro M. corti larvae (tetrathyridia) segmentation, we tested the effects of an inducing agent and some physical parameters in cultures. We found that a 5% CO2-95% N2 gas phase, an incubation temperature of 39 C (instead of 37 C), and a 24-hr pretreatment with trypsin (10(5) BAEE/ml, BAEE = Na-benzoil-L-arginine ethyl ester unit of trypsin activity) in Roswell Park Memorial Institute (RPMI) 1640 medium supplemented with 20% fetal bovine serum (FBS) are able to increase individually or synergistically the segmentation rate of tetrathyridia. A segmentation rate of up to 100% was achieved on day 4 of culture, when all these conditions were used simultaneously, in comparison with an average rate of 40% obtained not before day 11 in cultures without any inducing treatment. Fetal bovine serum is essential for segmentation, and a concentration of 20% was established as the standard for induction.     

Evaluation of the activity of filamentous fungi isolated from soils of the Pampa biome applied in the biological control of Tetranychus urticae (Acari: Tetranychidae) and Polyphagotarsonemus latus (Acari: Tarsonemidae)

Experimental and Applied Acarology - Tetranychus urticae Koch and Polyphagotarsonemus latus Banks are mite species considered capable of attaining pest levels, damaging a range of agricultural...     

MLL-SEPTIN gene fusions in hematological malignancies

The mixed lineage leukemia (MLL) locus is involved in more than 60 different rearrangements with a remarkably diverse group of fusion partners in approximately 10% of human leukemias. MLL rearrangements include chromosomal translocations, gene internal duplications, chromosome 11q deletions or inversions and MLL gene insertions into other chromosomes, or vice versa. MLL fusion partners can be classified into four distinct categories: nuclear proteins, cytoplasmatic proteins, histone acetyltransferases and septins. Five different septin genes (SEPT2, SEPT5, SEPT6, SEPT9, and SEPT11) have been identified as MLL fusion partners, giving rise to chimeric fusion proteins in which the N terminus of MLL is fused, in frame, to almost the entire open reading frame of the septin partner gene. The rearranged alleles result from heterogeneous breaks in distinct introns of both MLL and its septin fusion partner, originating distinct gene fusion variants. MLL-SEPTIN rearrangements have been repeatedly identified in de novo and therapy related myeloid neoplasia in both children and adults, and some clinicopathogenetic associations are being uncovered. The fundamental roles of septins in cytokinesis, membrane remodeling and compartmentalization can provide some clues on how abnormalities in the septin cytoskeleton and MLL deregulation could be involved in the pathogenesis of hematological malignancies.     

Design,synthesis, cholinesterase inhibition and molecular modelling study of novel tacrine hybrids with carbohydrate derivatives

A series of hybrids containing tacrine linked to carbohydrate-based moieties, such as d-xylose, d-ribose, and d-galactose derivatives, were synthesized by the nucleophilic substitution between 9-aminoalkylamino-1,2,3,4-tetrahydroacridines and the corresponding sugar-based tosylates. All compounds were found to be potent inhibitors of both acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) in the nanomolar IC₅₀ scale. Most of the d-xylose derivatives (6a-e) were selective for AChE and the compound 6e (IC₅₀?=?2.2?nM for AChE and 4.93?nM for BuChE) was the most active compound for both enzymes. The d-galactose derivative 8a was the most selective for AChE exhibiting an IC₅₀ ratio of 7.6 for AChE over BuChE. Only two compounds showed a preference for BuChE, namely 7a (d-ribose derivative) and 6b (d-xylose derivative). Molecular docking studies indicated that the inhibitors are capable of interacting with the entire binding cavity and the main contribution of the linker is to enable the most favorable positioning of the two moieties with CAS, PAS, and hydrophobic pocket to provide optimal interactions with the binding cavity. This finding is reinforced by the fact that there is no linear correlation between the linker size and the observed binding affinities. The majority of the new hybrids synthesized in this work do not violate the Lipinski's rule-of-five according to FAF-Drugs4, and do not demonstrated predicted hepatotoxicity according ProTox-II.     

Non-specific binding of Na+ and Mg2+ to RNA determined by force spectroscopy methods

RNA duplex stability depends strongly on ionic conditions, and inside cells RNAs are exposed to both monovalent and multivalent ions. Despite recent advances, we do not have general methods to quantitatively account for the effects of monovalent and multivalent ions on RNA stability, and the thermodynamic parameters for secondary structure prediction have only been derived at 1M [Na(+)]. Here, by mechanically unfolding and folding a 20 bp RNA hairpin using optical tweezers, we study the RNA thermodynamics and kinetics at different monovalent and mixed monovalent/Mg(2+) salt conditions. We measure the unfolding and folding rupture forces and apply Kramers theory to extract accurate information about the hairpin free energy landscape under tension at a wide range of ionic conditions. We obtain non-specific corrections for the free energy of formation of the RNA hairpin and measure how the distance of the transition state to the folded state changes with force and ionic strength. We experimentally validate the Tightly Bound Ion model and obtain values for the persistence length of ssRNA. Finally, we test the approximate rule by which the non-specific binding affinity of divalent cations at a given concentration is equivalent to that of monovalent cations taken at 100-fold concentration for small molecular constructs.