Conformation, protein recognition and repair of DNA interstrand and intrastrand cross-links of Antitumor trans-[PtCl(2)(NH(3))(thiazole)]

Replacement of one ammine in clinically ineffective trans-[PtCl(2)(NH(3))(2)] (transplatin) by a planar N-heterocycle, thiazole, results in significantly enhanced cytotoxicity. Unlike ‘classical’ cisplatin {cis-[PtCl(2)(NH(3))(2)]} or transplatin, modification of DNA by this prototypical cytotoxic transplatinum complex trans-[PtCl(2)(NH(3))(thiazole)] (trans-PtTz) leads to monofunctional and bifunctional intra or interstrand adducts in roughly equal proportions. DNA fragments containing site-specific bifunctional DNA adducts of trans-PtTz were prepared. The structural distortions induced in DNA by these adducts and their consequences for high-mobility group protein recognition, DNA polymerization and nucleotide excision repair were assessed in cell-free media by biochemical methods. Whereas monofunctional adducts of trans-PtTz behave similar to the major intrastrand adduct of cisplatin [J. Kasparkova, O. Novakova, N. Farrell and V. Brabec (2003) Biochemistry, 42, 792–800], bifunctional cross-links behave distinctly differently. The results suggest that the multiple DNA lesions available to trans-planaramine complexes may all contribute substantially to their cytotoxicity so that the overall drug cytotoxicity could be the sum of the contributions of each of these adducts. However, acquisition of drug resistance could be a relatively rare event, since it would have to entail resistance to or tolerance of multiple, structurally dissimilar DNA lesions

Effect of abdominal binding on respiratory mechanics during exercise in athletes with cervical spinal cord injury

West CR, Goosey-Tolfrey VL, Campbell IG, Romer LM. Effect of abdominal binding on respiratory mechanics during exercise in athletes with cervical spinal cord injury. J Appl Physiol 117: 36–45, 2014. First published May 22, 2014; doi:10.1152/japplphysiol.00218.2014.—We asked whether elastic binding of the abdomen influences respiratory mechanics during wheelchair propulsion in athletes with cervical spinal cord injury (SCI). Eight Paralympic wheelchair rugby players with motor-complete SCI (C5-C7) performed submaximal and maximal incremental exercise tests on a treadmill, both with and without abdominal binding. Measurements included pulmonary function, pressure-derived indices of respiratory mechanics, operating lung volumes, tidal flow-volume data, gas exchange, blood lactate, and symptoms. Residual volume and functional residual capacity were reduced with binding (77 18 and 81 11% of unbound, P 0.05), vital capacity was increased (114 9%, P 0.05), whereas total lung capacity was relatively well preserved (99 5%). During exercise, binding introduced a passive increase in transdiaphragmatic pressure, due primarily to an increase in gastric pressure. Active pressures during inspiration were similar across conditions. A sudden, sustained rise in operating lung volumes was evident in the unbound condition, and these volumes were shifted downward with binding. Expiratory flow limitation did not occur in any subject and there was substantial reserve to increase flow and volume in both conditions. V ˙ O2 was elevated with binding during the final stages of exercise (8 –12%, P 0.05), whereas blood lactate concentration was reduced (16 –19%, P 0.05). V ˙ O2/heart rate slopes were less steep with binding (62 35 vs. 47 24 ml/beat, P 0.05). Ventilation, symptoms, and work rates were similar across conditions. The results suggest that abdominal binding shifts tidal breathing to lower lung volumes without influencing flow limitation, symptoms, or exercise tolerance. Changes in respiratory mechanics with binding may benefit O2 transport capacity by an improvement in central circulatory function.This article has been made available through the Brunel Open Access Publishing Fund

Yeast mitochondrial HMG proteins: DNA-binding properties of the most evolutionarily divergent component of mitochondrial nucleoids

Comparative biochemical analysis of mtHMG proteins from distantly related yeast species revealed that they exhibit a preference for recombination/replication intermediates. We discuss how these biochemical characteristics relate to the role of mtHMG proteins in mtDNA compaction and evolution.Yeast mtDNA is compacted into nucleoprotein structures called mitochondrial nucleoids (mt-nucleoids). The principal mediators of nucleoid formation are mitochondrial high-mobility group (HMG)-box containing (mtHMG) proteins. Although these proteins are some of the fastest evolving components of mt-nucleoids, it is not known whether the divergence of mtHMG proteins on the level of their amino acid sequences is accompanied by diversification of their biochemical properties. In the present study we performed a comparative biochemical analysis of yeast mtHMG proteins from Saccharomyces cerevisiae (ScAbf2p), Yarrowia lipolytica (YlMhb1p) and Candida parapsilosis (CpGcf1p). We found that all three proteins exhibit relatively weak binding to intact dsDNA. In fact, ScAbf2p and YlMhb1p bind quantitatively to this substrate only at very high protein to DNA ratios and CpGcf1p shows only negligible binding to dsDNA. In contrast, the proteins exhibit much higher preference for recombination intermediates such as Holliday junctions (HJ) and replication forks (RF). Therefore, we hypothesize that the roles of the yeast mtHMG proteins in maintenance and compaction of mtDNA invivo are in large part mediated by their binding to recombination/replication intermediates. We also speculate that the distinct biochemical properties of CpGcf1p may represent one of the prerequisites for frequent evolutionary tinkering with the form of the mitochondrial genome in the CTG-clade of hemiascomycetous yeast species

Srs2 promotes Mus81-Mms4-mediated resolution of recombination intermediates

A variety of DNA lesions, secondary DNA structures or topological stress within the DNA template may lead to stalling of the replication fork. Recovery of such forks is essential for the maintenance of genomic stability. The structure-specific endonuclease Mus81–Mms4 has been implicated in processing DNA intermediates that arise from collapsed forks and homologous recombination. According to previous genetic studies, the Srs2 helicase may play a role in the repair of double-strand breaks and ssDNA gaps together with Mus81–Mms4. In this study, we show that the Srs2 and Mus81–Mms4 proteins physically interact in vitro and in vivo and we map the interaction domains within the Srs2 and Mus81 proteins. Further, we show that Srs2 plays a dual role in the stimulation of the Mus81–Mms4 nuclease activity on a variety of DNA substrates. First, Srs2 directly stimulates Mus81–Mms4 nuclease activity independent of its helicase activity. Second, Srs2 removes Rad51 from DNA to allow access of Mus81–Mms4 to cleave DNA. Concomitantly, Mus81–Mms4 inhibits the helicase activity of Srs2. Taken together, our data point to a coordinated role of Mus81–Mms4 and Srs2 in processing of recombination as well as replication intermediates

Srs2 mediates PCNA-SUMO-dependent inhibition of DNA repair synthesis

Completion of DNA replication needs to be ensured even when challenged with fork progression problems or DNA damage. PCNA and its modifications constitute a molecular switch to control distinct repair pathways. In yeast, SUMOylated PCNA (S-PCNA) recruits Srs2 to sites of replication where Srs2 can disrupt Rad51 filaments and prevent homologous recombination (HR). We report here an unexpected additional mechanism by which S-PCNA and Srs2 block the synthesis-dependent extension of a recombination intermediate, thus limiting its potentially hazardous resolution in association with a cross-over. This new Srs2 activity requires the SUMO interaction motif at its C-terminus, but neither its translocase activity nor its interaction with Rad51. Srs2 binding to S-PCNA dissociates Polδ and Polη from the repair synthesis machinery, thus revealing a novel regulatory mechanism controlling spontaneous genome rearrangements. Our results suggest that cycling cells use the Siz1-dependent SUMOylation of PCNA to limit the extension of repair synthesis during template switch or HR and attenuate reciprocal DNA strand exchanges to maintain genome stability. © 2013 European Molecular Biology Organization

The PCNA interaction protein box sequence in Rad54 is an integral part of its ATPase domain and is required for efficient DNA repair and recombination

Rad54 is an ATP-driven translocase involved in the genome maintenance pathway of homologous recombination (HR). Although its activity has been implicated in several steps of HR, its exact role(s) at each step are still not fully understood. We have identified a new interaction between Rad54 and the replicative DNA clamp, proliferating cell nuclear antigen (PCNA). This interaction was only mildly weakened by the mutation of two key hydrophobic residues in the highly-conserved PCNA interaction motif (PIP-box) of Rad54 (Rad54-AA). Intriguingly, the rad54-AA mutant cells displayed sensitivity to DNA damage and showed HR defects similar to the null mutant, despite retaining its ability to interact with HR proteins and to be recruited to HR foci in vivo. We therefore surmised that the PCNA interaction might be impaired in vivo and was unable to promote repair synthesis during HR. Indeed, the Rad54-AA mutant was defective in primer extension at the MAT locus as well as in vitro, but additional biochemical analysis revealed that this mutant also had diminished ATPase activity and an inability to promote D-loop formation. Further mutational analysis of the putative PIP-box uncovered that other phenotypically relevant mutants in this domain also resulted in a loss of ATPase activity. Therefore, we have found that although Rad54 interacts with PCNA, the PIP-box motif likely plays only a minor role in stabilizing the PCNA interaction, and rather, this conserved domain is probably an extension of the ATPase domain III

Estatinas: análise da compreensão de seus usuários sobre sua importância e reações adversas

O colesterol tem papel essencial no funcionamento do corpo humano. No entanto, quando seus níveis no sangue atingem valores elevados, essa substância pode se tornar um grande vilão da saúde. Para o tratamento do colesterol elevado, os profissionais de saúde frequentemente recorrem aos medicamentos da classe das estatinas. Essa pesquisa teve como objetivo investigar o conhecimento dos usuários que fazem uso de estatinas, tendo focado em sua importância e reações adversas. Para isso foi feito uma pesquisa exploratória, formulado um questionário para levantamento de dados com 19 perguntas e aplicado a 100 pessoas, via formulário, que fazem acompanhamento médico e tratamento medicamentoso. Destes participantes, a maioria foram mulheres (62%), a maioria está na faixa etária de 65 a 75 anos (32%) e a maioria tem renda de até 1 salário mínimo (42%). A maioria tem bons conhecimentos sobre o colesterol (92%) e sobre suas implicações (97%). Além disso, a maioria faz o tratamento com Sinvastatina (69%) de modo contínuo (86%). Poucos participantes relataram a necessidade de aumento da dose (11%) e de troca da medicação (22%). A maioria faz o tratamento sem ter suspendido ou abandonado (93%) e a maioria apresentou efeitos colaterais (82%), sendo a maioria a mialgia (64,6%). Por fim, a maioria daqueles que tiveram mialgia foram orientados a fazer uso de vitamina B (84,9%) por médicos (68,9%). Conclui-se que a população acometida pelo colesterol elevado tem bons conhecimentos sobre a doença e que as estatinas são medicamentos seguros e eficazes para o tratamento dessa doença

O uso de Zolpidem para tratamento de insônia

More than 73 million Brazilians suffer from insomnia, mainly in the country's large centers. Among the main causes of this problem are stress, anxiety, irregular sleeping habits, lifestyle and use of screens (cell phone, TV, etc.) at night. To treat this problem, many doctors prescribe zolpidem to their patients, as the medication is used to treat this sleep disorder. This research aimed to evaluate the use of zolpidem as a non-benzoadizepine sedative-hypnotic prescribed for insomnia. To this end, an exploratory research was carried out, a questionnaire for data collection with 12 questions was formulated and applied to 100 people, by form, who already use zolpidem. Of these participants, the majority were women (72%), the majority were between 25 and 35 years old (36%) and the majority had been using the medication for more than 1 year (58%). The majority reported an improvement in sleep quality after using the medication (97%) and the majority did not need to increase the medication dose (75%). Furthermore, the majority rarely forgot to administer the medication (76%), the majority never stopped administering it due to symptom improvement (71%) or worsening (92%) and the majority never administered the medication on their own (71% ). The majority administer another medication along with zolpidem (80%), the majority did not have any adverse reactions (74%), the majority did not attempt to wean off the medication (80%) and the majority never provided pills to people close to them (91% ). It is concluded that zolpidem is safe to treat sleep disorders with medical advice, and prolonged use should be avoided to avoid possible adverse reactions and dependence. The need for individualized assessment of each patient is evident. Keywords: 1. Zolpidem. 2. Insomnia. 3. Adverse reactions. 4. Medicine. 5. Sleep.Mais de 73 milhões de brasileiros sofrem com insônia, principalmente nos grandes centros do país. Entre as principais causas para esse problema estão o estresse, ansiedade, hábitos de sono irregulares, estilo de vida e uso de telas (celular, TV etc.) no período noturno. Para o tratamento desse problema, muitos médicos prescrevem o zolpidem para seus pacientes, visto que o medicamento serve para o tratamento desse distúrbio do sono. Essa pesquisa visou avaliar o uso do medicamento zolpidem como um sedativo-hipnótico não benzoadizepínico prescrito para insônia. Para isso foi feito uma pesquisa exploratória, formulado um questionário para levantamento de dados com 12 perguntas e aplicado a 100 pessoas, via formulário, que já fazem uso do zolpidem. Destes participantes, a maioria foram mulheres (72%), a maioria tem entre 25 e 35 anos (36%) e a maioria faz uso do medicamento a mais de 1 ano (58%). A maioria relatou melhoria na qualidade do sono após o uso do medicamento (97%) e a maioria não teve necessidade de aumento da dose do medicamento (75%). Além disso, a maioria raramente esqueceu de administrar o medicamento (76%), a maioria nunca deixou de administrar por melhora dos sintomas (71%) ou por piora (92%) e a maioria nunca administrou o medicamento por conta própria (71%). A maioria administra outro medicamento junto com o zolpidem (80%), a maioria não teve nenhuma reação adversa (74%), a maioria não tentou o desmame do medicamento (80%) e a maioria nunca forneceu comprimidos para pessoas próximas (91%). Conclui-se que o zolpidem é seguro para tratar distúrbios do sono com orientações médicas, devendo ser evitado o uso prolongado para evitar possíveis reações adversas e dependência. Fica evidente a necessidade de avaliação individualizada de cada paciente. Palavras-chave: 1. Zolpidem. 2. Insônia. 3. Reações adversas. 4. Medicamento. 5. Sono

Tuberculosis en gatos domésticos de la provincia de Santa Fe, Argentina

La tuberculosis en felinos es causada principalmente por Mycobacterium bovis (M. bovis), micobacteria zoonótica con amplio rango de hospedadores susceptibles. La provincia de Santa Fe, principal cuenca lechera del país, tiene la mayor cantidad de casos de tuberculosis zoonótica y animal. La tuberculosis animal por M. bovis es cada vez más comunicada en otras especies diferentes a los bovinos, como son los recientes reportes en felinos domésticos. La transmisión en los felinos puede ser tanto aerógena (entre gatos o por contacto con animales infectados) como digestiva (más común en felinos urbanos, por consumo de vísceras contaminadas). El objetivo de este trabajo es documentar casos de M. bovis en felinos de la provincia de Santa Fe, Argentina, y describir los diferentes genotipos presentes en esta población.Trabajo publicado en Cagliada, Maria del Pilar Lilia y Galosi, Cecilia Mónica (comps.). I Congreso de Microbiología Veterinaria. Libro de resúmenes. La Plata: Facultad de Ciencias Veterinarias, 2021.Facultad de Ciencias Veterinaria

RNA extraction from self-assembling peptide hydrogels to allow qPCR analysis of encapsulated cells

Self-assembling peptide hydrogels offer a novel 3-dimensional platform for many applications in cell culture and tissue engineering but are not compatible with current methods of RNA isolation; owing to interactions between RNA and the biomaterial. This study investigates the use of two techniques based on two different basic extraction principles: solution-based extraction and direct solid-state binding of RNA respectively, to extract RNA from cells encapsulated in four β-sheet forming self-assembling peptide hydrogels with varying net positive charge. RNA-peptide fibril interactions, rather than RNA-peptide molecular complexing, were found to interfere with the extraction process resulting in low yields. A column-based approach relying on RNA-specific binding was shown to be more suited to extracting RNA with higher purity from these peptide hydrogels owing to its reliance on strong specific RNA binding interactions which compete directly with RNA-peptide fibril interactions. In order to reduce the amount of fibrils present and improve RNA yields a broad spectrum enzyme solution—pronase—was used to partially digest the hydrogels before RNA extraction. This pre-treatment was shown to significantly increase the yield of RNA extracted, allowing downstream RT-qPCR to be performed