Functional consequences of mutating conserved SF2 helicase motifs in the Type III restriction endonuclease EcoP15I translocase domain

For efficient DNA hydrolysis, Type III restriction endonuclease EcoP15I interacts with two inversely oriented recognition sites in an ATP-dependent process. EcoP15I consists of two methylation (Mod) subunits and a single restriction (Res) subunit yielding a multifunctional enzyme complex able to methylate or to hydrolyse DNA. Comprehensive sequence alignments, limited proteolysis and mass spectroscopy suggested that the Res subunit is a fusion of a motor or translocase (Tr) domain of superfamily II helicases and an endonuclease domain with a catalytic PD…EXK motif. In the Tr domain, seven predicted helicase motifs (I, Ia, II–VI), a recently discovered Q-tip motif and three additional regions (IIIa, IVa, Va) conserved among Type III restriction enzymes have been identified that are predicted to be involved in DNA binding and ATP hydrolysis. Because DNA unwinding activity for EcoP15I (as for bona fide helicases) has never been found and EcoP15I ATPase rates are only low, the functional importance of the helicase motifs and regions was questionable and has never been probed systematically. Therefore, we mutated all helicase motifs and conserved regions predicted in Type III restriction enzyme EcoP15I and examined the functional consequences on EcoP15I enzyme activity and the structural integrity of the variants by CD spectroscopy. The resulting eleven enzyme variants all, except variant IVa, are properly folded showing the same secondary structure distribution as the wild-type enzyme. Classical helicase motifs I–VI are important for ATP and DNA cleavage by EcoP15I and mutations therein led to complete loss of ATPase and cleavage activity. Among the catalytically inactive enzyme variants three preserved the ability to bind ATP. In contrast, newly assigned motifs Q-tip, Ia and Va are not essential for EcoP15I activity and the corresponding enzyme variants were still catalytically active. DNA binding was only marginally reduced (2–7 fold) in all enzyme variants tested.     

Analysis of genome instability in breast cancer

Breast cancer is a heterogeneous disease, previously associated with genomic instability. Our aim was to analyze microsatellite markers in order to determine patterns and levels of instability, as well as possible correlations with histopathological parameters. Polymerase chain reaction was used to characterize microsatellite instability (MSI) and loss of heterozygosity (LOH) in 107 breast carcinomas at twelve microsatellite loci. Some of the markers were selected because of their relation to steroid hormone metabolism, which seems to be related to sporadic breast cancer risk. D5S346 and D17S250 markers showed a statistically significant frequency of MSI. LOH in D3S1611, D17S250, AR and ER-β were associated with some parameters of worse prognosis. Marker group analysis showed that CYP19, AR and ER-β were related to histological grade III, ER-negative and PR-negative cases. Our results suggest that marker group analysis may be preferred to the single marker strategy, being predictive of worst prognosis when single markers are unable to provide such information. A further evaluation of steroid metabolism genes and their association with low penetrance genes in breast cancer may be useful.     

Environmental factors associated with fish distribution in an urban neotropical river (Upper Tiet�� River Basin, S?o Paulo, Brazil)

The rivers and streams of the large urban centers in Southeast Brazil are increasingly being degraded, demanding expanded conservation efforts. This study was conducted in the Grande River, one of the main tributaries of the Billings Complex, a reservoir that is a strategic fresh water resource for the S?o Paulo metropolitan region. Water quality, habitat features and fish fauna were investigated at seven sites along the longitudinal gradient with the aim of identifying the distribution patterns and relative contributions of the environmental factors. The water samples and environmental characteristics were recorded, and fish were collected during the rainy (January to March) and dry seasons (July and August) of 2009. The water quality varied along the river, with higher values of conductivity, fecal coliforms and total phosphorus in the lower reach, indicating a strong influence of the urban area. Twenty-two fish species were recorded, two of which are considered endangered. A canonical correspondence analysis (CCA) indicated marked differences in species composition between the river??s upper and lower reaches, which was mainly attributed to vegetation cover and the presence of different meso-habitats, such as riffles and pools. Trychomycterus spp. and Astyanax paranae were associated with the upper reaches, while Astyanax fasciatus and Astyanax bockmanni, Cyphocharax modestus, Hoplias malabaricus and Hypostomus ancistroides occurred in the lower reaches. Despite the disturbance in water quality and riparian vegetation in the lower river section, no detectable changes in community structure were observed. However, the presence of some tolerant species, such as Astyanax fasciatus, Hoplosternum littorale and Hypostomus ancistroides, may indicate that the community is experiencing initial stages of disturbance.     

Influence of environmental variables on the growth and reproductive cycle of Crassostrea (Mollusca,Bivalvia) in Guaratuba Bay,Brazil

The production of oysters in Guaratuba Bay, in the state of Paraná, Brazil, is still a mixed activity of mariculture and extractivism. The sustainable development of this production requires the monitoring of environmental, genetic, reproductive, and zootechnical variables. This study evaluated the importance of these variables on Crassostrea oyster production. Data were obtained between September 2009 and February 2011 from six evaluations: water quality, oyster larvae in plankton, capture of oyster spat by artificial collectors, molecular identification of collected spat and larvae, continuous evaluation of the reproductive maturity stage of adult oysters, and zootechnical performance achieved by experimental production. Temperature, dissolved oxygen, and water transparency were the environmental factors that had the most influence on the reproductive cycle (e.g. gonadal development and spat capture) and on the growth of the oyster. The highest rates for spat uptake were recorded in the summer, coinciding with the peak of sexual maturity of adults. The growth of the oysters, in terms of the weight of the meat, was related to the gonadal stage.     

Antimicrobial effects of terpinen-4-ol against oral pathogens and its capacity for the modulation of gene expression

Abstract

This study evaluated the antibacterial activity of terpinen-4-ol against Streptococcus mutans and Lactobacillus acidophilus and its influence on gbpA (S. mutans) and slpA (L. acidophilus) gene expression. As measured by XTT assay, the concentrations of terpinen-4-ol that effectively inhibited the biofilm were 0.24% and 0.95% for S. mutans and L. acidophilus, respectively. Confocal microscopy revealed the presence of a biofilm attached to the enamel and dentin block surfaces with significant terpinen-4-ol effects against these microorganisms. The expression of the gbpA and slpA genes involved in adherence and biofilm formation was investigated using RT-PCR. Expression of these genes decreased after 15?min with 0.24% and 0.95% terpinen-4-ol in S. mutans and L. acidophilus, respectively. These findings demonstrate the antimicrobial activity of terpinen-4-ol and its ability to modulate the expression of gbpA and slpA genes, emphasizing the therapeutic capacity of terpinen-4-ol as an alternative to inhibit adherence in biofilm.     

Intermolecular interactions between DNA and methamphetamine,amphetamine, ecstasy and their major metabolites

In this work, we carried out a theoretical investigation regarding amphetamine-type stimulants, which can cause central nervous system degeneration, interacting with human DNA. These include amphetamine, methamphetamine, 3,4-Methylenedioxymethamphetamine (also known as ecstasy), as well as their main metabolites. The studies were performed through molecular docking and molecular dynamics simulations, where molecular interactions of the receptor–ligand systems, along with their physical–chemical energies, were reported. Our results show that 3,4-Methylenedioxymethamphetamine and 3,4-Dihydroxymethamphetamine (ecstasy) present considerable reactivity with the receptor (DNA), suggesting that these molecules may cause damage due to human-DNA. These results were indicated by free Gibbs change of bind (ΔG_bind) values referring to intermolecular interactions between the drugs and the minor grooves of DNA, which were predominant for all simulations. In addition, it was observed that 3,4-Dihydroxymethamphetamine (ΔG_bind = ?13.15 kcal/mol) presented greater spontaneity in establishing interactions with DNA in comparison to 3,4-Methylenedioxymethamphetamine (ΔG_bind = ?8.61 kcal/mol). Thus, according with the calculations performed our results suggest that the 3,4-Methylenedioxymethamphetamine and 3,4-Dihydroxymethamphetamine have greater probability to provide damage to human DNA fragments.     

Computational study of conformational changes in human 3-hydroxy-3-methylglutaryl coenzyme reductase induced by substrate binding

Abstract

The enzyme 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) is mainly involved in the regulation of cholesterol biosynthesis. HMGR catalyses the reduction of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) to mevalonate at the expense of two NADPH molecules in a two-step reversible reaction. In the present study, we constructed a model of human HMGR (hHMGR) to explore the conformational changes of HMGR in complex with HMG-CoA and NADPH. In addition, we analysed the complete sequence of the Flap domain using molecular dynamics (MD) simulations and principal component analysis (PCA). The simulations revealed that the Flap domain plays an important role in catalytic site activation and substrate binding. The apo form of hHMGR remained in an open state, while a substrate-induced closure of the Flap domain was observed for holo hHMGR. Our study also demonstrated that the phosphorylation of Ser872 induces significant conformational changes in the Flap domain that lead to a complete closure of the active site, suggesting three principal conformations for the first stage of hHMGR catalysis. Our results were consistent with previous proposed models for the catalytic mechanism of hHMGR.

Communicated by Ramaswamy H. Sarma     

Impact of unilateral spatial neglect on chronic patient's post-stroke quality of life

Abstract

Background: Unilateral spatial neglect (USN) is the prevalent feature in patients with right-sided stroke. It is diagnosed through the behavior inattention test (BIT) and has a negative impact on patients affecting both their functional capacity and quality of life.

Objective: Here, we aimed to evaluate the impact of USN on the quality of life of patients in the chronic phase of stroke.

Methods: This is a cross-sectional study with stroke patients with USN. After confirming the presence of stroke through neuroimaging examinations and of USN through the BIT, patients’ quality of life was evaluated by using the EUROQOL scale. Spearman’s correlation was used to validate the correlation between patients’ USN and quality of life, with a p?<?.05 representing significant results.

Results: Eighteen individuals were included. When correlating the value of each domain of the EUROQOL scale with the results of the BIT, we observed a negative correlation between mobility (r?=?–0.97; p?=?.000), self-care (r?=?–0.82; p?=?.013), usual activities (r?=?–0.87; p?=?.005); pain or discomfort (r?=?–0.88; p?=?.004), anxiety or depression (r?=?–0.97; p?=?.000), and EUROQOL total score (r?=?–0.97, p?=?.000).

Conclusion: After a correlation between the overall EUROQOL and BIT scores, we suggest that the higher the USN degree is in stroke patients, the worse their perceived quality of life tends to be.     

Polyamines affect the cellular growth and structure of pro‐embryogenic masses in Araucaria angustifolia embryogenic cultures through the modulation of proton pump activities and endogenous levels of polyamines

Polyamines (PAs) are abundant polycationic compounds involved in many physiological processes in plants, including somatic embryogenesis. This study investigates the role of PAs on cellular growth and structure of pro‐embryogenic masses (PEMs), endogenous PA and proton pump activities in embryogenic suspension cultures of Araucaria angustifolia. The embryogenic suspension cultures were incubated with putrescine (Put), spermidine (Spd), spermine (Spm) and the inhibitor methylglyoxal‐bis(guanylhydrazone) (MGBG), respectively (1 mM). After 24 h and 21 days, the cellular growth and structure of PEMs, endogenous PA contents and proton pump activities were analyzed. The addition of Spm reduced the cellular growth and promoted the development of PEMs in embryogenic cultures, which could be associated with a reduction in the activities of proton pumps, such as H⁺‐ATPase P‐ and V‐types and H⁺‐PPases, and alterations in the endogenous PA contents. Spm significantly affected the physiology of the A. angustifolia somatic embryogenesis suspension, as it potentially affects cellular growth and structure of PEMs through the modulation of proton pump activities. This work demonstrates the involvement of exogenous PAs in the modulation of cellular growth and structure of PEMs, endogenous PA levels and proton pump activities during somatic embryogenesis. To our knowledge, this study is the first to report a relationship between PAs and proton pump activities in these processes. The results obtained in this study offer new perspectives for studies addressing the role of PAs and proton pump on somatic embryogenesis in this species.