RESUMO
The use of force spectroscopy approaches performed with optical tweezers can be very useful in determining the binding modes and the physical chemistry of DNA interactions with ligands, from small drugs to proteins. Helminthophagous fungi, on the other hand, have important enzyme secretion mechanisms for various purposes, and the interactions between such enzymes and nucleic acids are very poorly studied. Therefore, the main goal of the present work was to investigate, at the molecular level, the mechanisms of interaction between fungal serine proteases and the double-stranded (ds) DNA molecule. Experimental assays performed with this single molecule technique consist in exposing different concentrations of the protease of this fungus to dsDNA until saturation while monitoring the changes on the mechanical properties of the macromolecular complexes formed, from where the physical chemistry of the interaction can be deduced. It was found that the protease binds strongly to the double-helix, forming aggregates and changing the persistence length of the DNA molecule. The present work thus allowed us to infer information at the molecular level on the pathogenicity of these proteins, an important class of biological macromolecules, when applied to a target specimen.
Assuntos
Ascomicetos , Serina Proteases , Serina Proteases/genética , Ascomicetos/genética , Serina Endopeptidases , DNARESUMO
This study aimed to isolate and identify yeasts present in the intestinal microbiota of Pacific white shrimp (Litopenaeus vannamei) cultivated in a tropical estuary and carry out in-vitro assessments regarding their probiotic and aflatoxin B1 (AFB1) adsorption capacity of isolated Saccharomyces cerevisiae strains. The isolation and identification of intestinal yeasts from 40 L. vannamei individuals were performed by molecular sequencing. Three S. cerevisiaestrains (C2B, C2D and C9) were chosen for probiotic potential assessments through homologous inhibition, self-aggregation, co-aggregation, antibacterial activity, gastrointestinal condition viability, and AFB1 adsorption analyses. The following species were identified: Candida spp., Candidatropicalis, Lodderomyces elongisporus, Rhodotorula spp., and S. cerevisiae. All isolated S. cerevisiae strains presented antibacterial activity, with the C9 strain displaying better performance in the antimicrobial activity, pH viability, and AFB1 adsorption assays. It was, thus, possible to isolate Candida spp., C. tropicalis, Rhodotorula spp. and S. cerevisiae from L. vannamei shrimp, and our study demonstrated for the first time that L. elongisporus may be present in the gut of this shrimp species in captive conditions. Furthermore, the isolated S. cerevisiae strains exhibited in-vitro probiotic and AFB1 adsorption potential.(AU)
Com este estudo, objetivaram-se isolar e identificar leveduras presentes na microbiota intestinal de Litopenaeus vannamei cultivados em estuário tropical e testar in vitro a capacidade probiótica e adsorvente de aflatoxina B1 (AFB1) por cepas de Saccharomyces cerevisiae isoladas. Foram adquiridos 40 camarões da espécie L. vannamei, fizeram-se o isolamento e a identificação de leveduras intestinais por sequenciamento molecular, e foram escolhidas três cepas de S. cerevisiae (C2B, C2D e C9) para avaliar o potencial probiótico. Realizaram-se os seguintes testes: inibição homóloga, autoagregação, coagregação, atividade antibacteriana, viabilidade às condições gastrointestinais e também a análise de adsorção de AFB1. Foram identificadas as seguintes espécies: Candida spp., Candidatropicalis, Lodderomyces elongisporus, Rhodotorula spp. e S. cerevisiae. As cepas de S. cerevisiae isoladas apresentaram atividade antibacteriana, e a cepa C9 obteve melhor desempenho nos testes de atividade antimicrobiana, na viabilidade em pH e na adsorção da AFB1 do que as demais. Concluiu-se que é possível isolar Candida spp., C. tropicalis, Rhodotorulasp. e S. cerevisiae, e o estudo demonstrou pela primeira vez que L. elongisporus pode estar presente no intestino em viveiros. As cepas de S. cerevisiae isoladas do intestinos de L. vannamei possuem potencial probiótico e adsorvente de AFB1 em testes in vitro.(AU)
Assuntos
Animais , Aflatoxina B1/efeitos adversos , Probióticos/efeitos adversos , Penaeidae/fisiologia , Leveduras/isolamento & purificação , Técnicas In Vitro , Adsorção/fisiologiaRESUMO
Croton heliotropiifolius Kunth, popularly known as "quince" and "velame," contains a high concentration of volatile oils in the leaves, and widely used in folk medicine as an antiseptic, analgesic, sedative, anti-inflammatory, spasmolytic and local anesthetic. The objectives of this investigation were to (1) identify the phytochemical compounds and (2) assess the cytogenotoxicity of the essential oil extracted from the leaves of C. heliotropiifolius Kunth. The oil was extracted utilizing hydrodistillation and phytochemical profile determined using gas chromatography and mass spectrometry (GCMS). In the toxicogenetics analysis, Allium cepa roots were exposed to 1% dimethylsulfoxide or methylmethanesulfonate (MMS, 10 µg/ml) negative and positive controls, respectively, and to C. heliotropiifolius oil at 6 concentrations (0.32; 1.6; 8; 40; 200 or 1000 µg/ml). The phytochemical profile exhibited 40 chromatographic bands, and 33 compounds identified. α-pinene (16.7%) and 1,8-cineole (13.81%) were identified as the major compounds. Some of these identified secondary metabolites displayed biological and pharmacological activities previously reported including antiseptic, analgesic, sedative, anti-inflammatory as well insecticidal, antiviral, anti-fungal actions. In the A. cepa test, C. heliotropiifolius leaves oil induced cytotoxicity at concentrations of 0.32, 1.6 or 200 µg/ml and genotoxicity at 200 or 1000 µg/ml as evidenced by increased presence of micronuclei and significant chromosomal losses. Based upon our observations data demonstrated that the essential oil of C. heliotropiifolius leaves contain monoterpene hydrocarbons, and oxygenated monoterpenes, sesquiterpenes, and oxygenated sesquiterpenes which are associated with cytotoxic and genotoxic responses noted in on A. cepa cells.
Assuntos
Anti-Infecciosos Locais , Croton , Óleos Voláteis , Óleos Voláteis/toxicidade , Folhas de Planta , Monoterpenos , Hipnóticos e SedativosRESUMO
Chloroquine (CLQ) and hydroxychloroquine (HCLQ) are compounds largely employed in the treatment of various human diseases for decades. Nevertheless, a number of intrinsic details concerning their mechanisms of action, especially at the molecular level, are still unknown or have presented controversial results in the literature. Using optical tweezers, here, we investigate at the single-molecule level the molecular mechanism of action of the drug CLQ in its intrinsic interaction with the double-stranded (ds)DNA molecule, one of its targets inside cells, determining the binding modes and the physicochemical (binding) parameters of the interaction. In particular, we show that the ionic strength of the surrounding medium strongly influences such interaction, changing even the main binding mode. In addition, the cytotoxicity of CLQ against three different cell lines was also investigated here, allowing one to evaluate and compare the effect of the drug on the cell viability. In particular, we show that CLQ is highly cytotoxic at a very low (a few micromolar) concentration range for all cell lines tested. These results were rigorously compared to the equivalent ones obtained for the closely related compound hydroxychloroquine (HCLQ), allowing a critical comparison between the action of these drugs at the molecular and cellular levels.
Assuntos
Cloroquina , Hidroxicloroquina , Cloroquina/química , Cloroquina/farmacologia , DNA/química , Humanos , Hidroxicloroquina/química , Hidroxicloroquina/farmacologia , Pinças ÓpticasRESUMO
It is well reported in the literature that caffeine, the most consumed alkaloid around the world, enhances the anticancer effects of the drug cisplatin by inhibiting DNA repair by the cellular machinery. Here, we perform single-molecule force spectroscopy assays with optical tweezers to show that caffeine enhances the toxicity not only of cisplatin but also of various different platinum-based drugs already at the molecular level, using samples containing only double-stranded (ds)DNA, platinum drugs, and the alkaloid in a simple phosphate buffer, that is, completely out of the complex environment found inside real living cells. In fact, our results show that caffeine acts as an allosteric catalyst which increases the effective equilibrium binding constant between DNA and the platinum drugs, also interfering in the cooperativity of the binding reactions. To the best of our knowledge, this is the first time that such a property of caffeine was demonstrated and characterized from a pure physicochemical perspective, outside the cellular environment. Thus, the present work provides new insights into the use of this alkaloid for current chemotherapeutic applications.
Assuntos
Antineoplásicos , Cisplatino , Antineoplásicos/química , Cafeína/farmacologia , Cisplatino/farmacologia , DNA/química , Análise EspectralRESUMO
Color change associated with significative positive improve in physical properties is a challenge in wood research. This study investigated the changes in the color of the Gmelina arborea wood which underwent a thermomechanical densification process. The process was performed by applying three different temperatures (140 °C, 160 °C and 180 °C) with thickness reduction of 20% and 40% using 2.5 MPa equivalent pressure. The color change of the pieces was analyzed through the CIEL * a * b * system. The relationship between the color and the equilibrium moisture content of the densified material was also analyzed. The process reduced the lightness and yellow hue of the wood, with increased red pigment resulting in darker coloration of the treated pieces. The higher temperature used resulted in more significant changes in wood tone and lower equilibrium moisture. The process proved to be effective to change the color and significantly reduce the equilibrium moisture content in wood samples where the density was increase by 20% and 50% compared to natural wood.
Assuntos
Temperatura Alta , Madeira , CorRESUMO
The objective of this study was to evaluate the production costs of processing logs from Eucalyptus grandis Hill ex Maiden and Eucalyptus saligna Sm, using two sawing pattern in a medium-sized sawmill. Alternating tangential sawing pattern was used for logs with a mean diameter of 33.0 cm and the radial sawing pattern for logs with a mean diameter of 47.0 cm. Based on the data obtained in the different sawing pattern, the fixed, variable and total costs were calculated and, subsequently, the average value for each of these costs. For the alternating tangential sawing pattern, the fixed cost represented 19.32% and the variable cost 80.68%. For the radial sawing pattern, the fixed cost represented 18.92% and the variable cost 81.08%. The radial sawing pattern presented lower average production costs, with a difference of 22.89 R$/m³, however there is no significant difference between the sawing pattern.
Assuntos
Eucalyptus , MadeiraRESUMO
Gluconeogenesis overstimulation due to hepatic insulin resistance is the best-known mechanism behind elevated glycemia in obese subjects with hepatic steatosis. This suggests that glucose production in fatty livers may differ from that of healthy livers, also in response to other gluconeogenic determinant factors, such as the type of substrate and modulators. Thus, the aim of this study was to investigate the effects of these factors on hepatic gluconeogenesis in cafeteria diet-induced obese adult rats submitted to a cafeteria diet at a young age. The livers of the cafeteria group exhibited higher gluconeogenesis rates when glycerol was the substrate, but lower rates were found when lactate and pyruvate were the substrates. Stearate or glucagon caused higher stimulations in gluconeogenesis in cafeteria group livers, irrespective of the gluconeogenic substrates. An increased mitochondrial NADH/NAD⺠ratio and a reduced rate of 14CO2 production from [14C] fatty acids suggested restriction of the citric acid cycle. The higher glycogen and lipid levels were possibly the cause for the reduced cellular and vascular spaces found in cafeteria group livers, likely contributing to oxygen consumption restriction. In conclusion, specific substrates and gluconeogenic modulators contribute to a higher stimulation of gluconeogenesis in livers from the cafeteria group.
Assuntos
Dieta/efeitos adversos , Ácidos Graxos/metabolismo , Fígado Gorduroso/induzido quimicamente , Glucagon/metabolismo , Gluconeogênese/efeitos dos fármacos , Animais , Ingestão de Energia , Comportamento Alimentar , Glucose/metabolismo , Ácido Láctico/administração & dosagem , Ácido Láctico/farmacologia , Masculino , Obesidade/induzido quimicamente , Consumo de Oxigênio , Ácido Pirúvico/administração & dosagem , Ácido Pirúvico/farmacologia , Ratos , Ratos WistarRESUMO
Melanoma accounts for only 4% of all skin cancers but is among the most lethal cutaneous neoplasms. Dacarbazine is the drug of choice for the treatment of melanoma in Brazil through the public health system mainly because of its low cost. However, it is an alkylating agent of low specificity and elicits a therapeutic response in only 20% of cases. Other drugs available for the treatment of melanoma are expensive, and tumor cells commonly develop resistance to these drugs. The fight against melanoma demands novel, more specific drugs that are effective in killing drug-resistant tumor cells. Dibenzoylmethane (1,3-diphenylpropane-1,3-dione) derivatives are promising antitumor agents. In this study, we investigated the cytotoxic effect of 1,3-diphenyl-2-benzyl-1,3-propanedione (DPBP) on B16F10 melanoma cells as well as its direct interaction with the DNA molecule using optical tweezers. DPBP showed promising results against tumor cells and had a selectivity index of 41.94. Also, we demonstrated the ability of DPBP to interact directly with the DNA molecule. The fact that DPBP can interact with DNA in vitro allows us to hypothesize that such an interaction may also occur in vivo and, therefore, that DPBP may be an alternative to treat patients with drug-resistant melanomas. These findings can guide the development of new and more effective drugs.
Assuntos
Antineoplásicos/química , Antineoplásicos/farmacologia , Chalconas/química , Chalconas/farmacologia , DNA de Neoplasias/química , DNA de Neoplasias/efeitos dos fármacos , Animais , Antineoplásicos/síntese química , Proliferação de Células/efeitos dos fármacos , Chalconas/síntese química , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Camundongos , Estrutura Molecular , Pinças Ópticas , Relação Estrutura-Atividade , Células Tumorais CultivadasRESUMO
As a model system to study the elasticity of bottle-brush polymers, we here introduce self-assembled DNA bottle brushes, consisting of a DNA main chain that can be very long and still of precisely defined length, and precisely monodisperse polypeptide side chains that are physically bound to the DNA main chains. Polypeptide side chains have a diblock architecture, where one block is a small archaeal nucleoid protein Sso7d that strongly binds to DNA. The other block is a net neutral, hydrophilic random coil polypeptide with a length of exactly 798 amino acids. Light scattering shows that for saturated brushes the grafting density is one side chain per 5.6 nm of DNA main chain. According to small-angle X-ray scattering, the brush diameter is D = 17 nm. By analyzing configurations of adsorbed DNA bottle brushes using AFM, we find that the effective persistence of the saturated DNA bottle brushes is Peff = 95 nm, but from force-extension curves of single DNA bottle brushes measured using optical tweezers we find Peff = 15 nm. The latter is equal to the value expected for DNA coated by the Sso7d binding block alone. The apparent discrepancy between the two measurements is rationalized in terms of the scale dependence of the bottle-brush elasticity using theory previously developed to analyze the scale-dependent electrostatic stiffening of DNA at low ionic strengths.
RESUMO
The Protium heptaphyllum species, also known as Almécega, produces an oily resin, used in folk medicine as an analgesic and anti-inflammatory agent, in healing, and as an expectorant, which is rich in pentacyclic triterpenes and essential oils. In this study, the essential oil obtained by hydrodistillation of Almécega's resin was analyzed by gas chromatography-triple quadrupole mass spectrometry and evaluated for chemical composition and vasorelaxant activity in rat superior mesenteric artery. The main constituents determined by gas chromatography-triple quadrupole mass spectrometry were limonene, p-cineole, and o-cymene. In intact rings precontracted with phenylephrine (Phe 1 µM), EOPh (3-750 µg/mL) induced relaxation, and the essential oil had a concentration-dependent vasorelaxant effect, without involvement of endothelial mediators.
Assuntos
Burseraceae/química , Óleos Voláteis/química , Analgésicos/química , Analgésicos/farmacologia , Animais , Anti-Inflamatórios/química , Anti-Inflamatórios/farmacologia , Cicloexenos/química , Cicloexenos/farmacologia , Células Endoteliais/efeitos dos fármacos , Cromatografia Gasosa-Espectrometria de Massas/métodos , Limoneno , Masculino , Óleos Voláteis/farmacologia , Fenilefrina/química , Fenilefrina/farmacologia , Ratos , Ratos Wistar , Resinas Vegetais/química , Terpenos/química , Terpenos/farmacologia , Vasodilatadores/química , Vasodilatadores/farmacologiaRESUMO
Croton zehntneri (Euphorbiaceae) is a native aromatic plant from Northeast region of Brazil. The monoterpenoid estragole (ESL) has been isolated by classical chromatographic methods from the essential oil (EO) of C. zehnteneri leaves and characterized by GC-FID and GC-MS, its antimicrobial and cytotoxic potentials being assessed. The analysis of the EO enabled the identification of 100% of the integrated constituents, of which yield was about 1.8%. The main components identified were: eucalyptol, estragole (84.7%) and spathulenol. The dosage of 50 µg/disk of ESL presented fairly significant zones of inhibition against Gram-positive bacteria and fungi. The ESL presented toxicity against Artemia salina with LC50 and LC90 of 4,54 and 8,47 µg mL-1. However, in tumor inhibition assays (human cells), there were no rewarding inhibition in any of the human cancer cell lines (MCF-7, HEP-2 and NCI-H292).