Surface modification of hydrophobic polymers for improvement of endothelial cell-surface interactions

The aim of this study is to improve the interaction of endothelial cells with polymers used in vascular prostheses. Polytetrafluoroethylene (PTFE; Teflon) films were treated by means of nitrogen and oxygen plasmas. Depending on the plasma exposure time, modified PTFE surfaces showed water-contact angles of 15¿58° versus 96° for unmodified PTFE. Electron spectroscopy in chemical analysis (ESCA) measurements revealed incorporation of both nitrogenand oxygen-containing groups into the PTFE surfaces, dependent on the plasma composition and exposure time. In-vitro biological evaluation of unmodified and modified PTFE surfaces showed that human endothelial cells, seeded from 20% human serum-containing culture medium, adhered well on to modified PTFE surfaces, but not on to unmodified films. Adhesion of endothelial cells on to expanded PTFE graft material (Gore-Tex) was also stimulated by plasma treatment of this substrate. On plasma-treated expanded PTFE, the adhering endothelial cells formed a monolayer, which covered the textured surface. The latter observation is important in view of the hemocompatibility of vascular grafts seeded with endothelial cells before implantation

A Further Unique Chondroitin Sulfate from the shrimp Litopenaeus vannamei with Antithrombin Activity that Modulates Acute Inflammation

The detailed structure of a further Chondroitin Sulfate from Litopenaeus vannamei shrimp (sCS) is described. The backbone structure was established by 1H/13C NMR, which identified 3-O-sulfated GlcA, 4-O-sulfated GalNAc, 6-O-sulfated GalNAc, and 4,6-di-O-sulfated GalNAc residues. GlcA is linked to GalNAc 4,6 di S and GlcA 3S is linked to GalNAc 4S, GalNAc 4,6 di-S and GalNAc6S residues. The anticoagulant properties of this sCS were evaluated by activated partial thromboplastin time, anti-IIa, anti-Xa and anti-heparin cofactor II-mediated activities, and sCS failed to stabilise antithrombin in a fluoresence shift assay. The anti-inflammatory effect of sCS was explored using a model of acute peritonitis, followed by leukocyte count and measurement of the cytokines, IL-1β, IL-6 and TNF-α. The compound showed low clotting effects, but high anti-IIa activity and HCII-mediated thrombin inhibition. Its anti-inflammatory effect was shown by leukocyte recruitment inhibition and a decrease in pro-inflammatory cytokine levels. Although the biological role of sCS remains unknown, its properties indicate that it is suitable for studies of multi-potent molecules obtained from natural sources

Benznidazole biotransformation and multiple targets in <i>Trypanosoma</i> cruzi revealed by metabolomics

&lt;b&gt;Background&lt;/b&gt;&lt;p&gt;&lt;/p&gt; The first line treatment for Chagas disease, a neglected tropical disease caused by the protozoan parasite Trypanosoma cruzi, involves administration of benznidazole (Bzn). Bzn is a 2-nitroimidazole pro-drug which requires nitroreduction to become active, although its mode of action is not fully understood. In the present work we used a non-targeted MS-based metabolomics approach to study the metabolic response of T. cruzi to Bzn.&lt;p&gt;&lt;/p&gt; &lt;b&gt;Methodology/Principal findings&lt;/b&gt;&lt;p&gt;&lt;/p&gt; Parasites treated with Bzn were minimally altered compared to untreated trypanosomes, although the redox active thiols trypanothione, homotrypanothione and cysteine were significantly diminished in abundance post-treatment. In addition, multiple Bzn-derived metabolites were detected after treatment. These metabolites included reduction products, fragments and covalent adducts of reduced Bzn linked to each of the major low molecular weight thiols: trypanothione, glutathione, γ-glutamylcysteine, glutathionylspermidine, cysteine and ovothiol A. Bzn products known to be generated in vitro by the unusual trypanosomal nitroreductase, TcNTRI, were found within the parasites, but low molecular weight adducts of glyoxal, a proposed toxic end-product of NTRI Bzn metabolism, were not detected.&lt;p&gt;&lt;/p&gt; &lt;b&gt;Conclusions/significance&lt;/b&gt;&lt;p&gt;&lt;/p&gt; Our data is indicative of a major role of the thiol binding capacity of Bzn reduction products in the mechanism of Bzn toxicity against T. cruzi

Os efeitos do cigarro e do consumo de café sobre a formação óssea e a integração óssea de implantes de hidroxiapatita

The present study aims to assess the effects of cigarette smoke inhalation and/or coffee consumption on bone formation and osseous integration of a dense hydroxyapatite (DHA) implant in rats. For this study, 20 male rats were divided into four groups (n = 5): CT (control) group, CE (coffee) group, CI (cigarette) group and CC (coffee + cigarette) group. During 16 weeks, animals in the CI group were exposed to cigarette smoke inhalation equivalent to 6 cigarettes per day; specimens in the CE group drank coffee as liquid diet; and rats in the CC group were submitted to both substances. In the 6th week a 5 mm slit in the parietal bone and a 4 mm slit in the tibia were performed on the left side: the former was left open while the latter received a DHA implant. As soon as surgeries were finished, the animals returned to their original protocols and after 10 weeks of exposure they were euthanised (ethically sacrificed) and the mentioned bones collected for histological processing. Data showed that exposure to cigarette smoke inhalation and coffee consumption did not interfere in weight gain and that solid and liquid diet consumption was satisfactory. Rats in the CC group showed a decrease in bone neoformation around the tibial DHA implant (31.8 ± 2.8) as well as in bone formation in the parietal slit (28.6 ± 2.2). On their own, cigarette smoke inhalation or coffee consumption also led to diminished bone neoformation around the implant and delayed the bone repair process in relation to the CT group. However, reduction in the bone repair process was accentuated with exposure to both cigarette smoke inhalation and coffee consumption in this study.O presente estudo teve como objetivo avaliar os efeitos do tabagismo e do consumo de café, isolada ou concomitantemente, sobre a formação óssea e a osseointegração de implantes hidroxiapatita densa. Foram utilizados 20 ratos machos, divididos em quatro grupos (n = 5): grupo CT (controle); grupo CA (café); grupo CI (cigarro), e grupo CC (cigarro + café). Durante 16 semanas, os animais do grupo CI foram expostos à fumaça de seis cigarros/dia; os animais do grupo CA consumiram café como dieta líquida, e os animais do grupo CC, ambas as substâncias. Após seis semanas de exposição, uma falha óssea de 5 mm foi produzida no osso parietal esquerdo e de 4 mm, na tíbia esquerda dos animais. A falha do parietal foi mantida aberta, enquanto na tíbia corpos cerâmicos de hidroxiapatita densa (HAD) foram implantados em cavidade produzida cirurgicamente. Após as cirurgias, os animais retornaram aos protocolos experimentais e, ao término de dez semanas, foram eutanasiados, sendo as tíbias e os parietais coletados para processamento histológico. A exposição à fumaça do cigarro e o consumo de café não interferiram no ganho de peso dos animais, e os consumos de dieta líquida e sólida foram satisfatórios entre os grupos. Os animais do grupo CC apresentaram menor volume de osso neoformado ao redor do implante de HAD na tíbia (31,8 ± 2,8) e menor osteogênese na falha produzida no osso parietal (28,6 ± 2,2). O café e o cigarro consumidos isoladamente provocam a diminuição do volume de osso ao redor do implante e o atraso no processo de reparação óssea. Observou-se que o consumo de café associado à exposição à fumaça do cigarro reduziu de forma acentuada o processo de reparação óssea, no presente estudo.17317

Os efeitos do cigarro e do consumo de café sobre a formação óssea e a integração óssea de implantes de hidroxiapatita

The present study aims to assess the effects of cigarette smoke inhalation and/or coffee consumption on bone formation and osseous integration of a dense hydroxyapatite (DHA) implant in rats. For this study, 20 male rats were divided into four groups (n = 5): CT (control) group, CE (coffee) group, CI (cigarette) group and CC (coffee + cigarette) group. During 16 weeks, animals in the CI group were exposed to cigarette smoke inhalation equivalent to 6 cigarettes per day; specimens in the CE group drank coffee as liquid diet; and rats in the CC group were submitted to both substances. In the 6th week a 5 mm slit in the parietal bone and a 4 mm slit in the tibia were performed on the left side: the former was left open while the latter received a DHA implant. As soon as surgeries were finished, the animals returned to their original protocols and after 10 weeks of exposure they were euthanised (ethically sacrificed) and the mentioned bones collected for histological processing. Data showed that exposure to cigarette smoke inhalation and coffee consumption did not interfere in weight gain and that solid and liquid diet consumption was satisfactory. Rats in the CC group showed a decrease in bone neoformation around the tibial DHA implant (31.8 ± 2.8) as well as in bone formation in the parietal slit (28.6 ± 2.2). On their own, cigarette smoke inhalation or coffee consumption also led to diminished bone neoformation around the implant and delayed the bone repair process in relation to the CT group. However, reduction in the bone repair process was accentuated with exposure to both cigarette smoke inhalation and coffee consumption in this study.O presente estudo teve como objetivo avaliar os efeitos do tabagismo e do consumo de café, isolada ou concomitantemente, sobre a formação óssea e a osseointegração de implantes hidroxiapatita densa. Foram utilizados 20 ratos machos, divididos em quatro grupos (n = 5): grupo CT (controle); grupo CA (café); grupo CI (cigarro), e grupo CC (cigarro + café). Durante 16 semanas, os animais do grupo CI foram expostos à fumaça de seis cigarros/dia; os animais do grupo CA consumiram café como dieta líquida, e os animais do grupo CC, ambas as substâncias. Após seis semanas de exposição, uma falha óssea de 5 mm foi produzida no osso parietal esquerdo e de 4 mm, na tíbia esquerda dos animais. A falha do parietal foi mantida aberta, enquanto na tíbia corpos cerâmicos de hidroxiapatita densa (HAD) foram implantados em cavidade produzida cirurgicamente. Após as cirurgias, os animais retornaram aos protocolos experimentais e, ao término de dez semanas, foram eutanasiados, sendo as tíbias e os parietais coletados para processamento histológico. A exposição à fumaça do cigarro e o consumo de café não interferiram no ganho de peso dos animais, e os consumos de dieta líquida e sólida foram satisfatórios entre os grupos. Os animais do grupo CC apresentaram menor volume de osso neoformado ao redor do implante de HAD na tíbia (31,8 ± 2,8) e menor osteogênese na falha produzida no osso parietal (28,6 ± 2,2). O café e o cigarro consumidos isoladamente provocam a diminuição do volume de osso ao redor do implante e o atraso no processo de reparação óssea. Observou-se que o consumo de café associado à exposição à fumaça do cigarro reduziu de forma acentuada o processo de reparação óssea, no presente estudo.Universidade José do Rosário VellanoUniversidade Federal de São Paulo (UNIFESP) Escola Paulista de MedicinaUniversidade Federal de AlfenasUniversidade Estadual de CampinasUniversidade José do Rosário Vellano Faculdade de MedicinaUNIFESP, EPMSciEL

The regulatory subunit of PKA-I remains partially structured and undergoes β-aggregation upon thermal denaturation

Background: The regulatory subunit (R) of cAMP-dependent protein kinase (PKA) is a modular flexible protein that responds with large conformational changes to the binding of the effector cAMP. Considering its highly dynamic nature, the protein is rather stable. We studied the thermal denaturation of full-length RIα and a truncated RIα(92-381) that contains the tandem cyclic nucleotide binding (CNB) domains A and B. Methodology/Principal Findings: As revealed by circular dichroism (CD) and differential scanning calorimetry, both RIα proteins contain significant residual structure in the heat-denatured state. As evidenced by CD, the predominantly α-helical spectrum at 25°C with double negative peaks at 209 and 222 nm changes to a spectrum with a single negative peak at 212-216 nm, characteristic of β-structure. A similar α→β transition occurs at higher temperature in the presence of cAMP. Thioflavin T fluorescence and atomic force microscopy studies support the notion that the structural transition is associated with cross-β-intermolecular aggregation and formation of non-fibrillar oligomers. Conclusions/Significance: Thermal denaturation of RIα leads to partial loss of native packing with exposure of aggregation-prone motifs, such as the B' helices in the phosphate-binding cassettes of both CNB domains. The topology of the β-sandwiches in these domains favors inter-molecular β-aggregation, which is suppressed in the ligand-bound states of RIα under physiological conditions. Moreover, our results reveal that the CNB domains persist as structural cores through heat-denaturation. © 2011 Dao et al

Contribution of microscopy for understanding the mechanism of action against trypanosomatids

Transmission electron microscopy (TEM) has proved to be a useful tool to study the ultrastructural alterations and the target organelles of new antitrypanosomatid drugs. Thus, it has been observed that sesquiterpene lactones induce diverse ultrastructural alterations in both T. cruzi and Leishmania spp., such as cytoplasmic vacuolization, appearance of multilamellar structures, condensation of nuclear DNA, and, in some cases, an important accumulation of lipid vacuoles. This accumulation could be related to apoptotic events. Some of the sesquiterpene lactones (e.g., psilostachyin) have also been demonstrated to cause an intense mitochondrial swelling accompanied by a visible kinetoplast deformation as well as the appearance of multivesicular bodies. This mitochondrial swelling could be related to the generation of oxidative stress and associated to alterations in the ergosterol metabolism. The appearance of multilamellar structures and multiple kinetoplasts and flagella induced by the sesquiterpene lactone psilostachyin C indicates that this compound would act at the parasite cell cycle level, in an intermediate stage between kinetoplast segregation and nuclear division. In turn, the diterpene lactone icetexane has proved to induce the external membrane budding on T. cruzi together with an apparent disorganization of the pericellar cytoskeleton. Thus, ultrastructural TEM studies allow elucidating the possible mechanisms and the subsequent identification of molecular targets for the action of natural compounds on trypanosomatids.Fil: Lozano, Esteban Sebastián. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Medicina y Biología Experimental de Cuyo; ArgentinaFil: Spina Zapata, Renata María. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos. Universidad Nacional de Cuyo. Facultad de Ciencias Médicas. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos; ArgentinaFil: Barrera, Patricia Andrea. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos. Universidad Nacional de Cuyo. Facultad de Ciencias Médicas. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos; ArgentinaFil: Tonn, Carlos Eugenio. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - San Luis. Instituto de Investigaciones en Tecnología Química. Universidad Nacional de San Luis. Facultad de Química, Bioquímica y Farmacia. Instituto de Investigaciones en Tecnología Química; ArgentinaFil: Sosa Escudero, Miguel Angel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos. Universidad Nacional de Cuyo. Facultad de Ciencias Médicas. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos; Argentin