AMIDO E HIPOCLORITO DE SÓDIO NO ENRAIZAMENTO IN VITRO DO ABACAXIZEIRO ‘GOLD’ E SEUS EFEITOS NA ACLIMATIZAÇÃO

Este trabalho teve como objetivo avaliar a substituição total ou parcial do ágar por amidos de milho e mandioca e da esterilizaçãofísica em autoclave pela esterilização química com hipoclorito de sódio (NaClO) no desenvolvimento de mudas deabacaxizeiro ‘Gold’ (Ananas comosus). Como agentes geleificantes,foram utilizados: M1: ágar (6,0 g L-1);M2: amido de milho (60,0 g L-1); M3: ágar (3,0 g L-1) + amido de milho (30,0 g L-1), e M4: ágar (3,0 g L-1) + amido de mandioca (30,0 g L-1). As esterilizações foram feitas por meio da autoclavagem a 121°C e da utilização de soluções de NaClO nas concentrações de 0,003% para o enxágue das vidrarias e 0,0003% para adição ao meio de cultivo. Após um mês de cultivo in vitro, não houve influência negativa do NaClO e dos agentes geleificantes no enraizamento das brotações para a maioria dos parâmetros avaliados. As mudas produzidas in vitro foram levadas para aclimatização em casa de vegetação, por 90 dias. Na aclimatização, as mudas oriundas dos tratamentos com esterilização química não diferiram das oriundas dos meios autoclavados em nenhum parâmetro avaliado, enquanto a combinação ágar + amido de milho proporcionou resultados inferiores entre os agentes geleificantes.

AMIDO DE MILHO E HIPOCLORITO DE SÓDIO NO ENRAIZAMENTO IN VITRO DO ABACAXIZEIRO VITÓRIA E SEU EFEITO NA ACLIMATIZAÇÃO

Este trabalho objetivou reduzir o custo de produção dos meios de cultura pela substituição do ágar por amido de milho e da esterilização física em autoclave pela esterilização química com hipoclorito de sódio (NaClO) em mudas de abacaxizeiro ‘Vitória’ propagadas in vitro. As brotações foram transferidas para meio de enraizamento composto pelos sais de MS, vitaminas de White, mioinositol e sacarose. O experimento foi instalado em DIC, em fatorial 3x2: meios geleificados com ágar (6,0 g L-1), amido de milho (60,0 g L-1) e ágar (3,0 g L-1) + amido de milho (30,0 g L-1), esterilizados em autoclave ou quimicamente com a fervura do meio e uso de NaClO a 0,05% para enxaguar a vidraria. Após um mês de enraizamento in vitro, parte das mudas foi avaliada quanto ao número de folhas e de raízes, e massas das matérias fresca e seca. O restante foi aclimatizado por 90 dias em casa de vegetação, ao final dos quais foram avaliados número de folhas, número de raízes, massa da matéria fresca e seca da parte aérea, raízes e total, área foliar, altura e diâmetro da roseta. A fervura do meio + esterilização química das vidrarias não diferiu da autoclavagem para a maioria dos parâmetros avaliados, enquanto o amido de milho proporcionou melhor desenvolvimento das mudas, tanto in vitro quanto durante a aclimatização.

Ligand binding to heparan sulfate proteoglycans induces their aggregation and distribution along actin cytoskeleton

Cell surface heparan sulfate proteoglycans (HSPGs) participate in molecular events that regulate cell adhesion, migration, and proliferation. The present study demonstrates that soluble heparin-binding proteins or cross-linking antibodies induce the aggregation of cell surface HSPGs and their distribution along underlying actin filaments. Immunofluorescence and confocal microscopy and immunogold and electron microscopy indicate that, in the absence of ligands, HSPGs are irregularly distributed on the fibroblast cell surface, without any apparent codistribution with the actin cytoskeleton. In the presence of ligand (lipoprotein lipase) or antibodies against heparan sulfate, HSPGs aggregate and colocalize with the actin cytoskeleton. Triton X-100 extraction and immunoelectron microscopy have demonstrated that in this condition HSPGs were clustered and associated with the actin filaments. Crosslinking experiments that use biotinylated lipoprotein lipase have revealed three major proteoglycans as binding sites at the fibroblast cell surface. These cross-linked proteoglycans appeared in the Triton X-100 insoluble fraction. Platinum/carbon replicas of the fibroblast surface incubated either with lipoprotein lipase or antiheparan sulfate showed large aggregates of HSPGs regularly distributed along cytoplasmic fibers. Quantification of the spacing between HSPGs by confocal microscopy confirmed that the nonrandom distribution of HSPG aggregates along the actin cytoskeleton was induced by ligand binding. When cells were incubated either with lipoprotein lipase or antibodies against heparan sulfate, the distance between immunofluorescence spots was uniform. In contrast, the spacing between HSPGs on fixed cells not incubated with ligand was more variable. This highly organized spatial relationship between actin and proteoglycans suggests that cortical actin filaments could organize the molecular machinery involved in signal transduction and molecular movements on the cell surface that are triggered by heparin-binding proteins.

Allosteric conversation in the androgen receptor ligand-binding domain surfaces

Androgen receptor (AR) is a major therapeutic target that plays pivotal roles in prostate cancer (PCa) and androgen insensitivity syndromes. We previously proposed that compounds recruited to ligand-binding domain (LBD) surfaces could regulate AR activity in hormone-refractory PCa and discovered several surface modulators of AR function. Surprisingly, the most effective compounds bound preferentially to a surface of unknown function [binding function 3 (BF-3)] instead of the coactivator-binding site [activation function 2 (AF-2)]. Different BF-3 mutations have been identified in PCa or androgen insensitivity syndrome patients, and they can strongly affect AR activity. Further, comparison of AR x-ray structures with and without bound ligands at BF-3 and AF-2 showed structural coupling between both pockets. Here, we combine experimental evidence and molecular dynamic simulations to investigate whether BF-3 mutations affect AR LBD function and dynamics possibly via allosteric conversation between surface sites. Our data indicate that AF-2 conformation is indeed closely coupled to BF-3 and provide mechanistic proof of their structural interconnection. BF-3 mutations may function as allosteric elicitors, probably shifting the AR LBD conformational ensemble toward conformations that alter AF-2 propensity to reorganize into subpockets that accommodate N-terminal domain and coactivator peptides. The induced conformation may result in either increased or decreased AR activity. Activating BF-3 mutations also favor the formation of another pocket (BF-4) in the vicinity of AF-2 and BF-3, which we also previously identified as a hot spot for a small compound. We discuss the possibility that BF-3 may be a protein-docking site that binds to the N-terminal domain and corepressors. AR surface sites are attractive pharmacological targets to develop allosteric modulators that might be alternative lead compounds for drug design.

Programmed death ligand 1 expression and tumor-infiltrating lymphocytes in glioblastoma.

BACKGROUND: Immune checkpoint inhibitors targeting programmed cell death 1 (PD1) or its ligand (PD-L1) showed activity in several cancer types. METHODS: We performed immunohistochemistry for CD3, CD8, CD20, HLA-DR, phosphatase and tensin homolog (PTEN), PD-1, and PD-L1 and pyrosequencing for assessment of the O6-methylguanine-methyltransferase (MGMT) promoter methylation status in 135 glioblastoma specimens (117 initial resection, 18 first local recurrence). PD-L1 gene expression was analyzed in 446 cases from The Cancer Genome Atlas. RESULTS: Diffuse/fibrillary PD-L1 expression of variable extent, with or without interspersed epithelioid tumor cells with membranous PD-L1 expression, was observed in 103 of 117 (88.0%) newly diagnosed and 13 of 18 (72.2%) recurrent glioblastoma specimens. Sparse-to-moderate density of tumor-infiltrating lymphocytes (TILs) was found in 85 of 117 (72.6%) specimens (CD3+ 78/117, 66.7%; CD8+ 52/117, 44.4%; CD20+ 27/117, 23.1%; PD1+ 34/117, 29.1%). PD1+ TIL density correlated positively with CD3+ (P < .001), CD8+ (P < .001), CD20+ TIL density (P < .001), and PTEN expression (P = .035). Enrichment of specimens with low PD-L1 gene expression levels was observed in the proneural and G-CIMP glioblastoma subtypes and in specimens with high PD-L1 gene expression in the mesenchymal subtype (P = 5.966e-10). No significant differences in PD-L1 expression or TIL density between initial and recurrent glioblastoma specimens or correlation of PD-L1 expression or TIL density with patient age or outcome were evident. CONCLUSION: TILs and PD-L1 expression are detectable in the majority of glioblastoma samples but are not related to outcome. Because the target is present, a clinical study with specific immune checkpoint inhibitors seems to be warranted in glioblastoma.

Protein pocket and ligand shape comparison and its application in virtual screening.

Understanding molecular recognition is one major requirement for drug discovery and design. Physicochemical and shape complementarity between two binding partners is the driving force during complex formation. In this study, the impact of shape within this process is analyzed. Protein binding pockets and co-crystallized ligands are represented by normalized principal moments of inertia ratios (NPRs). The corresponding descriptor space is triangular, with its corners occupied by spherical, discoid, and elongated shapes. An analysis of a selected set of sc-PDB complexes suggests that pockets and bound ligands avoid spherical shapes, which are, however, prevalent in small unoccupied pockets. Furthermore, a direct shape comparison confirms previous studies that on average only one third of a pocket is filled by its bound ligand, supplemented by a 50 % subpocket coverage. In this study, we found that shape complementary is expressed by low pairwise shape distances in NPR space, short distances between the centers-of-mass, and small deviations in the angle between the first principal ellipsoid axes. Furthermore, it is assessed how different binding pocket parameters are related to bioactivity and binding efficiency of the co-crystallized ligand. In addition, the performance of different shape and size parameters of pockets and ligands is evaluated in a virtual screening scenario performed on four representative targets.

Comparing assays for the measurement of AhR ligand activity

In this thesis (TFG) the results of the comparison of three assays for the measurement of AhR ligand activity are exposed. This study was part of a collaborative project aiming at the characterization of the AhR signaling activities of known naturally occurring compounds to explore the potential of using non-toxic compounds to treat inflammatory diseases via oral administration. The first goal of this project was to find an assay able to measure AhR-activity, so the comparison of different assays has been done in order to find the most convenient one according to the efficiency, sensitivity and precision. Moreover, other elements with operational nature such as price, toxicity of components or ease of use has been considered. From the use of compounds known from the literature to be AhR ligands, three assays have been tested: (1) P450-GloTM CYP1A2 Induction/Inhibition assay, (2) quantitative Polymerase Chain Reaction (qPCR) and (3) DR. CALUX® Bioassay. Moreover, a different experiment using the last assay was performed for the study in vivo of the transport of the compounds tested. The results of the TFG suggested the DR. CALUX® Bioassay as the most promising assay to be used for the screening of samples as AhR-ligands because it is quicker, easier to handle and less expensive than qPCR and more reproducible than the CYP1A2 Induction/Inhibition assay. Moreover, the use of this assay allowed having a first idea of which compounds are uptaken by the epithelial barrier and in with direction the transport happens.

Estimating the value of the metal-ligand bond dissociation enthalpy<D> (M-L) for adducts using empirical equations supported by TG data

In this work is presented and tested (for 106 adducts, mainly of the zinc group halides) two empirical equations supported in TG data to estimate the value of the metal-ligand bond dissociation enthalpy for adducts: <D> (M-O) = t i / g if t i < 420 K and <D> (M-O) = (t i / g ) - 7,75 . 10-2 . t i if t i > 420 K. In this empirical equations, t i is the thermodynamic temperature of the beginning of the thermal decomposition of the adduct, as determined by thermogravimetry, andg is a constant factor that is function of the metal halide considered and of the number of ligands, but is not dependant of the ligand itself. To half of the tested adducts the difference between experimental and calculated values was less than 5%. To about 80% of the tested adducts, the difference between the experimental (calorimetric) and the calculated (using the proposed equations) values are less than 15%.

Role of PheE15 gate in ligand entry and nitric oxide detoxification function of Mycobacterium tuberculosis truncated hemoglobin N

The truncated hemoglobin N, HbN, of Mycobacterium tuberculosis is endowed with a potent nitric oxide dioxygenase (NOD) activity that allows it to relieve nitrosative stress and enhance in vivo survival of its host. Despite its small size, the protein matrix of HbN hosts a two-branched tunnel, consisting of orthogonal short and long channels, that connects the heme active site to the protein surface. A novel dual-path mechanism has been suggested to drive migration of O(2) and NO to the distal heme cavity. While oxygen migrates mainly by the short path, a ligand-induced conformational change regulates opening of the long tunnel branch for NO, via a phenylalanine (PheE15) residue that acts as a gate. Site-directed mutagenesis and molecular simulations have been used to examine the gating role played by PheE15 in modulating the NOD function of HbN. Mutants carrying replacement of PheE15 with alanine, isoleucine, tyrosine and tryptophan have similar O(2)/CO association kinetics, but display significant reduction in their NOD function. Molecular simulations substantiated that mutation at the PheE15 gate confers significant changes in the long tunnel, and therefore may affect the migration of ligands. These results support the pivotal role of PheE15 gate in modulating the diffusion of NO via the long tunnel branch in the oxygenated protein, and hence the NOD function of HbN.

The saccharinate anion: a versatile and fascinating ligand in coordination chemistry

The saccharinate anion, obtained by deprotonation of the N-H moiety of saccharin (o-sulfobenzimide) is a very versatile and polyfunctional ligand in coordination chemistry. In this review the different forms of metal-to-ligand interactions involving this anion and some other coordination peculiarities are briefly discussed on the basis of some selected examples.

Building complexity in O2-binding copper complexes : site-selective metalation and intermolecular O2-binding at dicopper and heterometallic complexes derived from an unsymmetric ligand

A novel unsymmetric dinucleating ligand (LN3N4) combining a tridentate and a tetradentate binding sites linked through a m-xylyl spacer was synthesized as ligand scaffold for preparing homo- and dimetallic complexes, where the two metal ions are bound in two different coordination environments. Site-selective binding of different metal ions is demonstrated. LN3N4 is able to discriminate between CuI and a complementary metal (M′ = CuI, ZnII, FeII, CuII, or GaIII) so that pure heterodimetallic complexes with a general formula [CuIM′(LN3N4)]n+ are synthesized. Reaction of the dicopper(I) complex [CuI 2(LN3N4)]2+ with O2 leads to the formation of two different copper-dioxygen (Cu2O2) intermolecular species (O and TP) between two copper atoms located in the same site from different complex molecules. Taking advantage of this feature, reaction of the heterodimetallic complexes [CuM′(LN3N4)]n+ with O2 at low temperature is used as a tool to determine the final position of the CuI center in the system because only one of the two Cu2O2 species is formed

Estudo das propriedades e biodegradabilidade de blendas de poliéster/amido submetidas ao ataque microbiano

This work deals with the biodegradation of blends of poly(beta-hydroxybutyrate)/starch and poly(beta-hydroxybutyrate-co-hydroxyvalerate)/starch. The blends were obtained by evaporation of the solvent in the mixture of the polymers in chloroform. Tests were carried out in presence of micro-organisms which acted as biodegradation agents. The blends were consumed as carbon substrate and the production of CO2 was evaluated in the process. In addition, the polyesters' mechanical properties were reduced by the incorporation of starch in its structure. (¹H) NMR and infrared spectroscopy detected some characteristic polyester degradation groups in the polyesters' chemical structure, thus confirming the alteration suffered by it.

Modificação química e física do amido de quirera de arroz para aproveitamento na indústria de alimentos

The study evaluated the efficiency of chemical (phosphorylation) and physical (extrusion) modifications of the starch of broken rice. Results demonstrated a reduction in the moisture content of extruded and phosphorylated broken rice and an increase in the ash content of phosphorylated broken rice. Both phosphorylation and extrusion increased cold water binding capacity, swelling power, and solubility. Extruded and phosphorylated pastes were stable under refrigeration, but only extruded paste was stable when submitted to freezing. Phosphorylated paste had the lowest viscosity and the highest stability during heating, while the extruded one gelatinized without heating, but had higher losses during heating.

Modificação enzimática da farinha de arroz visando a produção de amido resistente

The aim of this work was to study the enzymatic modification on rice flour using lipase pancreatic and amyloglucosidase to obtain resistant starch. For this, Response Surface Methodology (RSM) was used to determine the best operating conditions for each enzyme. For lypase pancreatic, the highest value for resistant starch (45%) was achieved within 2 h reaction at pH 7 using an enzyme/substrate ratio of 4% (w/w) and Dp= 100/200 tyler. For amyloglucosidase, optima conditions corresponded to an enzyme/substrate ratio of 0,006 mL/g and Dp= 100/200 tyler at 45 ºC, yielding 57% of resistant starch in 2 h reaction. These results show the potential of using both enzymes to modified rice flour, increasing the resistant starch in about 5.7 folds in relation to the flour without treatment (resistant starch=10.6%).

Produção de poli(3-hidroxibutirato) por Cupriavidus Necator em meio hidrolisado de amido de arroz com suplementação de óleo de soja em diferentes temperaturas

Poly (3-hydroxybutyrate) (P(3HB)) is a biopolymer, completely biodegradable, which has similar properties to fuel-based polymers. However to make it economically competitive it is necessary the study of cheap sources of substrate. The influence of hydrolyzed rice starch supplemented with soybean oil at different temperatures (30, 35 and 40 °C) was studied in the production of P(3HB) by C. necator. The percentage of P(3HB) produced in the cultures at 30, 35 °C was 30, 39% and 35, 43% without and with supplementation of oil, respectively. The culture at 40 °C showed no production phase due to a possible oxygen limitation. These results demonstrate that hydrolyzed rice starch supplemented with soybean oil increases the yield of P(3HB) and temperature of 35 ºC is the most favorable for biopolymer production.