Quartz Crystal Microbalance monitoring the real-time binding of lectin with carbohydrate with high and low molecular mass

Quartz Crystal Microbalance (QCM) was used to monitor the mass changes on a quartz crystal surface containing immobilized lectins that interacted with carbohydrates. The strategy for lectin immobilization was developed on the basis of a multilayer system composed of Au-cystamine-glutaraldehyde-lectin. Each step of the immobilization procedure was confirmed by FTIR analysis. The system was used to study the interactions of Concanavalin A (ConA) with maltose and Jacalin with Fetuin. The real-time binding of different concentrations of carbohydrate to the immobilized lectin was monitored by means of QCM measurements and the data obtained allowed for the construction of Langmuir isotherm curves. The association constants determined for the specific interactions analyzed here were (6.4 +/- 0.2) X 10(4) M-1 for Jacalin-Fetuin and (4.5 +/- 0.1) x 10(2) M-1 for ConA-maltose. These results indicate that the QCM constitutes a suitable method for the analysis of lectin-carbohydrate interactions, even when assaying low molecular mass ligands such as disaccharides. Published by Elsevier B.V.

Lectins and/or xyloglucans/alginate layers as supports for immobilization of dengue virus particles

Formation of stable thin films of mixed xyloglucan (XG) and alginate (ALG) onto Si/SiO2 wafers was achieved under pH 11.6, 50 mM CaCl2, and at 70 degrees C. XG-ALG films presented mean thickness of (16 +/- 2) nun and globules rich surface, as evidenced by means of ellipsometry and atomic force microscopy (AFM), respectively. The adsorption of two glucose/mannose-binding seed (Canavalia ensiformis and Dioclea altissima) lectins, coded here as ConA and DAlt, onto XG-ALG surfaces took place under pH 5. Under this condition both lectins present positive net charge. ConA and DAIt adsorbed irreversibly onto XG-ALG forming homogenous monolayers similar to(4 +/- 1)nm thick. Lectins adsorption was mainly driven by electrostatic interaction between lectins positively charged residues and carboxylated (negatively charged) ALG groups. Adhesion of four serotypes of dengue virus, DENV (1-4), particles to XG-ALG surfaces were observed by ellipsometry and AFM. The attachment of dengue particles onto XG-ALG films might be mediated by (i) H bonding between E protein (located at virus particle surface) polar residues and hydroxyl groups present on XG-ALG surfaces and (ii) electrostatic interaction between E protein positively charged residues and ALG carboxylic groups. DENV-4 serotype presented the weakest adsorption onto XG-ALG surfaces, indicating that E protein on DENV-4 surface presents net charge (amino acid sequence) different from E proteins of other serotypes. All four DENV particles serotypes adsorbed similarly onto lectin films adsorbed. Nevertheless, the addition of 0.005 mol/L of mannose prevented dengue particles from adsorbing onto lectin films. XG-ALG and lectin layers serve as potential materials for the development of diagnostic methods for dengue. (c) 2008 Elsevier B.V. All rights reserved.

Effects of statins on matrix metalloproteinases and their endogenous inhibitors in human endothelial cells

Statins exert anti-inflammatory effects and downregulate matrix metalloproteinases (MMPs) expression, thus contributing to restore cardiovascular homeostasis in cardiovascular diseases. We aimed at comparing the effects of different statins (simvastatin, atorvastatin, and pravastatin) on MMP-2, MMP-9, tissue inhibitors of metalloproteinases (TIMP)-1, TIMP-2, and MMP-9/TIMP-1 and MMP-2/TIMP-2 ratios released by human umbilical vein endothelial cells (HUVEC) stimulated by phorbol myristate acetate (PMA). HUVECs were incubated with statins (0.1-10 mu M) for 12 h before stimulation with PMA 100 nM. Monolayers were used to perform cell viability assays and the supernatants were collected to determine MMPs and TIMPs levels by gelatin zymography and/or enzyme immunoassay. While treatment with PMA increased MMP-9 and TIMP-1 levels (by 556% and 159%, respectively; both P < 0.05), it exerted no effects on MMP-2 and TIMP-2 levels. Simvastatin and atorvastatin, but not pravastatin, attenuated PMA-induced increases in MMP-9 levels (P < 0.05). Only atorvastatin decreased baseline MMP-2 levels significantly (P < 0.05). We found no effects on TIMP-2 levels. Simvastatin and atorvastatin, but not pravastatin, decreased MMP-9/TIMP-1 ratio significantly (both P < 0.05), whereas atorvastatin and pravastatin, but not simvastatin, decreased MMP-2/TIMP-2 ratio significantly (both P < 0.05). Our data support the notion that statins with different physicochemical features exert variable effects on MMP/TIMP ratios (which reflect net MMP activity). Our results suggest that more lipophilic statins (simvastatin and atorvastatin), but not the hydrophilic statin pravastatin, downregulate net MMP-9 activity. However, atorvastatin and pravastatin may downregulate net MMP-2 activity. The clinical implications of the present findings deserve further investigation.

The structures of Langmuir-Blodgett films of fatty acids and their salts

Recent advances in several experimental techniques have enabled detailed structural information to be obtained for floating (Langmuir) monolayers and Langmuir-Blodgett films. These techniques are described briefly and their application to the study of films of fatty acids and their salts is discussed. Floating monolayers on aqueous subphases have been shown to possess a complex polymorphism with phases whose structures may be compared to those of smectic mesophases. However, only those phases that exist at high surface pressures are normally used in Langmuir-Blodgett (LB) deposition. In single LB monolayers of fatty acids and fatty acid salts the acyl chains are in the all-cans conformation with their long axes normal to the substrate. The in-plane molecular packing is hexagonal with long-range bond orientational order and short-range positional order: known as the hexatic-B structure. This structure is found irrespective of the phase of the parent floating monolayer. The structures of multilayer LB films are similar to the structures of their bulk crystals, consisting of stacked bilayer lamellae. Each lamella is formed from two monolayers of fatty acid molecules or ions arranged head to head and held together by hydrogen bonding between pairs of acids or ionic bonding through the divalent cations. With acids the acyl chains are tilted with respect to the substrate normal and have a monoclinic structure, whereas the salts with divalent cations may have the chains normal to the substrate or tilted. The in-plane structures are usually centred rectangular with the chains in the trans conformation and packed in a herringbone pattern, Multilayer films of the acids show only a single-step order-disorder transition at the malting point, This temperature tends to rise as the number of layers increases. Complex changes occur when multilayer films of the salts are heated. Disorder of the chains begins at low temperatures but the arrangement of the head groups does not alter until the melting temperature is reached, Slow heating to a temperature just below the melting temperature gives, with some salts, a radical change in phase. The lamellar structure disappears and a new phase consisting of cylindrical rods lying parallel to the substrate surface and stacked in a hexagonal pattern is formed, In each rod the cations are aligned along the central axis surrounded by the disordered acyl chains. (C) 2001 Elsevier Science B,V. All rights reserved.

Dual trafficking of Slit3 to mitochondria and cell surface demonstrates novel localization for Slit protein

Drosophila slit is a secreted protein involved in midline patterning. Three vertebrate orthologs of the fly slit gene, Slit1, 2, and 3, have been isolated. Each displays overlapping, but distinct, patterns of expression in the developing vertebrate central nervous system, implying conservation of function. However, vertebrate Slit genes are also expressed in nonneuronal tissues where their cellular locations and functions are unknown. In this study, we characterized the cellular distribution and processing of mammalian Slit3 gene product, the least evolutionarily conserved of the vertebrate Slit genes, in kidney epithelial cells, using both cellular fractionation and immunolabeling. Slit3, but not Slit2, was predominantly localized within the mitochondria. This localization was confirmed using immunoelectron microscopy in cell lines and in mouse kidney proximal tubule cells. In confluent epithelial monolayers, Slit3 was also transported to the cell surface. However, we found no evidence of Slit3 proteolytic processing similar to that seen for Slit2. We demonstrated that Slit3 contains an NH2-terminal mitochondrial localization signal that can direct a reporter green fluorescent protein to the mitochondria. The equivalent region from Slit1 cannot elicit mitochondrial targeting. We conclude that Slit3 protein is targeted to and localized at two distinct sites within epithelial cells: the mitochondria, and then, in more confluent cells, the cell surface. Targeting to both locations is driven by specific NH2-terminal sequences. This is the first examination of Slit protein localization in nonneuronal cells, and this study implies that Slit3 has potentially unique functions not shared by other Slit proteins.

Initiation of cell locomotility is a morphogenetic checkpoint in thyroid epithelial cells regulated by ERK and PI3-kinase signals

Epithelial locomotility is a fundamental determinant of tissue patterning that is subject to strict physiological regulation. The current, study sought to identify cellular signals that initiate cell migration in cultured thyroid epithelial cells. Porcine thyroid cells cultured as 3-dimensional follicles convert to 2-dimensional monolayers when deprived of agents that stimulate cAMP/PKA signaling. This morphogenetic event is driven by the activation of cell-on-substrate locomotility, providing a convenient assay for events that regulate the initiation of locomotion. In this system, the extracellular signal regulated kinase (ERK) pathway became activated as follicles converted to monolayer, as demonstrated by immunoblotting for activation-specific phosphorylation and nuclear accumulation of ERK. Inhibition of ERK activation using the drug PD98059 effectively prevented cells from beginning to migrate. PD98059 inhibited cell spreading, actin filament reorganization and the assembly of focal adhesions, cellular events that mediate the initiation of thyroid cell locomotility. Akt (PKB) signaling was also activated during follicle-to-monolayer conversion and the phosphoinositide 3-kinase (PI3-kinase) inhibitor, wortmannin, also blocked the initiation of cell movement. Wortmannin did not, however, block activation of ERK signaling. These findings, therefore, identify the ERK and PI3-kinase signaling pathways as important stimulators of thyroid cell locomotility. These findings are incorporated into a model where the initiation of thyroid cell motility constitutes a morphogenetic checkpoint regulated by coordinated changes in stimulatory (ERK, PI3-kinase) and tonic inhibitory (cAMP/PKA) signaling pathways. Cell Motil. Cytoskeleton 49:93-103, 2001. (C) 2001 Wiley-Liss, Inc.

Structural changes in mixed Langmuir-Blodgett films upon nanoparticle formation

The formation of CdS nanoparticles by reacting mixed Langmuir-Blodgett films of arachidic acid and either octadecylamine or dimethyldioctadecylammonium nitrate on a cadmium-containing subphase with hydrogen sulfide gas has resulted in the identification of a number of structural changes, observed using grazing incidence X-ray diffraction. In the case of octadecylamine, the structure after reaction is a hexagonal close-packed array of surfactant-stabilized nanoclusters, with a lattice constant of a = 17.65 Angstrom. In both octadecylamine and dimethyldioctadecylammonium nitrate films, the presence of a unit cell tilted at 38degrees to the plane of the substrate was found. Despite these changes, the average nanoparticle size was unaffected by the addition of either second component to the film.

A cassette ligation strategy with thioether replacement of three Gly-Gly peptide bonds: Total chemical synthesis of the 101 residue protein early pregnancy factor [psi(CH2S)(28-29,56-57,76-77)]

The 101 residue protein early pregnancy factor (EPF), also known as human chaperonin 10, was synthesized from four functionalized, but unprotected, peptide segments by a sequential thioether ligation strategy. The approach exploits the differential reactivity of a peptide-NHCH2CH2SH thiolate with XCH2CO-peptides, where X = Cl or I/Br. Initial model studies with short functionalized (but unprotected) peptides showed a significantly faster reaction of a peptide-NHCH2CH2SH thiolate with a BrCH2CO-peptide than with a CICH2CO-peptide, where thiolate displacement of the halide leads to chemoselective formation of a thioether surrogate for the Gly-Gly peptide bond. This rate difference was used as the basis of a novel sequential ligation approach to the synthesis of large polypeptide chains. Thus, ligation of a model bifunctional N-alpha-chloroacetyl, C-terminal thiolated peptide with a second N-alpha-bromoacetyl peptide demonstrated chemoselective bromide displacement by the thiol group. Further investigations showed that the relatively unreactive N-alpha-chloroacetyl peptides could be activated by halide exchange using saturated KI solutions to yield the highly reactive No-iodoacetyl peptides. These findings were used to formulate a sequential thioether ligation strategy for the synthesis of EPF, a 101 amino acid protein containing three Gly-Gly sites approximately equidistantly spaced within the peptide chain. Four peptide segments or cassettes comprising the EPF protein sequence (BrAc-[EPF 78-101] 12, ClAc-[EPF 58-75]-[NHCH2CH2SH] 13, ClAc-[EPF 30-55]-[NHCH2CH2SH] 14, and Ac-[EPF 1-27]-[NHCH2CH2SH] 15) of EPF were synthesized in high yield and purity using Boc SPPS chemistry. In the stepwise sequential ligation strategy, reaction of peptides 12 and 13 was followed by conversion of the N-terminal chloroacetyl functional group to an iodoacetyl, thus activating the product peptide for further ligation with peptide 14. The process of ligation followed by iodoacetyl activation was repeated to yield an analogue of EPF (EPF psi(CH2S)(28-29,56-57,76-77)) 19 in 19% overall yield.

beta-strand mimicking macrocyclic amino acids: Templates for protease inhibitors with antiviral activity

New amino acids are reported in which component macrocycles are constrained to mimic tripeptides locked in a beta-strand conformation. The novel amino acids involve macrocycles functionalized with both an N- and a C-terminus enabling addition of appendages at either end to modify receptor affinity, selectivity, or membrane permeability. We show that the cycles herein are effective templates within inhibitors of HIV-1 protease. Eleven compounds originating from such bifunctionalized cyclic templates are potent inhibitors of HIV-1 protease (Ki 0.3-50 nM; pH 6.5, I = 0.1 M). Unlike normal peptides comprising amino acids, five of these macrocycle-containing compounds are potent antiviral agents with sub-micromolar potencies (IC50 170-900 nM) against HIV-1 replication in human MT2 cells. The most active antiviral agents are the most lipophilic, with calculated values of LogD(6.5) greater than or equal to 4. All molecules have a conformationally constrained 17-membered macrocyclic ring that has been shown to structurally mimic a tripeptide segment (Xaa)-(Val/Ile)-(Phe/Tyr) of a peptide substrate in the extended conformation. The presence of two trans amide bonds and a para-substituted aromatic ring prevents intramolecular hydrogen bonds and fixes the macrocycle in the extended conformation. Similarly constrained macrocycles may be useful templates for the creation of inhibitors for the many other proteins and proteases that recognize peptide beta-strands.

Thin films of a tetracationic porphyrin

Langmuir-Blodgett films of the tetracationic porphyrin tetrakis( octadecyl-4-pyridinium) porphinatozinc(ii) bromide transferred from subphases containing different salts were studied using X-ray photoelectron spectroscopy (XPS) and X-ray reflectometry. In contrast to previous results at the air/water interface, we found that the porphyrin adopted a fixed conformation at the air/solid interface regardless of composition of the subphase or whether the films were transferred above or below the primary phase transition. This conformation was assigned to the formation of an interdigitated bilayer structure.

Conformational changes in SP-B as a function of surface pressure

X-ray reflectivity of bovine and sheep surfactant-associated protein B (SP-B) monolayers is used in conjunction with pressure-area isotherms and protein models to suggest that the protein undergoes changes in its tertiary structure at the air/water interface under the influence of surface pressure, indicating the likely importance of such changes to the phenomena of protein squeeze out as well as lipid exchange between the air-water interface and subphase structures. We describe an algorithm based on the well-established box- or layer-models that greatly assists the fitting of such unknown scattering-length density profiles, and which takes the available instrumental resolution into account. Scattering-length density profiles from neutron reflectivity of bovine SP-B monolayers on aqueous subphases are shown to be consistent with the exchange of a large number of labile protons as well as the inclusion of a significant amount of water, which is partly squeezed out of the protein monolayer at elevated surface pressures.

Multiple ordered phases in Langmuir-Blodgett films of cadmium arachidate at elevated temperatures

Using synchrotron X-ray grazing incidence diffraction, superlattice structures have been observed to develop in Langmuir-Blodgett films of cadmium arachidate as the temperature is raised. The previously reported superstructure in the stacked lamellae at room temperature changes at about 70 degreesC and there are further changes at about 90 and 103 degreesC before the major phase transition from stacked lamellae to hexagonally packed rods occurs at 107 degreesC (Langmuir 1997, 13, 1602). Between 70 and 103 degreesC there is a 1 x 10 one-dimensional in-plane superstructure, which is commensurate with the local structure and has an interlayer shift along [01] by a distance of b (of the local structure) at lower temperatures, and a further shift at about 90 degreesC. At lower (

New syntheses of potential biologically active xanthones and benzoxanthones

10º Encontro Nacional de Química Orgânica e 1º Simpósio Luso-Brasileiro de Química Orgânica, Lisboa, 4-6 Setembro de 2013.

Electroanalysis of urinary L-dopa using tyrosinase immobilized on gold nanoelectrode ensembles

The performance of an amperometric biosensor constructed by associating tyrosinase (Tyr) enzyme with the advantages of a 3D gold nanoelectrode ensemble (GNEE) is evaluated in a flow-injection analysis (FIA) system for the analysis of l-dopa. GNEEs were fabricated by electroless deposition of the metal within the pores of polycarbonate track-etched membranes. A simple solvent etching procedure based on the solubility of polycarbonate membranes is adopted for the fabrication of the 3D GNEE. Afterward, enzyme was immobilized onto preformed self-assembled monolayers of cysteamine on the 3D GNEEs (GNEE-Tyr) via cross-linking with glutaraldehyde. The experimental conditions of the FIA system, such as the detection potential (−0.200 V vs. Ag/AgCl) and flow rates (1.0 mL min−1) were optimized. Analytical responses for l-dopa were obtained in a wide concentration range between 1 × 10−8 mol L−1 and 1 × 10−2 mol L−1. The limit of quantification was found to be 1 × 10−8 mol L−1 with a resultant % RSD of 7.23% (n = 5). The limit of detection was found to be 1 × 10−9 mol L−1 (S/N = 3). The common interfering compounds, namely glucose (10 mmol L−1), ascorbic acid (10 mmol L−1), and urea (10 mmol L−1), were studied. The recovery of l-dopa (1 × 10−7 mol L−1) from spiked urine samples was found to be 96%. Therefore, the developed method is adequate to be applied in the clinical analysis.

Agentes fitoquímicos como radio-sensibilizadores na terapêutica em medicina nuclear

Mestrado em Medicina Nuclear. Área de especialização: Radiofarmácia.