Standardisation and comparison of methods employed for microbial cell surface hydrophobicity and charge determination

Whilst there are a number of methods available to characterise the cell surface hydrophobicity (CSH) and cell surface charge (CSC) of microorganisms, there is still debate concerning the correlation of results between individual methods. In this study, the techniques of bacterial adherence to hydrocarbons (BATH) and hydrophobic interaction chromatography (HTC) were used to measure CSH. Electrostatic interaction chromatography (ESIC) and zeta potential (ZP) measurements were used to determine CSC. To allow meaningful comparisons between the BATH and HIC tests, between ESIC and ZP and also between CSH and CSC, the buffer systems employed in each test were standardised (phosphate buffered saline, pH 7.3, 0.01 mM). Isolates of Staphylococcus epidermidis derived from microbial biofilm were used as the test organism in this study. The isolates examined exhibited primarily medium to high CSH and a highly negative CSC. Good correlation of CSH measurement was observed between the BATH and HIC tests (r = 0.89). Good correlation was observed between ESIC (anionic exchange column) and ZP measurements. No correlations were observed between isolate CSC and either increased or decreased CSH. It is recommended that whenever comparisons of various methods to determine either CSC or CSH (by partitioning methods), the buffer systems should remain constant throughout to achieve consistency of results.

THE EFFECTS OF 3 NON-ANTIBIOTIC, ANTIMICROBIAL AGENTS ON THE SURFACE HYDROPHOBICITY OF CERTAIN MICROORGANISMS EVALUATED BY DIFFERENT METHODS

The effects of three non-antibiotic, antimicrobial agents (taurolidine, chlorhexidine acetate and providone-iodine) on the surface hydrophobicity of the clinical strains Escherichia coli, Staphylococcus saprophyticus, Staphylococcus epidermidis and Candida albicans were examined. Three recognized techniques for hydrophobicity measurements, Bacterial Adherence to Hydrocarbons (BATH), the Salt Aggregation Test (SAT) and Hydrophobic Interaction Chromatography (HIC) were compared. At concentrations reported to interfere with microbial-epithelial cell adherence, all three agents altered the cell surface hydrophobicity. However, these effects failed to exhibit a uniform relationship. Generally, taurolidine and povidone-iodine treatments decreased the hydrophobicity of the strains examined whereas chlorhexidine acetate effects depended upon the micro-organism treated. Subsequently, the exact contribution of altered cell surface hydrophobicity to the reported microbial anti-adherence effects is unclear. Comparison of the three techniques revealed a better correlation between the results obtained with the BATH test and HIC than the results obtained with the BATH and SAT or SAT and HIC. However, these differences may be due to the inaccuracy associated with the visual assessment of results employed by the SAT.

Études de type structure fonction du couplage électromécanique et de la coopérativité sous-unitaire chez les canaux potassiques dépendants du voltage

Les canaux potassiques voltage-dépendants forment des tétramères dont chaque sous-unité comporte six segments transmembranaires (S1 à S6). Le pore, formé des segments S5-S6 de chaque sous-unité, est entouré de quatre domaines responsables de la sensibilité au potentiel membranaire, les senseurs de voltage (VS; S1-S4). Lors d’une dépolarisation membranaire, le mouvement des résidus chargés situés dans le VS entraine un mouvement de charges détectable en électrophysiologie, le courant de « gating ». L’activation du VS conduit à l'ouverture du pore, qui se traduit par un changement de conformation en C-terminal du segment S6. Pour élucider les principes qui sous-tendent le couplage électromécanique entre ces deux domaines, nous avons étudié deux régions présumées responsables du couplage chez les canaux de type Shaker K+, soit la région carboxy-terminale du segment S6 et le lien peptidique reliant les segments transmembranaire S4-S5 (S4-5L). Avec la technique du « cut-open voltage clamp fluorometry » (COVCF), nous avons pu déterminer que l’interaction inter-sous-unitaire RELY, formée par des acides aminés situés sur le lien S4-5L et S6 de deux sous-unités voisines, est impliquée dans le développement de la composante lente observée lors du retour des charges de « gating » vers leur état de repos, le « OFF-gating ». Nous avons observé que l’introduction de mutations dans la région RELY module la force de ces interactions moléculaires et élimine l’asymétrie observée dans les courants de « gating » de type sauvage. D’ailleurs, nous démontrons que ce couplage inter-sous-unitaire est responsable de la stabilisation du pore dans l’état ouvert. Nous avons également identifié une interaction intra-sous-unitaire entre les résidus I384 situé sur le lien S4-5L et F484 sur le segment S6 d’une même sous-unité. La déstabilisation de cette interaction hydrophobique découple complètement le mouvement des senseurs de voltage et l'ouverture du pore. Sans cette interaction, l’énergie nécessaire pour activer les VS est moindre en raison de l’absence du poids mécanique appliqué par le pore. De plus, l’abolition du couplage électromécanique élimine également le « mode shift », soit le déplacement de la dépendance au voltage des charges de transfert (QV) vers des potentiels hyperpolarisants. Ceci indique que le poids mécanique du pore imposé au VS entraine le « mode shift », en modulant la conformation intrinsèque du VS par un processus allostérique.

Inhibition of Mycobacterium tuberculosis tyrosine phosphatase PtpA by synthetic chalcones: Kinetics, molecular modeling, toxicity and effect on growth

Tuberculosis (TB) is a major cause of morbidity and mortality throughout the world, and it is estimated that one-third of the world`s population is infected with Mycobacterium tuberculosis. Among a series of tested compounds, we have recently identified five synthetic chalcones which inhibit the activity of M. tuberculosis protein tyrosine phosphatase A (PtpA), an enzyme associated with M. tuberculosis infectivity. Kinetic studies demonstrated that these compounds are reversible competitive inhibitors. In this work we also carried out the analysis of the molecular recognition of these inhibitors on their macromolecular target, PtpA, through molecular modeling. We observed that the predominant determinants responsible for the inhibitory activity of the chalcones are the positions of the two methoxyl groups at the A-ring, that establish hydrogen bonds with the amino acid residues Arg17, His49, and Thr12 in the active site of PtpA, and the substitution of the phenyl ring for a 2-naphthyl group as B-ring, that undergoes p stacking hydrophobic interaction with the Trp48 residue from PtpA. Interestingly, reduction of mycobacterial survival in human macrophages upon inhibitor treatment suggests their potential use as novel therapeutics. The biological activity, synthetic versatility, and low cost are clear advantages of this new class of potential tuberculostatic agents. (C) 2010 Elsevier Ltd. All rights reserved.

Purification and partial characterization of an aminopeptidase from the midgut tissue of Dysdercus peruvianus

The surface of midgut cells in Hemiptera is ensheathed by a lipoprotein membrane (the perimicrovillar membrane), which delimits a closed compartment with the microvillar membrane, the so-called perimicrovillar space. In Dysdercus peruvianus midgut perimicrovillar space a soluble aminopeptidase maybe involved in the digestion of oligopeptides and proteins ingested in the diet. This D. peruvianus aminopeptidase was purified to homogeneity by ion-exchange chromatography on an Econo-Q column, hydrophobic interaction chromatography on phenyl-agarose column and preparative polyacrylamide gel electrophoresis. The results suggested that there is a single molecular species of aminopeptidase in D. peruvianus midgut. Molecular mass values for the aminopeptidase were estimated to be 106 kDa (gel filtration) and 55 kDa (SDS-PAGE), suggesting that the enzyme occurs as a dimer under native conditions. Kinetic data showed that D. peruvianus aminopeptidase hydrolyzes the synthetic substrates LpNA, RpNA, A beta NA and AsnMCA (K(m)s 0.65, 0.14, 0.68 and 0.74 mM, respectively). The aminopeptidase activity upon LpNA was inhibited by EDTA and 1,10-phenanthroline, indicating the importance of metal ions in enzyme catalysis. One partial sequence of BLAST-identified aminopeptidase was found by random sequencing of the D. peruvianus midgut cDNA library. Semi-quantitative RT-PCR analysis showed that the aminopeptidase genes were expressed throughout the midgut epithelium, in the epithelia of V1, V2 and V3. Malphigian tubules and fat body, but it was not expressed in the salivary glands. These results are important in furthering our understanding of the digestive process in this pest species. (c) 2010 Elsevier Inc. All rights reserved.

Adsorption of anionic amphiphilic polyelectrolytes onto amino-terminated solid surfaces

The adsorption behavior of several amphiphilic polyelectrolytes of poly(maleic anhydride-alt-styrene) functionalized with naphthyl and phenyl groups, onto amino-terminated silicon wafer has been studied by means of null- ellipsometry, atomic force microscopy (AFM) and contact angle measurements. The maximum of adsorption, Gamma(plateau), varies with the ionic strength, the polyelectrolyte structure and the chain length. Values of Gamma(plateau) obtained at low and high ionic strengths indicate that the adsorption follows the ""screening-reduced adsorption"" regime. Large aggregates were detected in solution by means of dynamic light scattering and fluorescence measurements. However. AFM indicated the formation of smooth layers and the absence of aggregates. A model based on a two-step adsorption behavior was proposed. In the first one, isolated chains in equilibrium with the aggregates in solution adsorbed onto amino-terminated surface. The adsorption is driven by electrostatic interaction between protonated surface and carboxylate groups. This first layer exposes naphtyl or phenyl groups to the solution. The second layer adsorption is now driven by hydrophobic interaction between surface and chains and exposes carboxylate groups to the medium, which repel the forthcoming chain by electrostatic repulsion. Upon drying some hydrophobic naphtyl or phenyl groups might be oriented to the air, as revealed by contact angle measurements. Such amphiphilic polyelectrolyte layers worked well for the building-up of multilayers with chitosan. (C) 2010 Elsevier Ltd. All rights reserved.

Lysozyme binding to poly(4-vinyl-N-alkylpyridinium bromide)

The adsorption behavior of polycations at ionic strengths (1) ranging from 0.001 to 0.1 onto silicon wafers was studied by means of ellipsometry, contact angle measurements and atomic force microscopy (AFM). Polycations chosen were bromide salts of poly(4-vinylpyridine) N-alkyl quaternized with linear aliphatic chains of 2 and 5 carbon atoms, QPVP-C2 and QPVP-C5, respectively. Under 1 0.001 the reduction of screening effects led to low adsorbed amounts of QPVP-C2 or QPVP-C5 (1.0 +/- 0.1 mg/m(2)), arising from the adsorption of extended chains. Upon increasing l to 0.1, screening effects led to conformational changes of polyelectrolyte chains ill Solution and to higher adsorbed amount values (1.9 +/- 0.2 mg/m(2)). Advancing contact angle theta(a) measurements performed with water drops onto QPVP-C2 and QPVP-C5 adsorbed layers varied from (45 +/- 2)degrees to (50 +/- 5)degrees, evidencing the exposure of both hydrophobic alkyl groups and charged moieties. The adsorption of lysozyme (LYZ) molecules to QPVP-C5 layers was more pronounced than to QPVP-C2 films. Antimicrobial effect of LYZ bound to QPVP-C2 or QPVP-C5 layers or to Si wafers was evaluated with enzymatic assays using Micrococcus luteus as Substrates. The adsorption behavior of QPVP-C2 and QPVP-C5 at the water-air interface was studied by means Of surface tension measurements. Only QPVP-C5 was able to reduce water Surface tension. Mixtures of LYZ and QPVP-C5 were more efficient in reducing Surface tension than pure LYZ solution, evidencing co-adsorption at liquid-air interface. Moreover, antimicrobial action observed for mixtures of LYZ and QPVP-C5 was more pronounced than that measured for pure LYZ. Hydrophobic interaction between LYZ and QPVP-C5 ill Solution seems to drive the binding and to preserve LYZ secondary structure. (c) 2008 Elsevier Inc. All rights reserved.

Labaditin, a cyclic peptide with rich biotechnological potential: preliminary toxicological studies and structural changes in water and lipid membrane environment

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Síntese e avaliação farmacológica de protótipos candidatos à fármacos para o tratamento dos sintomas da anemia falciforme

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Imobilização de lipases por adsorção e ligação covalente em derivados de agarose e quitosana e sua aplicação em biocatálise

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Obtenção e estudo de polímeros supramoleculares derivados de ácidos carboxílicos-meglumina em fase aquosa

Pós-graduação em Química - IQ

Purificação, imobilização e caracterização bioquímica de lipase produzida por Penicillium sect. Gracilenta CBMAI 1583 em cultivo submerso

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Caracterização biofísica da interação entre a proteína M2-1 do Vírus Sincicial Respiratório Humano (HRSV) com quercetina

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Síntese de polímeros supramoleculares derivados dos ácidos palmítico e mirístico com meglumina e suas caracterizações em meio aquoso

Pós-graduação em Química - IQ

Biochemical Aspects of a Serine Protease from Caesalpinia echinata Lam. (Brazilwood) Seeds: A Potential Tool to Access the Mobilization of Seed Storage Proteins

Several proteins have been isolated from seeds of leguminous, but this is the first report that a protease was obtained from seeds of Caesalpinia echinata Lam., a tree belonging to the Fabaceae family. This enzyme was purified to homogeneity by hydrophobic interaction and anion exchange chromatographies and gel filtration. This 61-kDa serine protease (CeSP) hydrolyses H-D-prolyl-L-phenylalanyl-L-arginine-p-nitroanilide (K-m 55.7 mu M) in an optimum pH of 7.1, and this activity is effectively retained until 50 degrees C. CeSP remained stable in the presence of kosmotropic anions (PO43-, SO42-, and CH3COO-) or chaotropic cations (K+ and Na+). It is strongly inhibited by TLCK, a serine protease inhibitor, but not by E-64, EDTA or pepstatin A. The characteristics of the purified enzyme allowed us to classify it as a serine protease. The role of CeSP in the seeds cannot be assigned yet but is possible to infer that it is involved in the mobilization of seed storage proteins.