Evaluación del grado de queratinización y el recuento de AgNORs en citología exfoliativa de mucosa oral normal de individuos fumadores y no fumadores

Objetivos. En individuos fumadores con mucosa oral clínicamente sana, se han observado cambios citológicos como una mayor queratinización, existiendo también reportes de un mayor grado de actividad nucleolar. En estos estudios, las células para frotis se han obtenido por medio de espátula de madera. Nuestro objetivo es evaluar la profundidad de muestras citológicas de mucosa oral obtenidas con cepillo para frotis (endobrush) y comparar el grado de queratinización y la actividad nucleolar en pacientes fumadores y no fumadores. Diseño del estudio. Se obtuvieron frotis de mucosa oral de borde de lengua clínicamente normal de 30 individuos fumadores y 30 no fumadores, utilizando espátula de madera y endobrush. Las muestras fueron teñidas con Papanicolaou y con la tinción AgNORs. Resultados. Con la espátula de madera se obtuvo un mayor porcentaje de células epiteliales superficiales anucleadas (P= 0.016) y con el endobrush se obtuvieron células más profundas (tipo intermedias) (P= 0.035). Los individuos fumadores presentaron un mayor porcentaje de células superficiales anucleadas con ambas técnicas, diferencia que fue estadísticamente significativa con la técnica endobrush (P=0.005). El promedio de AgNORs en las células nucleadas fue mayor en los individuos fumadores (3.83) que en los no fumadores (2.79) (P= 0.003). Conclusiones. El endobrush permite obtener células de estratos más profundos. Los individuos fumadores con mucosa clínicamente normal presentan un mayor porcentaje de células queratinizadas y una mayor actividad nucleolar, sugiriendo que el consumo de cigarrillo influye en la actividad celular de la mucosa del borde de lengua.Objetive.In smokers with clinically normal buccal mucosa, cytological changes such as increased keratinization, and higher nucleolar activity have been observed. In these studies the cells for cytological smears were obtained with a wooden spatula. Our objectives were to evaluate the depth of cytological smears of oral mucosa obtained with both a brush (endobrush) and a wooden spatula, and to compare the degree of keratinization and the nucleolar activity in smokers and non-smokers. Design. We obtained cytological smears of clinically normal lateral tongues of 30 smokers and 30 non-smokers using both a wooden spatula and endobrush. The samples were dyed with Papanicolaou and the AgNORs. Results. With the wooden spatula we found a greater percentage of enucleated superficial epithelial cells (P = 0.016) and deeper cells were obtained with an endobrush (intermediate cells) (P =0.035). The smokers showed a greater percentage of enucleated superficial cells with both techniques, however this difference was significantly greater with Endobrush (P=0.005). The average of AgNORs in the nucleated cells was greater in smokers (3.83) than in non-smokers (2.79) (P=0.003). Conclusion. The Endobrush allows the clinician to obtain deeper cells of buccal mucosa. Smokers with clinically normal mucosa show a greater percentage of keratinized cells and a greater nucleolar activity, suggesting that cigarette smoking influences the cellular activity of the mucosa of the lateral tongue

Structure of the dimeric N-glycosylated form of fungal β-N-acetylhexosaminidase revealed by computer modeling, vibrational spectroscopy, and biochemical studies

<p>Abstract</p> <p>Background</p> <p>Fungal β-<it>N</it>-acetylhexosaminidases catalyze the hydrolysis of chitobiose into its constituent monosaccharides. These enzymes are physiologically important during the life cycle of the fungus for the formation of septa, germ tubes and fruit-bodies. Crystal structures are known for two monomeric bacterial enzymes and the dimeric human lysosomal β-<it>N</it>-acetylhexosaminidase. The fungal β-<it>N</it>-acetylhexosaminidases are robust enzymes commonly used in chemoenzymatic syntheses of oligosaccharides. The enzyme from <it>Aspergillus oryzae </it>was purified and its sequence was determined.</p> <p>Results</p> <p>The complete primary structure of the fungal β-<it>N</it>-acetylhexosaminidase from <it>Aspergillus oryzae </it>CCF1066 was used to construct molecular models of the catalytic subunit of the enzyme, the enzyme dimer, and the <it>N</it>-glycosylated dimer. Experimental data were obtained from infrared and Raman spectroscopy, and biochemical studies of the native and deglycosylated enzyme, and are in good agreement with the models. Enzyme deglycosylated under native conditions displays identical kinetic parameters but is significantly less stable in acidic conditions, consistent with model predictions. The molecular model of the deglycosylated enzyme was solvated and a molecular dynamics simulation was run over 20 ns. The molecular model is able to bind the natural substrate – chitobiose with a stable value of binding energy during the molecular dynamics simulation.</p> <p>Conclusion</p> <p>Whereas the intracellular bacterial β-<it>N</it>-acetylhexosaminidases are monomeric, the extracellular secreted enzymes of fungi and humans occur as dimers. Dimerization of the fungal β-<it>N</it>-acetylhexosaminidase appears to be a reversible process that is strictly pH dependent. Oligosaccharide moieties may also participate in the dimerization process that might represent a unique feature of the exclusively extracellular enzymes. Deglycosylation had only limited effect on enzyme activity, but it significantly affected enzyme stability in acidic conditions. Dimerization and <it>N</it>-glycosylation are the enzyme's strategy for catalytic subunit stabilization. The disulfide bridge that connects Cys⁴⁴⁸with Cys⁴⁸³stabilizes a hinge region in a flexible loop close to the active site, which is an exclusive feature of the fungal enzymes, neither present in bacterial nor mammalian structures. This loop may play the role of a substrate binding site lid, anchored by a disulphide bridge that prevents the substrate binding site from being influenced by the flexible motion of the loop.</p

Genotypic Characterization of Sulfur-Oxidative Desulfurizing Bacterial Strains Isolated from Mexican Refineries

We performed a genotypic characterization of new sulfur selective dibenzothiophene (DBT) desulfurizing strains which have been isolated from hydrocarbon contaminated soils originating from Mexican refineries. We compared them to two reference strains (strains Rhodococcus erythropolis IGTS8 and R. erythropolis X309). These reference strains have been extensively studied and are protected by patents as well as all biodesulfurization (BDS) processes derived from them. The aim of the present study was to use molecular tools to characterize these new isolates and determine whether they were different from the other patented strains. The characterization methods included 16S rDNA gene partial sequencing, PCR amplification of fragments associated to the desulfurization phenotype, restriction analysis and randomly amplified polymorphic DNA (RAPD). Partial 16S rDNA gene sequence analysis showed that the isolated strains were different from R. erythropolis IGTS8 although they contained the same desulfurizing dszABC genes. The RAPD patterns were also different from those of the two reference strains u ed in this study confirming that the isolated bacteria were different from the already patented desulfurizing strains R. erythropolis IGTS8 and X309

A Hierarchical Approach to Cooperativity in Macromolecular and Self-Assembling Binding Systems

The study of complex macromolecular binding systems reveals that a high number of states and processes are involved in their mechanism of action, as has become more apparent with the sophistication of the experimental techniques used. The resulting information is often difficult to interpret because of the complexity of the scheme (large size and profuse interactions, including cooperative and self-assembling interactions) and the lack of transparency that this complexity introduces into the interpretation of the indexes traditionally used to describe the binding properties. In particular, cooperative behaviour can be attributed to very different causes, such as direct chemical modification of the binding sites, conformational changes in the whole structure of the macromolecule, aggregation processes between different subunits, etc. In this paper, we propose a novel approach for the analysis of the binding properties of complex macromolecular and self-assembling systems. To quantify the binding behaviour, we use the global association quotient defined as Kc = [occupied sites]/([free sites] L), L being the free ligand concentration. Kc can be easily related to other measures of cooperativity (such as the Hill number or the Scatchard plot) and to the free energies involved in the binding processes at each ligand concentration. In a previous work, it was shown that Kc could be decomposed as an average of equilibrium constants in two ways: intrinsic constants for Adair binding systems and elementary constants for the general case. In this study, we show that these two decompositions are particular cases of a more general expression, where the average is over partial association quotients, associated with subsystems from which the system is composed. We also show that if the system is split into different subsystems according to a binding hierarchy that starts from the lower, microscopic level and ends at the higher, aggregation level, the global association quotient can be decomposed following the hierarchical levels of macromolecular organisation. In this process, the partial association quotients of one level are expressed, in a recursive way, as a function of the partial quotients of the level that is immediately below, until the microscopic level is reached. As a result, the binding properties of very complex macromolecular systems can be analysed in detail, making the mechanistic explanation of their behaviour transparent. In addition, our approach provides a model-independent interpretation of the intrinsic equilibrium constants in terms of the elementary ones