Estandarización de un protocolo de extracción y caracterización del material proteico del esmalte dental erupcionado

Introducción: El 95% del peso del esmalte dental erupcionado sano corresponde a material inorgánico, 4% agua y 1% a materia orgánica (con un 0,03 a 0,1% de trazas de proteínas estructurales). En el caso del esmalte con defectos del desarrollo - como la fluorosis dental - existe evidencia de la retención de material proteico, despertando la necesidad del estudio del proteoma del esmalte en estas condiciones. Limitaciones inherentes a los tejidos duros, como su bajo contenido proteico encerrado en una matriz mineral, dificultan los procesos de extracción y caracterización de proteínas. El presente estudio, busca hacer una adaptación metodológica de los procedimientos de extracción de proteínas en esmalte dental erupcionado humano, para su posible aplicación al estudio del proteoma del esmalte con defectos del desarrollo.Objetivo: Estandarizar una técnica de extracción y caracterización del material proteico del esmalte de dientes erupcionados.Materiales y métodos: Se recolectaron dientes sanos con extracción indicada y se realizaron: -cortes de secciones longitudinales de 550 μm, - separación mecánica de esmalte/dentina y - pulverización de esmalte dental. El pulverizado se sometió a desmineralización/precipitación de proteínas con TCA 12%. El extracto fue separado por electroforesis SDS-PAGE y caracterizado por LC-MS/MS.Resultados: el procedimiento fue estandarizado. No se evidenciaron bandas de proteína después de la electroforesis SDS-PAGE, pero se identificaron y caracterizaron 138 péptidos, correspondientes a 13 proteínas, 3 de ellas específicas del esmalte (Amelogenina X, Amelogenina Y, Ameloblastina).Conclusiones: por primera vez en Colombia, se estandarizan y se adaptan métodos de extracción y caracterización de proteínas del esmalte dental, abriendo las puertas al estudio del proteoma de este tejid

Туров и его историко-культурное наследие

Материалы IV Республик. науч. конф. студентов, магистрантов и аспирантов, Гомель, 12 мая 2011 г

Novel sample-substrates for the determination of new psychoactive substances in oral fluid by desorption electrospray ionization-high resolution mass spectrometry

A reliable screening and non invasive method based on the use of microextraction by packed sorbent coupled with desorption electrospray ionization-high resolution mass spectrometry was developed and validated for the detection of new psychoactive substances in oral fluid. The role of different sample substrates in enhancing signal intensity and stability was evaluated by testing the performances of two polylactide-based materials, i.e. non-functionalized and functionalized with carbon nanoparticles, and a silica-based material compared to commercially available polytetrafluorethylene supports. The best results were achieved by using the nonfunctionalized polylactide substrates to efficiently ionize compounds in positive ionization mode, whereas the silica coating proved to be the best choice for operating in negative ionization mode. LLOQs in the low μg/L, a good precision with CV% always lower than 16% and RR% in the 83(±4)-120(±2)% range, proved the suitability of the developed method for the determination of the analytes in oral fluid. Finally, the method was applied for screening oral fluid samples for the presence of psychoactive substances during private parties, revealing mephedrone in only one sample out of 40 submitted to analysis

A collaborative evaluation of LC-MS/MS based methods for BMAA analysis: soluble bound BMAA found to be an important fraction.

Exposure to β-Ν-methylamino-l-alanine (BMAA) might be linked to the incidence of amyotrophic lateral sclerosis, Alzheimer's disease and Parkinson's disease. Analytical chemistry plays a crucial role in determining human BMAA exposure and the associated health risk, but the performance of various analytical methods currently employed is rarely compared. A CYANOCOST initiated workshop was organized aimed at training scientists in BMAA analysis, creating mutual understanding and paving the way towards interlaboratory comparison exercises. During this workshop, we tested different methods (extraction followed by derivatization and liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) analysis, or directly followed by LC-MS/MS analysis) for trueness and intermediate precision. We adapted three workup methods for the underivatized analysis of animal, brain and cyanobacterial samples. Based on recovery of the internal standard D3BMAA, the underivatized methods were accurate (mean recovery 80%) and precise (mean relative standard deviation 10%), except for the cyanobacterium Leptolyngbya. However, total BMAA concentrations in the positive controls (cycad seeds) showed higher variation (relative standard deviation 21%-32%), implying that D3BMAA was not a good indicator for the release of BMAA from bound forms. Significant losses occurred during workup for the derivatized method, resulting in low recovery ( < 10%). Most BMAA was found in a trichloroacetic acid soluble, bound form and we recommend including this fraction during analysis

Structural Basis for Dityrosine-Mediated Inhibition of α-Synuclein Fibrillization

[Image: see text] α-Synuclein (α-Syn) is an intrinsically disordered protein which self-assembles into highly organized β-sheet structures that accumulate in plaques in brains of Parkinson’s disease patients. Oxidative stress influences α-Syn structure and self-assembly; however, the basis for this remains unclear. Here we characterize the chemical and physical effects of mild oxidation on monomeric α-Syn and its aggregation. Using a combination of biophysical methods, small-angle X-ray scattering, and native ion mobility mass spectrometry, we find that oxidation leads to formation of intramolecular dityrosine cross-linkages and a compaction of the α-Syn monomer by a factor of √2. Oxidation-induced compaction is shown to inhibit ordered self-assembly and amyloid formation by steric hindrance, suggesting an important role of mild oxidation in preventing amyloid formation

Biochemical and biophysical aspects of molecular recognition and signalling by neurotrophins

Neurotrophins are members of a family of structurally and functionally relatedneurotrophic factors that control the development and maintenance of the nervoussystem. There are currently 5 members which make up this family: nerve growth factor(NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), neurotrophin-4(NT-4) and neurotrophin-6 (NT-6), which so far is only described in fish. Neurotrophinmediated effects, (e.g. cell survival and differentiation) are caused by ligand specificbinding to the trk family of tyrosine kinase receptors. NGF binds to TrkA, BDNF andNT 4 bind TrkB and NT-3 preferentially binds to TrkC. Due to the similarity in activitiesbetween NT-6 and NGF, the former is believed to act through a fish homologue of TrkA.Furthermore, neurotrophins bind with equal affinities to the p75 neurotrophin receptor(p75NTR) which is related to members of the tumour necrocis factor (TNF) receptorsuperfamily. Members of this family were initially grouped according to similaritiesin their extracellular domains. However, recent sequence analysis revealed that somemembers also share similar intracellular domain of about 80 residues involved inapoptosis, called the "death domain" (DD). Using site-directed mutagenesis and various biological and biochemical assaysthe critical domains and residues determining neurotrophin specificity towards bindingand receptor activation were identified. Results defined discontinous stretches ofamino acids in the primary structure of these proteins which, upon inspection ofthe prototypic three dimensional structure of NGF, delineated a continuous surfaceextending approximately parallel to the two-fold symmetry axis of the molecule. Usinginformation from this structure-function analysis, chimaeric neurotrophins with novelproperties were constructed such as a heparin-binding NGF and a multifunctional pan-neurotrophin-1(PNT-1). To gain insight on the possible mechanisms of p75NTR signalling, the structureof the p75 intracellular domain (p75ICD) was determined using nuclear magnetic resonance(NMR) spectroscopy. The only structured region in our ICD construct was the conservedDD module which reveals a novel fold (shared only with Fas receptor) consisting oftwo perpendicular sets of three helices packed into a globular structure. A surfacearea devoid of charged residues (hydrophobic patch) in the DD indicated a potentialsite of interaction with downstream targets. Using the Selectively Infective Phage(SIP) display technique, several peptides binding to the ICD were selected. A peptide(CFFRGGFFNHNPRYC) that interacted with the DD, was further studied by NMR and wasfound to bind to the above-mentioned hydrophobic patch. These selected peptides shouldprovide leads to the natural targets of the p75ICD and also should prove to be usefulreagents in probing the signalling mechanism of this receptor. Taken together, this study combines genetic, biochemical and biophysical approachesto understand the molecular basis of recognition and signalling by neurotrophins,and would help in the design of agonsists and antagonists that could mediate in disorderslike neurodegeneration

Advances in MS-Based Analytical Methods: Innovations and Future Trends

2 pages.-- Editorial.-- This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly citedPeer reviewe

Amyloid-β oligomers are captured by the DNAJB6 chaperone : Direct detection of interactions that can prevent primary nucleation

A human molecular chaperone protein, DnaJ heat shock protein family (Hsp40) member B6 (DNAJB6), efficiently inhibits amyloid aggregation. This inhibition depends on a unique motif with conserved serine and threonine (S/T) residues that have a high capacity for hydrogen bonding. Global analysis of kinetics data has previously shown that DNAJB6 especially inhibits the primary nucleation pathways. These observations indicated that DNAJB6 achieves this remarkably effective and sub-stoichiometric inhibition by interacting not with the monomeric unfolded conformations of the amyloid-β symbol (Aβ) peptide but with aggregated species. However, these pre-nucleation oligomeric aggregates are transient and difficult to study experimentally. Here, we employed a native MS-based approach to directly detect oligomeric forms of Aβ formed in solution. We found that WT DNAJB6 considerably reduces the signals from the various forms of Aβ (1-40) oligomers, whereas a mutational DNAJB6 variant in which the S/T residues have been substituted with alanines does not. We also detected signals that appeared to represent DNAJB6 dimers and trimers to which varying amounts of Aβ are bound. These data provide direct experimental evidence that it is the oligomeric forms of Aβ that are captured by DNAJB6 in a manner which depends on the S/T residues. We conclude that, in agreement with the previously observed decrease in primary nucleation rate, strong binding of Aβ oligomers to DNAJB6 inhibits the formation of amyloid nuclei