RESUMEN
Mildly denaturing conditions induce bovine alpha-crystallin, the major structural lens protein, to self-assemble into fibrillar structures in vitro. The natural dipeptide l-carnosine has been shown to have potential protective and therapeutic significance in many diseases. Carnosine derivatives have been proposed as potent agents for ophthalmic therapies of senile cataracts and diabetic ocular complications. Here we report the inhibitory effect induced by the peptide (l- and d-enantiomeric form) on alpha-crystallin fibrillation and the almost complete restoration of the chaperone activity lost after denaturant and/or heat stress. Scanning force microscopy (SFM), thioflavin T, and a turbidimetry assay have been used to determine the morphology of alpha-crystallin aggregates in the presence and absence of carnosine. DSC and a near-UV CD assay evidenced that the structural precursors of amyloid fibrils are polypeptide chain segments that lack stable structural elements. Moreover, we have found a disassembling effect of carnosine on alpha-crystallin amyloid fibrils. Finally, we show the ability of carnosine to restore most of the lens transparency in organ-cultured rat lenses exposed to similar denaturing conditions that were used for the in vitro experiments.
Asunto(s)
Amiloide/química , Carnosina/química , Catarata/metabolismo , alfa-Cristalinas/química , Animales , Rastreo Diferencial de Calorimetría , Carnosina/farmacología , Bovinos , Dicroismo Circular , Femenino , Microscopía de Fuerza Atómica , Chaperonas Moleculares/metabolismo , Técnicas de Cultivo de Órganos , Ratas , Ratas Sprague-Dawley , Espectrometría de Fluorescencia , Espectrofotometría Ultravioleta , Estereoisomerismo , alfa-Cristalinas/antagonistas & inhibidoresRESUMEN
alpha-Crystallin in its native state is a large, heterogeneous, low-molecular weight (LMW) aggregate that under certain conditions may progressively became part of insoluble high-molecular weight (HMW) systems. These systems are supposed to play a relevant role in eye lens opacification and vision impairment. In this paper, we report the effects of trehalose on alpha-crystallin aggregates. The role of trehalose in alpha-crystallin stress tolerance, chaperone activity and thermal stability is studied. The results show that trehalose stabilizes the alpha-crystallin native structure, inhibits alpha-crystallin aggregation, and disaggregates preformed LMW systems not affecting its chaperone activity.
Asunto(s)
Trehalosa/farmacología , alfa-Cristalinas/efectos de los fármacos , Benzotiazoles , Dicroismo Circular , Microscopía de Fuerza Atómica , Estructura Cuaternaria de Proteína , Espectrometría de Fluorescencia , Tiazoles , alfa-Cristalinas/química , alfa-Cristalinas/fisiologíaRESUMEN
The verification of the cDNA-deduced sequence of the high molecular weight glutenin subunit 1Bx7 in Chinese Spring cultivar was achieved by direct matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) analysis of the tryptic fragments. The published sequence of the 1Bx7 subunit contains 5 Lys and 15 Arg residues but, due to the presence of three Arg-Pro bonds, which are generally resistant to cleavage by trypsin, or cleaved to a very limited extent by trypsin, 19 peptides can be predicted. The identification of the tryptic fragments was achieved by direct MALDI-MS analysis by using three different matrices (DHB, SA and HCCA) in combination with the most compatible sample preparation procedures in order to obtain the maximum sequence coverage. MALDI analysis of the 1Bx7 tryptic digest resulted in the identification of the expected peptides and additional fragments arising from non-specific cleavages; the fragments that were not detected are peptides with low mass (from 147.2 to 317.4), so we obtained a sequence coverage of 98.8%. The results reported here also indicated that the sequence of the 1Bx7 subunit from cv. Chinese Spring is different from the cDNA-deduced sequence reported in the literature; in particular, a possible insertion of the hexapeptide QPGQGQ within the sequence Gln630-Tyr725 was suggested. Finally, it is possible to rule out glycosylation of the 1Bx7 subunit, or any other post-translational modification, to within the detection limits of the method.
Asunto(s)
Glútenes/análisis , Proteómica/instrumentación , Proteómica/métodos , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Triticum/química , Análisis de los Alimentos/instrumentación , Mapeo Peptídico/instrumentación , Mapeo Peptídico/métodos , Triticum/clasificaciónRESUMEN
Starch enzymatic degradation caused by endogenous hydrolases is studied by in situ NMR spectroscopy on a set of hard and soft wheat flours. The results obtained by two different techniques (HR-MAS and (1)H NMR in solution) are analyzed in terms of a Michaelis-Menten kinetic phenomenological model taking into account the presence of endogenous enzymes and their eventual inactivation. The parameters resulting from the best fit of all experimental data to the kinetic model equations are submitted to a multivariate statistical analysis to assess the role of the oligosaccharides release in distinguishing between hard and soft wheats.
Asunto(s)
Harina/análisis , Hidrolasas/metabolismo , Espectroscopía de Resonancia Magnética , Almidón/metabolismo , Triticum/química , CinéticaRESUMEN
A totally protected di-O-benzyl derivative of triacetonlactose dimethyl acetal was transformed into a 4'-hexeno disaccharide by elimination of acetone with t-BuOK in DMF and subsequently in 5'-C-methoxy derivative by oxidation with MCPBA in methanol as a solvent. The hydrolysis of this latter compound affords 2,6-di-O-benzyl-L-arabino-aldohexosos-5-ulose, which by intramolecular aldol condensation with DBU gives an inosose that was stereoselectively reduced to epi-inositol. Therefore our synthetic strategy offers a new and simple method to transform lactose into carbocyclic monosaccharide analogues.