Probing the role of tryptophans in Aequorea victoria green fluorescent proteins with an expanded genetic code

The expanded genetic code in combination with site-directed mutagenesis was used to probe spectroscopic and structural roles of tryptophan (Trp) residues in Aequorea victoria green fluorescent proteins (avGFPs). Nine different halogen-, chalcogen-, and methyl-containing Trp isosteric analogues and surrogates were incorporated into avGFPs containing indole moieties in, and outside of, the chromophore, by the use of the selective pressure incorporation method. Such isosteric replacements introduced minimal local geometry changes in indole moieties, often to the level of single atomic exchange ('atomic mutation') and do not affect three-dimensional structures of avGFPs but induce changes in spectral properties. Our approach offers a new platform to re-evaluate issues like resonance transfer, mechanisms of chromophore formation and maturation, as well as the importance of local geometry and weak sulphur-aromatic interactions for avGFP spectral properties and structural stability. The library of novel tailor-made avGFP mutants and variants generated in this work has demonstrated not only the potentials of the expanded genetic code to study spectroscopic functions, but also a new approach to generate tailor-made proteins with interesting and useful spectral properties.     

Expansion of the genetic code enables design of a novel "gold" class of green fluorescent proteins

Much effort has been dedicated to the design of significantly red shifted variants of the green fluorescent protein (GFP) from Aequoria victora (av). These approaches have been based on classical engineering with the 20 canonical amino acids. We report here an expansion of these efforts by incorporation of an amino substituted variant of tryptophan into the "cyan" GFP mutant, which turned it into a "gold" variant. This variant possesses a red shift in emission unprecedented for any avFP, similar to "red" FPs, but with enhanced stability and a very low aggregation tendency. An increasing number of non-natural amino acids are available for chromophore redesign (by engineering of the genetic code) and enable new general strategies to generate novel classes of tailor-made GFP proteins.     

Complexes of aluminium(III) with glucose-6-phosphate in aqueous solutions

The interaction of aluminium(III) with glucose-6-phosphate (GP: LH2) in aqueous solutions has been studied from pH 1 to pH 8, by pH-potentiometry and multinuclear (31P, 27Al, 13C) NMR spectroscopy. Various mononuclear species (MLH2, MLH, ML, ML2H, ML2 and MLH(-3)) and dinuclear complexes M2L2H-n (n=1-4) are formed in the system. NMR clearly indicates that GP is already bound to Al(III) at pH 1. The potentiometric speciation results are confirmed and completed by spectroscopic experiments. Many peaks are observed in the 31P NMR spectra suggesting the formation of isomeric species. An attempt to assign the signals to the corresponding complexes is made, allowing a discussion about their structure. Interestingly enough no metal ion-induced deprotonation and coordination of the alcoholic-OH functions have been observed.     

Interaction of acidic poly-amino acids with octacalcium phosphate

Octacalcium phosphate (OCP) hydrolysis into hydroxyapatite (HA) has been investigated in aqueous solutions at different concentrations of poly-L-aspartate (PASP) and poly-L-glutamate (PGLU). In the absence of the polyelectrolytes, the transformation of OCP into HA is complete in 48 h. Both poly-L-aspartate and poly-L-glutamate inhibit OCP hydrolysis. However, PGLU displays a greater inhibiting effect, as a result of the different extent of phase transformation obtained at the same polyelectrolyte concentration. The inhibition takes place through polyelectrolyte adsorption on the (100) face of OCP crystals, which prevents the splitting of OCP crystals along their c-axis and the transformation into the final very long, needle-like, apatitic crystals.     

Aluminum speciation studies in biological fluids. Part 9. A quantitative investigation of aluminum(III)-glutamate complex equilibria and their potential implications for aluminum metabolism and toxicity

As a nonessential element, aluminum is likely to be toxic both at low usual dietary levels in the long run (chronic toxicity) and at high therapeutic levels in shorter periods of time (acute toxicity). In both situations, aluminum toxicity is a direct function of aluminum bioavailability, which is itself dependent on Al(3+) solubility and charge neutralization. Dietary acids, by their intrinsic acidity and coordinating capacity, can extend the pH range, thus the section of the gastrointestinal tract, within which the Al(3+) ion remains soluble, and also help Al(3+) diffusion across the intestinal epithelium through the formation of neutral complex species. The present work examines the impact of glutamic acid, an essential amino acid also widely used in industrial food and drinks, on aluminum speciation in the gastrointestinal tract and blood plasma. Complex formation between the Al(3+) ion and glutamate has first been investigated through potentiometric titrations, complex stoichiometries being then checked by ESI mass spectrometry and NMR measurements. A series of mono- and polynuclear species has been characterized, whose influence on aluminum distribution in vivo has been assessed by computer simulation. The capacity of glutamate to maintain Al(3+) ions in solution under normal dietary conditions is predicted to be intermediate between glycine-like amino acids and succinate on the one hand, and tartrate and malate on the other hand, its Al(3+) neutralization effect being similar to that of succinate, tartrate and malate. These results, which point to a potential aggravating role of glutamate on aluminum gastrointestinal absorption, substantiate recent observations made on rats. In spite of the moderate effect expected from glutamate on aluminum bioavailability under most aluminum-based therapies investigated, attention is therefore called to the risk of glutamic acid ingestion simultaneously to any aluminum therapeutic form. Incidentally, the former implication of 'the' aluminum glutamate complex in the transfer of aluminum through the blood-brain barrier of aluminum loaded rats may effectively be attributed to one of the species characterized here, but is of no significance at all to aluminum contamination in humans, even at most extreme levels.     

Carbohydrate and amino acid metabolism in Tuber borchii mycelium during glucose utilization: a (13)C NMR study

The metabolism of [1-13C]glucose in the vegetative mycelium of the ectomycorrhizal ascomycete Tuber borchii was studied in order to characterize the biochemical pathways for the assimilation of glucose and amino acid biosynthesis. The pathways were characterized using nuclear magnetic resonance spectroscopy in conjunction with [1-13C]glucose labeling. The enzymes of mannitol cycle and ammonium assimilation were also evaluated. The majority of the 13C label was incorporated into mannitol and this polyol was formed via a direct route from absorbed glucose. Amino acid biosynthesis was also an important sink of assimilated carbon and 13C was mainly incorporated into alanine and glutamate. From this intramolecular 13C enrichment, it is concluded that pyruvate, arising from [1-13C]glucose catabolism, was used by alanine aminotransferase, pyruvate dehydrogenase and pyruvate carboxylase before entering the Krebs cycle. The transfer of 13C-labeled mycelium on [12C]glucose showed that mannitol, alanine, and glutamate carbon were used to synthesize glutamine and arginine that likely play a storage role.     

Towards a framework for the evolutionary genomics of Kinetoplastids: what kind of data and how much?

The current status of kinetoplastids phylogeny and evolution is discussed in view of the recent progresses on genomics. Some ideas on a potential framework for the evolutionary genomics of kinetoplastids are presented.     

The effect of Cyclosporine A chronic administration on the antioxidant pattern of rat liver mitochondria: structural and functional consequences

Cyclosporine A (CsA) plays a pivotal role in controlling Ca2+ movement in the cell modulating also the mitochondrial permeability transition pore. We investigated if chronic administration of CsA may have some effects on the lipophilic and hydrophilic antioxidant pattern of rat liver mitochondria and on their morphological structure. It seems that CsA administration does not statistically affect the redox status of the antioxidants investigated and their amounts (vitamin E, CoQ9, CoQ10, glutathione, uric acid and ascorbic acid) despite the variety of effects that this treatment produces at physiological and morphological levels. However, some kind of derangement could occur in the liver biochemical machinery since CsA treatment induces a markedly increased variability in antioxidant contents.     

The role of intravascular ultrasound imaging in vascular brachytherapy

Intracoronary brachytherapy has recently emerged as a new therapy to prevent restenosis. Initial experimental work was achieved in animal models and the results were assessed by histomorphometry. Initial clinical trials used angiography to guide dosimetry and to assess efficacy. Intravascular ultrasound (IVUS) permits tomographic examination of the vessel wall, elucidating the true morphology of the lumen and transmural components, which cannot be investigated on the lumenogram obtained by angiography. This paper reviews the use of IVUS in the clinical studies of brachytherapy conducted to date. IVUS allows clinicians to make a thorough assessment of the remodeling of the vessel and appears to have a major role to play in facilitating understanding of the underlying mechanisms of action in this emerging field. The authors propose that state-of-the-art IVUS techniques should be employed to further knowledge of the mechanisms of action of brachytherapy in atherosclerotic human coronary arteries.