Nanoporous surfaces as harvesting agents for mass spectrometric analysis of peptides in human plasma

Silica-based nanoporous surfaces have been developed in order to capture low molecular weight peptides from human plasma. Harvested peptides were subjected to mass spectrometric analysis by using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) as a means of detecting and assessing the bound molecules. Peptide profiles consisting of about 70 peaks in the range 800-10,000 m/z were generated. The method could allow detection of small peptides at ng/mL concentration levels, either in standard solutions or in plasma. The same molecular cutoff effect was observed for mixtures of standard proteins and peptides incubated with silicon-based nanoporous surfaces.     

Structural and functional analysis of the two haemoglobins of the antarctic seabird Catharacta maccormicki characterization of an additional phosphate binding site by molecular modelling.

The amino-acid sequence and the oxygen-binding properties of the two haemoglobins of the Antarctic seabird south polar skua have been investigated. The two haemoglobins showed peculiar functional features, which were probably acquired to meet special needs in relation to the extreme environmental conditions. Both haemoglobins showed a weak alkaline Bohr effect which, during prolonged flight, may protect against sudden and uncontrolled stripping of oxygen in response to acidosis. We suggest that a weak Bohr effect in birds may reflect adaptation to extreme life conditions. The values of heat of oxygenation suggest different functional roles of the two haemoglobins. The experimental evidence suggests that both haemoglobins may bind phosphate at two distinct binding sites. In fact, analysis of the molecular models revealed that an additional phosphate binding site, formed by residues NA1alpha, G6alpha and HC3alpha, is located between the two alpha chains. This additional site may act as an entry/leaving site, thus increasing the probability of capturing phosphate and transferring it to the main binding site located between the two beta chains by means of a site-site migratory mechanism, thereby favouring the release of oxygen. It is suggested that most haemoglobins possess an additional phosphate binding site, having such a role in oxygen transport.     

Supramolecular assemblies of carotenoids.

Carotenoid self-assemblies were formed by aqueous dilution of ethanolic solutions. The four 3',6'-epimers of capsanthol ((all-E,3R,5'R)-3,3',6'-trihydroxy-beta,kappa-carotene) give rise to right- and left-handed card-pack and head-to-tail types of self-assemblies detected by exciton couplets appearing in the CD spectra. Slow kinetics of formation followed for some of the aggregates indicate the complexity of the process. The exciton signals do not appear from equimolar mixtures of related compounds that produce identical type of aggregates of opposite sense on their own. Transformation of self-assembly may reflect the population of kappa-ring rotamers.     

Oxidative stress and phytochelatin characterisation in bread wheat exposed to cadmium excess.

In this work, we first investigated if the bread wheat (Triticum aestivum L.) cv. Albimonte can be defined as "shoot cadmium excluder"--by comparing the cadmium (Cd) content in leaves and roots and by calculating the shoot-to-root Cd concentration ratio. Furthermore, we evaluated if the exposure to Cd excess could generate oxidative stress in leaves and roots of this cv., in terms of hydrogen peroxide (H(2)O(2)) accumulation, NAD(P)H oxidation rate, and variations in reduced glutathione (GSH) content and peroxidase (POD, EC 1.11.1.7) activity. Finally, we surveyed possible quali- quantitative differences in thiol-peptide compound pattern between roots and leaves, in order to verify whether phytochelatins (PCs) and related thiol-peptides could contribute in limiting the Cd-induced oxidative stress. Unambiguous characterisation of PCs and related forms present in the root samples was obtained by electrospray ionisation mass spectrometry (ESI-MS) and ESI-tandem MS (ESI-MS/MS). Our results indicate that in leaves the stress generated by the low accumulation of Cd (due to a moderate translocation in planta) seems to be counteracted by the antioxidant response and by the PC biosynthesis. On the contrary, in roots, in spite of the elevated presence of PCs and related thiol-peptide-compounds, the excess of Cd causes a decline in the antioxidant protection of the organ, with the consequent generation of considerable amounts of H(2)O(2), a direct agent of oxidative stress.     

Impairing the bioenergetic status and the biogenesis of mitochondria triggers mitophagy in yeast

Autophagy, a highly regulated programme found in almost all eukaryotes, is mainly viewed as a catabolic process that degrades nonessential cellular components into molecular building blocks, subsequently available for biosynthesis at a lesser expense than de novo synthesis. Autophagy is largely known to be regulated by nutritional conditions. Here we show that, in yeast cells grown under nonstarving conditions, autophagy can be induced by mitochondrial dysfunction. Electron micrographs and biochemical studies show that an autophagic activity can result from impairing the mitochondrial electrochemical transmembrane potential. Furthermore, mitochondrial damage-induced autophagy results in the preferential degradation of impaired mitochondria (mitophagy), before leading to cell death. Mitophagy appears to rely on classical macroautophagy machinery while being independent of cellular ATP collapse. These results suggest that in this case, autophagy can be envisioned either as a process of mitochondrial quality control, or as an ultimate cellular response triggered when cells are overwhelmed with damaged mitochondria.     

In vitro binding of leukotriene B4 (LTB4) to human serum albumin: evidence from spectroscopic, molecular modeling, and competitive displacement studies

Circular dichroism (CD) and UV absorption spectroscopy were utilized for the first time to investigate the interaction between leukotriene B4 (LTB4) and human serum albumin (HSA) in vitro. The weak intrinsic CD signal of LTB4 was enhanced fivefold in the presence of HSA. The red-shifted, hypochromic, and reduced vibrational fine structure of the ligand/protein UV absorption spectrum indicated complexation of the two molecules in solution. Results obtained from CD titration experiments were subjected to non-linear regression analysis to estimate the binding parameters (Ka = 6.7 x 10(4) M(-1), n = 1). Palmitic acid strongly decreased the induced CD signal of the LTB4/HSA complex, suggesting the role of a high-affinity fatty acid HSA binding site in the leukotriene complexation. Molecular modeling calculations based on the crystal structure of HSA predicted that the long-chain fatty acid site that overlaps with drug binding site II in subdomain IIIA was the most likely binding location for LTB4. Using the drug site II-specific marker ligand rac-ibuprofen, this prediction was confirmed with induced-CD displacement measurements. To the authors' knowledge, the current study represents the first demonstration of binding of LTB4 to HSA in vitro and has implications for the biological transport of this important pro-inflammatory mediator in vivo.     

Increasing mitochondrial substrate-level phosphorylation can rescue respiratory growth of an ATP synthase-deficient yeast

In a previous study we have identified Fmc1p, a mitochondrial protein involved in the assembly/stability of the yeast F0F1-ATP synthase at elevated temperatures. The deltafmc1 mutant was shown to exhibit a severe phenotype of very slow growth on respiratory substrates at 37 degrees C. We have isolated ODC1 as a multicopy suppressor of the fmc1 deletion restoring a good respiratory growth. Odc1p expression level was estimated to be at least 10 times higher in mitochondria isolated from the deltafmc1/ODC1 transformant as compared with wild type mitochondria. Interestingly, ODC1 encodes an oxodicarboxylate carrier, which transports alpha-ketoglutarate and alpha-ketoadipate or any other transported tricarboxylic acid cycle intermediate in a counter-exchange through the inner mitochondrial membrane. We show that the suppression of the respiratory-growth-deficient fmc1 by the overexpressed Odc1p was not due to a restored stable ATP synthase. Instead, the rescuing mechanism involves an increase in the flux of tricarboxylic acid cycle intermediate from the cytosol into the mitochondria, leading to an increase in the alpha-ketoglutarate oxidative decarboxylation, resulting in an increase in mitochondrial substrate-level-dependent ATP synthesis. This mechanism of metabolic bypass of a defective ATP synthase unravels the physiological importance of intramitochondrial substrate-level phosphorylations. This unexpected result might be of interest for the development of therapeutic solutions in pathologies associated with defects in the oxidative phosphorylation system.     

Ligand loop effects on the free energy change of redox and pH-dependent equilibria in cupredoxins probed on amicyanin variants

In this work, we have determined the thermodynamic parameters of the reduction of four different variants of Thiobacillus versutus amicyanin by electrochemical techniques. In addition, the thermodynamic parameters were determined of the low-pH conformational change involving protonation of the C-terminal histidine ligand and the concomitant dissociation of this histidine from the Cu(I) ion. In these variants, the native C-terminal loop containing the Cys, His, and Met copper ligands has been replaced with the corresponding polypeptide segments of Pseudomonas aeruginosa azurin, Populus nigra plastocyanin, Alcaligenes faecalis S-6 pseudoazurin, and Thiobacillus ferrooxidans rusticyanin. For the reduction reaction, each loop invariably holds an entropic "memory" of the mother protein. The thermodynamics of the low-pH transition vary in a fashion that is species-dependent. When present, the memory effect again shows a large entropic component. In particular, loop elongation tends to favor the formation of the Cu(I)-His bond (hence disfavors His protonation, yielding lower pK(a) values) probably due to an increased flexibility of the loop in the reduced state. Overall, it appears that both reduction and low-pH transition are loop-responsive processes. The spacing between the ligands mostly affects the change in the conformational freedom that accompanies the reaction.