Three acidic residues are at the active site of a beta-propeller architecture in glycoside hydrolase families 32, 43, 62, and 68

Multiple-sequence alignment of glycoside hydrolase (GH) families 32, 43, 62, and 68 revealed three conserved blocks, each containing an acidic residue at an equivalent position in all the enzymes. A detailed analysis of the site-directed mutations so far performed on invertases (GH32), arabinanases (GH43), and bacterial fructosyltransferases (GH68) indicated a direct implication of the conserved residues Asp/Glu (block I), Asp (block II), and Glu (block III) in substrate binding and hydrolysis. These residues are close in space in the 5-bladed beta-propeller fold determined for Cellvibrio japonicus alpha-L-arabinanase Arb43A [Nurizzo et al., Nat Struct Biol 2002;9:665-668] and Bacillus subtilis endo-1,5-alpha-L-arabinanase. A sequence-structure compatibility search using 3D-PSSM, mGenTHREADER, INBGU, and SAM-T02 programs predicted indistinctly the 5-bladed beta-propeller fold of Arb43A and the 6-bladed beta-propeller fold of sialidase/neuraminidase (GH33, GH34, and GH83) as the most reliable topologies for GH families 32, 62, and 68. We conclude that the identified acidic residues are located at the active site of a beta-propeller architecture in GH32, GH43, GH62, and GH68, operating with a canonical reaction mechanism of either inversion (GH43 and likely GH62) or retention (GH32 and GH68) of the anomeric configuration. Also, we propose that the beta-propeller architecture accommodates distinct binding sites for the acceptor saccharide in glycosyl transfer reaction.     

Hypoxia/reoxygenation and vitamin C intake influence NO synthesis and antioxidant defenses of neutrophils

Oxidative stress induced by hypoxia/reoxygenation mediates the pathophysiological consequence of ischemia/reperfusion and human diseases. Diving apnea could be a good model of oxidative stress induced by hypoxia/reoxygenation. We studied the influence of vitamin C diet supplementation on the response of neutrophil antioxidant defenses, NO production, and redox status to diving apnea. Seven professional apnea divers participated in a double-blind cross study. Divers were assigned to either vitamin C-supplemented (1 g/d for a week) or placebo groups. Blood samples were taken under basal conditions, immediately after diving apnea for 4 h and after 1 h of recovery. Plasma vitamin C increased only in the supplemented group after diving and was maintained high in recovery. Diving apnea decreased neutrophil GSH/GSSG ratio in both groups, but maintained protein carbonyl derivates. Neutrophil catalase activity and levels and glutathione peroxidase activity were lower in the supplemented group than in the placebo group after diving. iNOS and nitrite levels decreased only in the supplemented group after diving and recovery. Diving apnea induced oxidative stress and initiated neutrophil reactions that resemble the acute-phase immune response with increased myeloperoxidase activity in neutrophils. Diet supplementation with vitamin C reduced neutrophil iNOS levels and NO production.     

Self-incompatibility genotypes in almond re-evaluated by PCR,stylar ribonucleases,sequencing analysis and controlled pollinations

As part of the almond breeding programme at IRTA, we investigated the S genotypes of several cultivars using a combination of RNase zymograms, testcrosses, pollen-tube growth analysis and molecular identification by PCR analysis. For some of the cultivars examined, discrepancies appeared between their S alleles as reported in the literature and those found in this investigation, leading to a re-evaluation of their S genotypes. Analysis of the stylar ribonucleases (RNases), which are known to correlate with S alleles, of cvs. Achaak, Ardechoise, Desmayo Largueta, Ferrastar, Gabaix, Garbí, Glorieta, Languedoc, Primorskiy and Texas revealed inconsistencies with respect to the S₅ and S₁₀ alleles. However, PCR with the conserved primer pair AS1II/AmyC5R failed to detect any of these inconsistencies. When the S alleles from Desmayo Largueta, Gabaix, Primorskiy and Texas were sequenced, Texas and Primorskiy were found to carry the reported S₅ allele, while Desmayo Largueta and Gabaix carried a new allele, which has been tentatively denoted as S₂₅ This new S allele, previously reported to be S₁₀, was also identified in Achaak, Ardechoise and Ferrastar. The proposed new S genotypes are Achaak (S₂S₂₅), Ardechoise (S₁S₂₅), Desmayo Largueta (S₁S₂₅), Ferrastar (S₂S₂₅) and Gabaix (S₁₀S₂₅). The S alleles of Garbí, Glorieta, Languedoc, Texas and Primorskiy remain as reported in the literature. Testcrosses in the field and laboratory confirmed the new S genotypes. One cultivar (Gabaix) could be assigned to the existing cross-incompatibility group O of unique genotypes, and two new groups were established (XVI and XVII) consisting of two cultivars each. The clarification of these S alleles will be useful in almond breeding programmes and for planning new commercial orchards in the future.     

Spectral analysis and fingerprinting for biomedia characterisation

Classical culture media, as well as domestic and/or industrial wastewater treated by biological processes, have a complex composition. The on-line and/or in situ determination of some substances is possible, but expensive, as sample collection and pre-treatment are often necessary with strict rules of sterility. More global methods can be used to detect rapidly "accidents" such as the appearance of an undesirable by-product in a fermentation broth or of a toxic substance in wastewater. These methods combine a "hard" part, for sensing, and a "soft" part, for data treatment. Among potential "hard" candidates, spectroscopy can be the basis for non-invasive and non-destructive measuring systems. Some of them have been already tested in situ: ultra-violet-visible, infra-red (mid or near), fluorescence (mono-dimensional, two-dimensional or synchronous), dielectric, while others, more sophisticated, such as mass spectrometry, coupled or not to pyrolysis, nuclear magnetic resonance and Raman spectroscopy, have been proposed. All these methods provide spectra, i.e. large sets of data, from which meaningful information should be rapidly extracted, either for analysis or fingerprinting. The recourse to data-mining techniques (the "soft" part) such as principal components analysis, projection on latent structures or artificial neural networks, is a necessary step for that task. A review of techniques, mostly based on spectroscopy, with examples taken in the bioengineering field in general is proposed.     

Wild-type Escherichia coli producing microcins B17, D93, J25, and L; cloning of genes for microcin L production and immunity

For the first time, an Escherichia coli strain producing four microcins (Mcc), B17, D93, J25, and L, and showing immunity to Mcc V was isolated and characterized. Each of the gene clusters encoding the production of Mcc B17, D93, and L was cloned separately. The gene cluster for Mcc L was cloned within a 13.5-kb HindIII-SalI fragment, which includes the Mcc V immunity gene, cvi.     

Zinc-induced decrease of the thermal stability and regeneration of rhodopsin

Zinc is present at high concentrations in the photoreceptor cells of the retina where it has been proposed to play a role in the visual phototransduction process. In order to obtain more information about this role, the study of the effect of zinc on several properties of the visual photoreceptor rhodopsin has been investigated. A specific effect of Zn(2+) on the thermal stability of rhodopsin, obtained from bovine retinas and solubilized in dodecyl maltoside detergent, in the dark is reported. The thermal stability of rhodopsin in its ground state (dark state) is clearly reduced with increasing Zn(2+) concentrations (0-50 microm Zn(2+)). The thermal bleaching process is accelerated in the presence of Zn(2+) with k rate constants, at 55 degrees C, of 0.028 +/- 0.002 min(-1) (0 microm Zn(2+)) and 0.056 +/- 0.003 min(-1) (50 microm Zn(2+)), corresponding to t(12) values of 24.4 +/- 1.6 min and 11.8 +/- 0.1 min, respectively. Thermodynamic parameters derived from Arrhenius plots show a significant E(a) increase at 50 microm Zn(2+) for the process, with deltaG++ decrease and increase in deltaH++ and deltaS++ possibly reflecting conformational rearrangements and reordering of water molecules. The stability of the metarhodopsin II intermediate is also decreased and changes in the metarhodopsin II decay pathway are also detected. The extent of rhodopsin regeneration in vitro is also reduced by zinc. These effects, specific for zinc, are also seen for rhodopsin in native disc membranes, and may be relevant to the suggested role of Zn(2+) in normal and pathological retinal function.     

A graphical method for the analysis of anisotropic rotational diffusion in proteins

A graphical method for the analysis of relaxation data is presented. It allows a fast estimation of the range of values of the components of the axially symmetric rotational diffusion tensor that are compatible with the experimental relaxation data. The graphical method clearly shows the contribution of different experimental relaxation parameters to the measured anisotropy. In particular, for proteins with moderate anisotropy, data from at least two N-H bonds forming angles close to 0° and 90° with respect to the principal axis of the rotational diffusional tensor are needed. For very anisotropic systems, combination of different relaxation parameters from a single residue is enough to characterize the local anisotropy.     

Gas-chromatography/mass-spectrometry analysis of human skin constituents as heptafluorobutyrate derivatives with special reference to long-chain bases

The composition of the constituents (monosaccharides, long-chain bases, and fatty acids) found in an ethanol extract of the human skin could be determined, without time-consuming steps of purification, after acid-catalyzed anhydrous methanolysis, followed by the formation of volatile derivatives with heptafluorobutyric anhydride and gas-chromatography/mass-spectrometry analysis. Despite the extreme heterogeneity of such extracts, the electron impact analysis of the constituents allowed qualitative and quantitative determinations of monosaccharides, long-chain bases, fatty acids, and alkyl-glycerols. Throughout the different long-chain bases, sphingenines (Sphes), sphinganines, phytosphingosines, and 6-hydroxy-Sphes (6oh-Sphes) can be identified and quantified. Long-chain bases with a chain-length up to 28 carbon atoms can be identified through specific fragmentation patterns in the electron impact mode. Particular attention was drawn to the behavior of compounds of the family of 6oh-Sphes upon acid-catalyzed methanolysis.