Unraveling the mechanisms of tryptophan fluorescence quenching in the triosephosphate isomerase from Giardia lamblia

In the native state several proteins exhibit a quenching of fluorescence of their tryptophans. We studied triosephosphate isomerase from Giardia lamblia (GlTIM) to dissect the mechanisms that account for the quenching of fluorescence of its Trp. GlTIM contains four Trp per monomer (Trp75, Trp162, Trp173, and Trp196) distributed throughout the 3D structure. The fluorescence of the denatured enzyme is 3-fold higher than that of native GlTIM. To ascertain the origin of this phenomenon, single and triple mutants of Trp per Phe were made. The intrinsic fluorescence was determined, and the data were interpreted on the basis of the crystal structure of the enzyme. Our data show that the fluorescence of all Trp residues is quenched through two different mechanisms. In one, fluorescence is quenched by aromatic-aromatic interactions due to the proximity and orientation of the indole groups of Trp196 and Trp162. The magnitude of the quenching of fluorescence in Trp162 is higher than in the other three Trp. Fluorescence quenching is also due to energy transfer to the charged residues that surround Trp 75, 173 and 196. Further analysis of the fluorescence of GlTIM showed that, among TIMs from other parasites, Trp at position 12 exhibits rather unique properties.     

Putting the stress on senescence

Cultivation of primary cells over many generations eventually results in a reproducible loss of proliferative potential that has been termed 'replicative senescence'. Recent work has revealed the heterogeneity of senescence. Importantly, the analysis of the various aspects and types of senescence has turned out to be very informative about numerous in vivo processes, and particularly about carcinogenesis.     

EPR and redox characterization of iron-sulfur centers in nitrate reductases A and Z from Escherichia coli. Evidence for a high-potential and a low-potential class and their relevance in the electron-transfer mechanism.

The redox properties of the iron-sulfur centers of the two nitrate reductases from Escherichia coli have been investigated by EPR spectroscopy. A detailed study of nitrate reductase A performed in the range +200 mV to -500 mV shows that the four iron-sulfur centers of the enzyme belong to two classes with markedly different redox potentials. The high-potential group comprises a [3Fe-4S] and a [4Fe-4S] cluster whose midpoint potentials are +60 mV and +80 mV, respectively. Although these centers are magnetically isolated, they are coupled by a significant anticooperative redox interaction of about 50 mV. The [4Fe-4S]1+ center occurs in two different conformations as shown by its composite EPR spectrum. The low-potential group contains two [4Fe-4S] clusters with more typical redox potentials (-200 mV and -400 mV). In the fully reduced state, the three [4Fe-4S]1+ centers are magnetically coupled, leading to a broad featureless spectrum. The redox behaviour of the high-pH EPR signal given by the molybdenum cofactor was also studied. The iron-sulfur centers of the second nitrate reductase of E. coli, nitrate reductase Z, exhibit essentially the same characteristics than those of nitrate reductase A, except that the midpoint potentials of the high-potential centers appear negatively shifted by about 100 mV. From the comparison between the redox centers of nitrate reductase and of dimethylsulfoxide reductase, a correspondence between the high-potential iron-sulfur clusters of the two enzymes can be proposed.     

Evidence for cooperative effects in the binding of polyvalent metal ions to pure phosphatidylcholine bilayer vesicle surfaces

An internal NMR monitor for the study of lanthanide ion (Ln³⁺) binding to phospholipid bilayer membranes has been developed. The dimethylphosphate anion, DMP^?, forms labile complexes with Ln³⁺ in aqueous solution and in solutions also containing bilayer dispersions. The hyperfine shift in the DMP^? resonance induced by Pr³⁺ ions has been used to determine the overall thermodynamic formation constants for the Pr(DMP)²⁺ and Pr(DMP)₂⁺ complexes: 81 (M^?1) and 349 (M^?2) at 52°C; the limiting hyperfine shift (³¹P) at 52°C is 91.5 ppm downfield. These parameters, applied to the observed DMP^? hyperfine shift in the presence of the membrane, establish both the free Pr³⁺ concentration and the amount of Pr³⁺ bound to the phospholipid surface. Extensive data for the binding of Pr³⁺ to the outer surfaces of sonicated vesicles yield a limiting hyperfine shift per Pr³⁺ of 181.6 ppm downfield for the dipalmitoylphosphatidylcholine ³¹P resonance at 52°C, clearly demonstrating that the binding stoichiometry is two DPPCs per Pr³⁺. A Hill analysis indicates that the binding data are more anti-cooperative than a realistic Langmuir isotherm, yet more cooperative than a Stern isotherm incorporating electrostatic considerations at the Debye-Hückel level. Fittings to specific models lead to a cooperative model in which tense (T) sites, with low affinity for Pr³⁺, present in the absence of metal ions, quickly give way to relaxed (R) sites (two DPPCs per site), with much higher affinity for Pr³⁺, as the amount of Pr³⁺ bound to the surface increases. The intrinsic equilibrium constants for the binding of Pr³⁺ to DPPC vesicles are 2 M^?1 and 3 000 M^?1 for the T and R sites, respectively, at 52°C. The distribution coefficient between these sites ([R]/[T]) in the absence of Ln³⁺ is 0.14 at 52°C. We picture the binding site conversion as a head-group conformational change involving mostly the choline moiety. Sketchy results for binding on the inside vesicle surface indicate that the overall affinity for Pr³⁺ is significantly greater and suggest that the site stoichiometry may be different.     

Characterization and mapping of the pyrophosphorolytic activity of the phage phi 29 DNA polymerase. Involvement of amino acid motifs highly conserved in alpha-like DNA polymerases.

The phi 29 DNA polymerase, an alpha-like DNA polymerase, shows an inorganic pyrophosphate-dependent degradative activity with similar requirements to the corresponding one of Escherichia coli DNA polymerase I: (a) it requires a high concentration of inorganic pyrophosphate and is reversed by polymerization; (b) like DNA polymerization, it needs a duplex DNA with protruding 5' single-strand; (c) it acts in the 3' to 5' direction releasing free dNTPs, thus, it can be considered as the reversal of polymerization; (d) although a correctly base-paired 3' primer terminus is the preferred substrate, the pyrophosphorolytic activity is able to remove mismatched 3' ends. In agreement with the structural and functional model previously proposed for the phi 29 DNA polymerase, the analysis of point mutations has revealed that the pyrophosphorolytic activity, like the polymerization activity, is located at the C-terminal portion of the molecule, involving the amino acid motif YCDTD, highly conserved in alpha-like DNA polymerases. Furthermore, the analysis of phi 29 DNA polymerase mutants indicates that pyrophosphorolysis, like DNA polymerization, also requires an efficient translocation of the enzyme along the template.     

Fungal Olecranon Bursitis in an Immunocompetent Patient by <Emphasis Type="Italic">Knoxdaviesia dimorphospora</Emphasis> sp. nov.: Case Report and Review

Bursitis is a common medical condition that can occur either with or without infection. We present a case of fungal olecranon bursitis in an immunocompetent individual caused by the new species Knoxdaviesia dimorphospora. It is a dematiaceous filamentous fungus characterized by the production of two different conidia: hyaline and cylindrical, which rise up from phialidic conidiogenous cells located in the upper part of differentiated and unbranched conidiophores, and pale brown and ellipsoidal conidia produced by phialidic conidiogenous cells which are born directly on hyphae. In addition to its morphological peculiarities, the novelty of the fungus was confirmed by sequence analysis of the internal transcribed spacer (ITS) regions and D1/D2 domains of the 28S of the nuclear rRNA gene. The fungal infection was confirmed by cytological examination and repeated cultures. The infection was resolved by surgical debridement and drainage, and the patient presented a complete functional recovery 3 months later. The in vitro antifungal susceptibility to this new human opportunist is provided, terbinafine being the drug with the most potent activity.     

Effect of dietary supplementation with a highly pure and concentrated docosahexaenoic acid (DHA) supplement on human sperm function

The aim of this study was to evaluate the possible beneficial effects of diet supplementation with a highly concentrated and purified docosahexaenoic acid (DHA) formula on human sperm function. We performed a prospective, randomized, double blind, placebo-controlled intervention study. One-hundred eighty human semen samples from sixty infertile patients recruited in a private assisted reproduction center were included. All samples were examined according to World Health Organization guidelines. We analyzed macroscopic and microscopic sperm parameters, oxidative stress, apoptosis, lipid peroxidation, mitochondrial membrane potential and DNA fragmentation before and after supplementation with different DHA daily doses (0.5, 1 and 2?g) or placebo for 1 and 3 months. No differences were found in traditional sperm parameters except for progressive sperm motility, with a significant increase after DHA ingestion after the first month with 1 or 2?g doses and after 3 months with 0.5?g of DHA. This effect was more evident in asthenozoospermic patients. No differences were found in any molecular semen parameter except oxidative stress, in which a slight benefit was observed after DHA treatment. In conclusion, this study support previous indications that highlight the importance of DHA supplementation as a means of improving sperm quality in asthenozoospermic men.     

What is going on at the Molí del Salt site? A zooarchaeological approach to the last hunter-gatherers from South Catalonia

Abstract

Small game seems to have increased during the Upper Palaeolithic to the detriment of large game on the Iberian Peninsula. The economical and socio-cultural factors associated with this ecological shift represent a widely discussed topic. The present work attempts to elucidate the subsistence strategies occurring through the Late Pleistocene in Iberia using the example of the Molí del Salt (Tarragona, Spain), an archaeological site located in the NE of the Iberian Peninsula. The taphonomical analysis of faunal remains shows a high incidence of human activity on different taxonomical groups, although the European rabbit (Oryctolagus cuniculus) stands out. This taxon presents cut-marks related to various processing activities (e.g. skinning and defleshing) and intentional bone breakage to access marrow. The abundance of specimens with human-induced damage enables us to make inferences regarding the procurement strategies and the occupational patterns at the site, where long and stable occupations seem to have occurred.