Hydrogen/deuterium exchange studies of native rabbit MM-CK dynamics

Creatine kinase (CK) isoenzymes catalyse the reversible transfer of a phosphoryl group from ATP onto creatine. This reaction plays a very important role in the regulation of intracellular ATP concentrations in excitable tissues. CK isoenzymes are highly resistant to proteases in native conditions. To appreciate localized backbone dynamics, kinetics of amide hydrogen exchange with deuterium was measured by pulse-labeling the dimeric cytosolic muscle CK isoenzyme. Upon exchange, the protein was digested with pepsin, and the deuterium content of the resulting peptides was determined by liquid chromatography coupled to mass spectrometry (MS). The deuteration kinetics of 47 peptides identified by MS/MS and covering 96% of the CK backbone were analyzed. Four deuteration patterns have been recognized: The less deuterated peptides are located in the saddle-shaped core of CK, whereas most of the highly deuterated peptides are close to the surface and located around the entrance to the active site. Their exchange kinetics are discussed by comparison with the known secondary and tertiary structures of CK with the goal to reveal the conformational dynamics of the protein. Some of the observed dynamic motions may be linked to the conformational changes associated with substrate binding and catalytic mechanism.     

An improved method for typing of plasma α1-protease inhibitor (PI1) variants in sheep

Summary
Sheep plasma α₁-protease inhibitor (PI1) variants were typed by analysing neuraminidase treated samples by isoelectric focusing in ultra-thin polyacrylamide gels followed by staining for trypsin inhibition. The 10 different PI1 pheno-types observed were shown to be controlled by four codominant alleles. The probability of sire exclusion provided by PI1 polymorphism was about 0.45 in both of the studied milk sheep breeds (Latxa and Karrantzar) of the Basque Country.     

Polymorphism in Cepaea Hortensis in marginal populations in Spain

Cepaea hortensis (Mull) is found in Spain only in the western part of the Iberian mountain range. This is the southernmost limit of its range. The most frequent colour is yellow, brown being very rare. The most frequent band patterns are 12345, fused banded and unbanded. There are associations between maximum and minimum temperatures and pink and effectively unbanded yellow phenotypes. The populations may be grouped into two zones, one located in the north and the other in the south of the sampled area. Visual predation was not observed. The marginal populations studied by us were compared with others from Northern Europe, and it was observed that pink snails are more frequent at the southern limit, and unbanded ones at the northern limit. This was interpreted as a climatic selection.     

Blood parameters and metabolites in the teleost fish Colossoma macropomum exposed to sulfide or hypoxia

Juvenile tambaqui, Colossoma macropomum, were exposed to sulfide and hypoxia for 12, 24, 48 and 96 h. Hemoglobin concentrations, red blood cell counts, and mean cell hemoglobin, were higher at 12 h in fish exposed to hypoxia. However, control fish and those exposed to sulfide and hypoxia had lower red blood cell count, hemoglobin concentration and hematocrit at 96 h. Methemoglobin was higher than in the controls, probably due to the hypoxemia induced by these stressors. Sulfhemoglobin was not detected in significant amounts in the blood of fish exposed to sulfide (in vivo), yet hemoglobin converted into sulfhemoglobin at 1-15 mM sulfide in vitro. Anaerobic metabolism seemed to be an important mechanism for adapting to sulfide exposure and blood pH returned to control values after 24 h of sulfide, preventing acidosis. The high sulfide tolerance in tambaqui is associated with its high tolerance to hypoxia.     

Functional morphology of gills and respiratory area of two active rheophilic fish species, Plagioscion squamosissimus and Prochilodus scrofa

Measurements of gill dimensions were carried out on two ecologically distinct active rheophilic teleost species, the curvina Plagioscion squamosissimus and the curimbatá Prochilodus scrofa , and were analysed in relation to body mass according to the equation Y=aW^b . The gill respiratory areas of P. squamosissimus and P. scrofa were large as expected for active fish and increased with increasing body mass ( b =0.70 and 0.72, respectively) showing no significant difference between them. However, the large respiratory area of both species was realized in a different way revealing an adaptation to the plasticity of head components related to feeding habits. Consequently, significant differences were found between the number and average length of gill filaments and the bilateral area of secondary lamellae. The respiratory area of P. scrofa was due mainly to larger bilateral surface area of the secondary lamellae and its growth coefficient ( b =0.51) that was significantly higher ( P <0.05) than that found for P. squamosissimus ( b = 0.36). The frequency of secondary lamellae mm⁻¹ of filament was similar in both species (22 ± 2 on one side of gill filament). The dimensions of gill components and the respiratory area of these species suggest a complex interaction between head form, and feeding habits related to the functional morphology of the gills to meet the oxygen requirements of each species.     

The Leptospira interrogans lexA gene is not autoregulated

Footprinting and mutagenesis experiments demonstrated that Leptospira interrogans LexA binds the palindrome TTTGN(5)CAAA found in the recA promoter but not in the lexA promoter. In silico analysis revealed that none of the other canonical SOS genes is under direct control of LexA, making the leptospiral lexA gene the first described which is not autoregulated.     

Physiological responses to sulfide toxicity by the air-breathing catfish, Hoplosternum littorale (Siluriformes, Callichthyidae)

Hemolytic anemia accompanied by changes in the immunology system is one of the sulfide intoxication harmful effects on Hoplosternum littorale. Hematological parameters are considered as effective indicators of stress caused by this hydrogen sulfide. During sulfide exposure, H. littorale neither alters the methemoglobin concentration nor forms sulfhemoglobin in the presence of high levels of dissolved sulfide in the water. Cytochrome c oxidase shows little activity in the gills and blood of H. littorale when exposed to sulfide. Alternative metabolic routes are suggested through which the accumulation of pyruvate leads to the formation of an end product other than lactate.     

The Zebrafish as a New Model for the In Vivo Study of Shigella flexneri Interaction with Phagocytes and Bacterial Autophagy

Autophagy, an ancient and highly conserved intracellular degradation process, is viewed as a critical component of innate immunity because of its ability to deliver cytosolic bacteria to the lysosome. However, the role of bacterial autophagy in vivo remains poorly understood. The zebrafish (Danio rerio) has emerged as a vertebrate model for the study of infections because it is optically accessible at the larval stages when the innate immune system is already functional. Here, we have characterized the susceptibility of zebrafish larvae to Shigella flexneri, a paradigm for bacterial autophagy, and have used this model to study Shigella-phagocyte interactions in vivo. Depending on the dose, S. flexneri injected in zebrafish larvae were either cleared in a few days or resulted in a progressive and ultimately fatal infection. Using high resolution live imaging, we found that S. flexneri were rapidly engulfed by macrophages and neutrophils; moreover we discovered a scavenger role for neutrophils in eliminating infected dead macrophages and non-immune cell types that failed to control Shigella infection. We observed that intracellular S. flexneri could escape to the cytosol, induce septin caging and be targeted to autophagy in vivo. Depletion of p62 (sequestosome 1 or SQSTM1), an adaptor protein critical for bacterial autophagy in vitro, significantly increased bacterial burden and host susceptibility to infection. These results show the zebrafish larva as a new model for the study of S. flexneri interaction with phagocytes, and the manipulation of autophagy for anti-bacterial therapy in vivo.     

Structure of the yeast vacuolar ATPase

The subunit architecture of the yeast vacuolar ATPase (V-ATPase) was analyzed by single particle transmission electron microscopy and electrospray ionization (ESI) tandem mass spectrometry. A three-dimensional model of the intact V-ATPase was calculated from two-dimensional projections of the complex at a resolution of 25 angstroms. Images of yeast V-ATPase decorated with monoclonal antibodies against subunits A, E, and G position subunit A within the pseudo-hexagonal arrangement in the V1, the N terminus of subunit G in the V1-V0 interface, and the C terminus of subunit E at the top of the V1 domain. ESI tandem mass spectrometry of yeast V1-ATPase showed that subunits E and G are most easily lost in collision-induced dissociation, consistent with a peripheral location of the subunits. An atomic model of the yeast V-ATPase was generated by fitting of the available x-ray crystal structures into the electron microscopy-derived electron density map. The resulting atomic model of the yeast vacuolar ATPase serves as a framework to help understand the role the peripheral stalk subunits are playing in the regulation of the ATP hydrolysis driven proton pumping activity of the vacuolar ATPase.