Low-Intensity Focused Ultrasound Modulates Monkey Visuomotor Behavior

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Identification and characterization of new protein chemoattractants in the frog skin secretome

The vomeronasal organ is a chemosensory organ present in most vertebrates and involved in chemical communication. In the last decade, the deciphering of the signal transduction process of this organ has progressed. However, less is known about the vomeronasal organ ligands and their structure-function relationships. Snakes possess a highly developed vomeronasal system that is used in various behaviors such as mating, predator detection, or prey selection, making this group a suitable model for study of the vomeronasal chemoreception. In this work, we used a proteomics approach to identify and characterize proteins from frog cutaneous mucus proteome involved in prey recognition by snakes of the genus Thamnophis. Herein we report the purification and characterization of two proteins isolated from the frog skin secretome that elicit the vomeronasal organ-mediated predatory behavior of Thamnophis marcianus. These proteins are members of the parvalbumin family, which are calcium-binding proteins generally associated to muscular and nervous tissues. This is the first report that demonstrates parvalbumins are not strictly restricted to intracellular compartments and can also be isolated from exocrine secretions. Purified parvalbumins from frog muscle and mucus revealed identical chemoattractive properties for T. marcianus. Snake bioassay revealed the Ca(2+)/Mg(2+) dependence of the bioactivity of parvalbumins. So parvalbumins appear to be new candidate ligands of the vomeronasal organ.     

Mitochondrial NADH fluorescence is enhanced by complex I binding

Mitochondrial NADH fluorescence has been a useful tool in evaluating mitochondrial energetics both in vitro and in vivo. Mitochondrial NADH fluorescence is enhanced several-fold in the matrix through extended fluorescence lifetimes (EFL). However, the actual binding sites responsible for NADH EFL are unknown. We tested the hypothesis that NADH binding to Complex I is a significant source of mitochondrial NADH fluorescence enhancement. To test this hypothesis, the effect of Complex I binding on NADH fluorescence efficiency was evaluated in purified protein, and in native gels of the entire porcine heart mitochondria proteome. To avoid the oxidation of NADH in these preparations, we conducted the binding experiments under anoxic conditions in a specially designed apparatus. Purified intact Complex I enhanced NADH fluorescence in native gels approximately 10-fold. However, no enhancement was detected in denatured individual Complex I subunit proteins. In the Clear and Ghost native gels of the entire mitochondrial proteome, NADH fluorescence enhancement was localized to regions where NADH oxidation occurred in the presence of oxygen. Inhibitor and mass spectroscopy studies revealed that the fluorescence enhancement was specific to Complex I proteins. No fluorescence enhancement was detected for MDH or other dehydrogenases in this assay system, at physiological mole fractions of the matrix proteins. These data suggest that NADH associated with Complex I significantly contributes to the overall mitochondrial NADH fluorescence signal and provides an explanation for the well established close correlation of mitochondrial NADH fluorescence and the metabolic state.     

Molecular structure and metal-binding properties of the periplasmic CopK protein expressed in Cupriavidus metallidurans CH34 during copper challenge

The copK gene is localized on the pMOL30 plasmid of Cupriavidus metallidurans CH34 within the complex cop cluster of genes, for which 21 genes have been identified. The expression of the corresponding periplasmic CopK protein is strongly upregulated in the presence of copper, leading to a high periplasmic accumulation. The structure and metal-binding properties of CopK were investigated by NMR and mass spectrometry. The protein is dimeric in the apo state with a dissociation constant in the range of 10^- 5 M estimated from analytical ultracentrifugation. Mass spectrometry revealed that CopK has two high-affinity Cu(I)-binding sites per monomer with different Cu(I) affinities. Binding of Cu(II) was observed but appeared to be non-specific. The solution structure of apo-CopK revealed an all-β fold formed of two β-sheets in perpendicular orientation with an unstructured C-terminal tail. The dimer interface is formed by the surface of the C-terminal β-sheet. Binding of the first Cu(I)-ion induces a major structural modification involving dissociation of the dimeric apo-protein. Backbone chemical shifts determined for the 1Cu(I)-bound form confirm the conservation of the N-terminal β-sheet, while the last strand of the C-terminal sheet appears in slow conformational exchange. We hypothesize that the partial disruption of the C-terminal β-sheet is related to dimer dissociation. NH-exchange data acquired on the apo-protein are consistent with a lower thermodynamic stability of the C-terminal sheet. CopK contains seven methionine residues, five of which appear highly conserved. Chemical shift data suggest implication of two or three methionines (Met54, Met38, Met28) in the first Cu(I) site. Addition of a second Cu(I) ion further increases protein plasticity. Comparison of the structural and metal-binding properties of CopK with other periplasmic copper-binding proteins reveals two conserved features within these functionally related proteins: the all-β fold and the methionine-rich Cu(I)-binding site.     

Structure and dynamics of the membrane-embedded domain of LmrAinvestigated by coupling polarized ATR-FTIR spectroscopy and (1)H/(2)H exchange

Bacterial LmrA, an integral membrane protein of Lactococcus lactis, confers multidrug resistance by mediating active extrusion of a wide variety of structurally unrelated compounds. Similar to its eucaryotic homologue P-gp, this protein is a member of the ATP-binding cassette (ABC) superfamily. Different predictive models, based on hydropathy profiles, have been proposed to describe the structure of the ABC transporters in general and of LmrA in particular. We used polarized attenuated total reflection infrared spectroscopy, combined with limited proteolysis, to investigate the secondary structure and the orientation of the transmembrane segments of LmrA. We bring the first experimental evidence that the membrane-embedded domain of LmrA is composed of transmembrane-oriented alpha-helices. Furthermore, a new approach was developed in order to provide information about membrane domain dynamics. Monitoring the infrared linear dichroism spectra in the course of (1)H/(2)H exchange allowed to focus the recording of exchange rates on the membrane-embedded region of the protein only. This approach revealed an unusual structural dynamics, indicating high flexibility in this antibiotic binding and transport region.     

Neutrophil chemotaxis and superoxide production are induced by cross-linking FcgammaRII receptors

Neutrophils express two types of receptor for the Fc region of IgG, FcgammaRII and FcgammaRIIIB. Via these receptors, neutrophils bind IgG complexes that contain more than one IgG molecule. This binding activates functional processes, such as the respiratory burst and chemotaxis. Neutrophils were treated with biotinylated anti-Fc receptor monoclonal antibodies and chemotaxis toward streptavidin, a cross-linking agent, was determined. Cross-linking FcgammaRII and not FcgammaRIIIB induced neutrophil chemotaxis. Superoxide production in response to immobilized anti-Fc receptor antibodies was also examined. Anti-FcgammaRII Fab bound to ELISA plates induced superoxide production, while anti-FcgammaRIIIB Fab did not. Pretreatment of neutrophils with anti-FcgammaRII Fab reduced superoxide generated by immobilized anti-FcgammaRII antibody. The data demonstrate that FcgammaRII and not FcgammaRIIIB are responsible for neutrophil chemotaxis and superoxide production upon Fc receptor activation.     

Purification and characterization of two voltage-dependent anion channel isoforms from plant seeds

Mitochondria were isolated from imbibed seeds of lentil (Lens culinaris) and Phaseolus vulgaris. We copurified two voltage-dependent anion channel from detergent solubilized mitochondria in a single purification step using hydroxyapatite. The two isoforms from P. vulgaris were separated by chromatofocusing chromatography in 4 M urea without any loss of channel activity. Channel activity of each isoform was characterized upon reconstitution into diphytanoyl phosphatidylcholine planar lipid bilayers. Both isoforms form large conductance channels that are slightly anion selective and display cation selective substates.     

Spoligotyping of mycobacterium tuberculosis isolates obtained from patients in the Samara region

The spoligotyping of 138 M. tuberculosis isolates obtained on the territory of the Samara region made it possible to classify them as belonging to 27 groups. The largest cluster including 94 strains (68.1%) belonged to Peking spoligotype. As shown with the use of the commercial system INNO-LiPA, 78 out of 134 strains (58.2%) had mutations in different sites of gene pro B. The analysis of the markers of resistance to rifampicin in Peking spoligotype revealed that 65 (83.9%) out of 90 such cultures had mutations in gene pro B, i.e. potentially these strains had multiple antimicrobial resistance. This work demonstrated the prevalence of Peking spoligotype in a prison antituberculosis colony (81.6%).     

Global analysis of the Ralstonia metallidurans proteome: prelude for the large-scale study of heavy metal response

A proteome map of Ralstonia metallidurans strain CH34 was constructed using two-dimensional (2-D) gel electrophoresis in combination with automated Edman degradation and mass spectrometry (MS). R. metallidurans CH34 is the type-strain of a family of highly related strains characterized by their multiple resistance to millimolar amounts of heavy metals, conferred by two large plasmids. The protein content of this bacterium grown in minimal medium was separated by 2-D gel electrophoresis using various pH gradients. Protein identification was carried out via N-terminal amino acid sequencing, matrix assisted laser desorption/ionisation-time of flight-mass spectrometry (MALDI-TOF-MS) and tandem MS. So far, 224 different proteins were characterized from 352 protein spots. Although the proteome map is still not complete, one could appraise the importance of proteomics for genome analyses through (i). the identification of previously undetected open reading frames, (ii). the identification of proteins not encoded by the already sequenced genome fragments, (iii). the characterization of protein-encoding genes spanning two different contigs, enabling their merging, and (iv). the precise delineation of the N-terminus of several proteins. Finally, this map will prove a useful tool in the identification of proteins differentially expressed in the presence of different heavy metals.