A thermodynamic and structural study of myelin basic protein in lipid membrane models

Myelin basic protein (MBP) is a major protein of the myelin membrane in the central nervous system. It is believed to play a relevant role in the structure and function of the myelin sheath and is a candidate autoantigen in demyelinating processes such as multiple sclerosis. MBP has many features typical of soluble proteins but is capable of strongly interacting with lipids, probably via a conformation change. Its structure in the lipid membrane as well as the details of its interaction with the lipid membrane are still to be resolved. In this article we study the interaction of MBP with Langmuir films of anionic and neutral phospholipids, used as experimental models of the lipid membrane. By analyzing the equilibrium surface pressure/area isotherms of these films, we measured the protein partition coefficient between the aqueous solution and the lipid membrane, the mixing ratio between protein and lipid, and the area of the protein molecules inserted in the lipid film. The penetration depth of MBP in the lipid monolayer was evaluated by x-ray reflectivity measurements. The mixing ratio and the MBP molecular area decrease as the surface pressure increases, and at high surface pressure the protein is preferentially located at the lipid/water interface for both anionic and neutral lipids. The morphology of MBP adsorbed on lipid films was studied by atomic force microscopy. MBP forms bean-like structures and induces a lateral compaction of the lipid surface. Scattered MBP particles have also been observed. These particles, which are 2.35-nm high, 4.7-nm wide, and 13.3-nm long, could be formed by protein-lipid complexes. On the basis of their size, they could also be either single MBP molecules or pairs of c-shaped interpenetrating molecules.     

The leucokinin-like peptide receptor from the cattle fever tick,Rhipicephalus microplus,is localized in the midgut periphery and receptor silencing with validated double-stranded RNAs causes a reproductive fitness cost

The cattle fever tick, Rhipicephalus microplus (Canestrini) (Acari: Ixodidae), is a one-host tick that infests primarily cattle in tropical and sub-tropical regions of the world. This species transmits deadly cattle pathogens, especially Babesia spp., for which a recombinant vaccine is not available. Therefore, disease control depends on tick vector control. Although R. microplus was eradicated in the USA, tick populations in Mexico and South America have acquired resistance to many of the applied acaricides. Recent acaricide-resistant tick reintroductions detected in the U.S. underscore the need for novel tick control methods. The octopamine and tyramine/octopamine receptors, both G protein-coupled receptors (GPCR), are believed to be the main molecular targets of the acaricide amitraz. This provides the proof of principle that investigating tick GPCRs, especially those that are invertebrate-specific, may be a feasible strategy for discovering novel targets and subsequently new anti-tick compounds. The R. microplus leucokinin-like peptide receptor (LKR), also known as the myokinin- or kinin receptor, is such a GPCR. While the receptor was previously characterized in vitro, the function of the leucokinin signaling system in ticks remains unknown. In this work, the LKR was immunolocalized to the periphery of the female midgut and silenced through RNA interference (RNAi) in females. To optimize RNAi experiments, a dual-luciferase system was developed to determine the silencing efficiency of LKR-double stranded RNA (dsRNA) constructs prior to testing those in ticks placed on cattle. This assay identified two effective dsRNAs. Silencing of the LKR with these two validated dsRNA constructs was verified by quantitative real time PCR (qRT-PCR) of female tick dissected tissues. Silencing was significant in midguts and carcasses. Silencing caused decreases in weights of egg masses and in the percentages of eggs hatched per egg mass, as well as delays in time to oviposition and egg hatching. A role of the kinin receptor in tick reproduction is apparent.     

Cyclic AMP-dependent protein kinase controls energy interconversion during the catalytic cycle of the yeast copper-ATPase

The pathogenesis of human Menkes and Wilson diseases depends on alterations in copper transport. Some reports suggest that intracellular traffic of copper might be regulated by kinase-mediated phosphorylation. However, there is no evidence showing the influence of kinase-related processes in coupled ATP hydrolysis/copper transport cycles. Here, we show that cyclic AMP-dependent protein kinase (PKA) regulates Ccc2p, the yeast Cu(I)-ATPase, with PKA-mediated phosphorylation of a conserved serine (Ser258) being crucial for catalysis. Long-range intramolecular communication between Ser258 and Asp627 (at the catalytic site) modulates the key pumping event: the conversion of the high-energy to the low-energy phosphorylated intermediate associated with copper release.     

Role of xanthine oxidase activation and reduced glutathione depletion in rhinovirus induction of inflammation in respiratory epithelial cells

Rhinoviruses are the major cause of the common cold and acute exacerbations of asthma and chronic obstructive pulmonary disease. We previously reported rapid rhinovirus induction of intracellular superoxide anion, resulting in NF-kappaB activation and pro-inflammatory molecule production. The mechanisms of rhinovirus superoxide induction are poorly understood. Here we found that the proteolytic activation of the xanthine dehydrogenase/xanthine oxidase (XD/XO) system was required because pretreatment with serine protease inhibitors abolished rhinovirus-induced superoxide generation in primary bronchial and A549 respiratory epithelial cells. These findings were confirmed by Western blotting analysis and by silencing experiments. Rhinovirus infection induced intracellular depletion of reduced glutathione (GSH) that was abolished by pretreatment with either XO inhibitor oxypurinol or serine protease inhibitors. Increasing intracellular GSH with exogenous H2S or GSH prevented both rhinovirus-mediated intracellular GSH depletion and rhinovirus-induced superoxide production. We propose that rhinovirus infection proteolytically activates XO initiating a pro-inflammatory vicious circle driven by virus-induced depletion of intracellular reducing power. Inhibition of these pathways has therapeutic potential.     

Ca binding to sarcoplasmic reticulum ATPase phosphorylated by Pi reveals four thapsigargin-sensitive Ca sites in the presence of ADP

Sarcoplasmic reticulum (SR) Ca²⁺-ATPase was phosphorylated by P_i at pH 8.0 in the presence of dimethyl sulfoxide (Me₂SO). Under these conditions, it was possible to measure transient ⁴⁵Ca²⁺ binding to the phosphoenzyme. Binding reached 1.2 Ca²⁺ per phosphoenzyme (E-PCa_x) within 10 min in 30% Me₂SO, 20 mM MgCl₂ and 0.1 mM P_i and the phosphoenzyme only decreased by 23% during this period. This Ca²⁺ binding was abolished by thapsigargin, showing that it is associated with functional sites of the Ca²⁺-ATPase. At 40% Me₂SO, simultaneous addition of Ca²⁺ and ADP increased Ca²⁺ binding up to almost four Ca²⁺ per phosphoenzyme (ADPE-PCa_y), revealing a species bearing simultaneously four Ca²⁺ sites. Both E-PCa_x and ADPE-PCa_y were further identified as distinct species by (2′,3′-O-2(2,4,6-trinitrophenyl)adenosine 5′-triphosphate) fluorescence, which revealed long-range modifications in the Ca²⁺-transport sites induced by ADP binding to E-P. In addition, E-PCa_x was shown to be a functional intermediate of the cycle leading to ATP synthesis provided that Me₂SO was diluted. These findings indicate that more than two functional Ca²⁺-sites exist on the functional Ca²⁺-ATPase unit, and that the additional sites become accessible upon ADP addition. This is compatible with a four-site model of the SR Ca²⁺-ATPase allowing simultaneous binding of Ca²⁺ at lumenal and cytosolic sites. The stoichiometries for Ca²⁺ binding found here could either be interpreted as binding of four Ca²⁺ on a Ca²⁺-ATPase monomer considered as the functional unit or as binding of two Ca²⁺ per monomer of a functional dimer.     

Role of IFN-γ +874 T/A single nucleotide polymorphism in the tuberculosis outcome among Brazilians subjects

Several genetic cytokine gene variants have been associated with host susceptibility to infectious diseases, including tuberculosis. Based upon the importance of IFN-γ in protective immunity against Mycobacterium tuberculosis, and the functional role of the IFN-γ + 874T/A single nucleotide polymorphism in IFN-γ production, we genotyped 93 Brazilian tuberculosis patients and 266 asymptomatic health care workers, including 150 individuals with a positive tuberculin skin test, and analyzed the possible association of the +874A low IFN-γ producer allele with tuberculosis occurrence. Using multivariable logistic regression models, genotype and allele frequencies of the mutant + 874A (low IFN-γ producer) allele were significantly associated with tuberculosis disease. Heterozygous carriers had a 25% increased chance, while individuals presenting the A/A homozygous genotype had an over two-fold risk of having active tuberculosis (95% CI, 1.16–5.91, P = 0.03). Despite the mixed ethnicity observed in Brazilian populations, the present data agree with observations reported in other populations and thus demonstrate that the functional +874T/A IFN-γ gene polymorphism is associated with tuberculosis in different populations.     

Exposure to waterborne copper and high temperature induces the formation of reactive oxygen species and causes mortality in the Amazonian fish <Emphasis Type="Italic">Hoplosternum littorale</Emphasis>

Hoplosternum littorale is an Amazon fish that lives in urban areas surrounded by polluted igarapés, where elevated copper concentrations eventually occur. The central goal of this study was to evaluate the associated effects of high temperature and copper contamination on survival time and biochemical responses of the Amazonian fish species H. littorale. We exposed fish to two nominal dissolved copper concentrations (50 and 500 µg l^?1) and combined temperatures of 28 and 34°C. Our findings showed that the combination of these variables affects the survival time of this species. The activity of the biotransformation enzymes ethoxyresorufin-O-deethylase and glutathione-S-transferase showed no alterations in fish within all treatments. The increase of reactive oxygen species and the decrease in potential total antioxidant capacity promoted the imbalance in the antioxidant system. An induction in superoxide dismutase activity occurred in fish exposed to copper concentrations of 50 and 500 µg l^?1 at both temperatures, suggesting liver impairments. Thus, we suggest that H. littorale is sensitive to copper, and this sensitivity is increased further with exposure to high temperatures, particularly in the survival time and reactive oxygen species formation of this fish species.     

An atomic force microscopy investigation of protein crystal surface topography

Tapping mode atomic force microscopy was employed to study the surface structure of different protein crystals in a liquid environment. The (101) face of hen egg-white lysozyme crystals and the (111) face of horse spleen ferritin crystals were studied. On the (101) face of lysozyme crystals we observed islands delimitated by micro-steps and elongated in the [010] direction. The elongation direction coincides with the preferential growth direction predicted by a growth model reported in the literature. The islands observed on the ferritin (111) face are also delimitated by micro-steps but have circular symmetry. Sectioning of the images allowed us to measure the step heights. The surface free energy was estimated from the growth step morphology. Molecular resolution was achieved for ferritin crystals, showing a hexagonal surface packing, as expected for the molecular lattice of a (111) face in a fcc crystal.