Osteopenia due to enhanced cathepsin K release by BK channel ablation in osteoclasts

Background

The process of bone resorption by osteoclasts is regulated by Cathepsin K, the lysosomal collagenase responsible for the degradation of the organic bone matrix during bone remodeling. Recently, Cathepsin K was regarded as a potential target for therapeutic intervention of osteoporosis. However, mechanisms leading to osteopenia, which is much more common in young female population and often appears to be the clinical pre-stage of idiopathic osteoporosis, still remain to be elucidated, and molecular targets need to be identified.

Methodology/Principal Findings

We found, that in juvenile bone the large conductance, voltage and Ca²⁺-activated (BK) K⁺ channel, which links membrane depolarization and local increases in cytosolic calcium to hyperpolarizing K⁺ outward currents, is exclusively expressed in osteoclasts. In juvenile BK-deficient (BK^−/−) female mice, plasma Cathepsin K levels were elevated two-fold when compared to wild-type littermates. This increase was linked to an osteopenic phenotype with reduced bone mineral density in long bones and enhanced porosity of trabecular meshwork in BK^−/− vertebrae as demonstrated by high-resolution flat-panel volume computed tomography and micro-CT. However, plasma levels of sRANKL, osteoprotegerin, estrogene, Ca²⁺ and triiodthyronine as well as osteoclastogenesis were not altered in BK^−/− females.

Conclusion/Significance

Our findings suggest that the BK channel controls resorptive osteoclast activity by regulating Cathepsin K release. Targeted deletion of BK channel in mice resulted in an osteoclast-autonomous osteopenia, becoming apparent in juvenile females. Thus, the BK^−/− mouse-line represents a new model for juvenile osteopenia, and revealed the BK channel as putative new target for therapeutic controlling of osteoclast activity.     

Regulation of inflammatory chemokine receptors on blood T cells associated to the circulating versus liver chemokines in dengue fever

Little is known about the role of chemokines/chemokines receptors on T cells in natural DENV infection. Patients from DENV-2 and -3- outbreaks were studied prospectively during the acute or convalescent phases. Expression of chemokine receptor and activation markers on lymphocyte subpopulations were determined by flow cytometry analysis, plasma chemokine ligands concentrations were measured by ELISA and quantification of CCL5/RANTES(+) cells in liver tissues from fatal dengue cases was performed by immunochemistry. In the acute DENV-infection, T-helper/T-cytotoxic type-1 cell (Th1/Tc1)-related CCR5 is significantly higher expressed on both CD4 and CD8 T cells. The Th1-related CXCR3 is up-regulated among CD4 T cells and Tc2-related CCR4 is up-regulated among CD8 T cells. In the convalescent phase, all chemokine receptor or chemokine ligand expression tends to reestablish control healthy levels. Increased CCL2/MCP-1 and CCL4/MIP-1β but decreased CCL5/RANTES levels were observed in DENV-patients during acute infection. Moreover, we showed an increased CD107a expression on CCR5 or CXCR3-expressing T cells and higher expression of CD29, CD44(HIGH) and CD127(LOW) markers on CCR4-expressing CD8 T cells in DENV-patients when compared to controls. Finally, liver from dengue fatal patients showed increased number of cells expressing CCL5/RANTES in three out of four cases compared to three death from a non-dengue patient. In conclusion, both Th1-related CCR5 and CXCR3 among CD4 T cells have a potential ability to exert cytotoxicity function. Moreover, Tc1-related CCR5 and Tc2-related CCR4 among CD8 T cells have a potential ability to exert effector function and migration based on cell markers evaluated. The CCR5 expression would be promoting an enhanced T cell recruitment into liver, a hypothesis that is corroborated by the CCL5/RANTES increase detected in hepatic tissue from dengue fatal cases. The balance between protective and pathogenic immune response mediated by chemokines during dengue fever will be discussed.     

Selective antibacterial activity of the cationic peptide PaDBS1R6 against Gram-negative bacteria

Infections caused by Gram-negative bacteria, Escherichia coli and Pseudomonas aeruginosa foremost among them, constitute a major worldwide health problem. Bioinformatics methodologies are being used to rationally design new antimicrobial peptides, a potential alternative for treating these infections. One of the algorithms used to develop antimicrobial peptides is the Joker, which was used to design the peptide PaDBS1R6. This study evaluates the antibacterial activities of PaDBS1R6 in vitro and in vivo, characterizes the peptide interaction to target membranes, and investigates the PaDBS1R6 structure in contact with mimetic vesicles. Moreover, we demonstrate that PaDBS1R6 exhibits selective antimicrobial activity against Gram-negative bacteria. In the presence of negatively charged and zwitterionic lipids the structural arrangement of PaDBS1R6 transits from random coil to α-helix, as characterized by circular dichroism. The tertiary structure of PaDBS1R6 was determined by NMR in zwitterionic dodecylphosphocholine (DPC) micelles. In conclusion, PaDBS1R6 is a candidate for the treatment of nosocomial infections caused by Gram-negative bacteria, as template for producing other antimicrobial agents.     

Morphological and Chemical Differentiation between Tunisian Populations of Pinus halepensis,Pinus brutia,and Pinus pinaster

The lipid fraction of seeds from different pine species and populations was studied regarding total lipid content, fatty acid profile and vitamin E composition. The investigated seeds contained a high percentage of lipid (13.6 to 31.5 %). Lipid fractions were found to be rich in vitamin E, which varied significantly among species and populations. P. halepensis (Ph−Hn) showed the highest content of vitamin E (256.3 mg/kg of seeds) and the uppermost content of α-tocopherol (44 mg/kg). However, P. halepensis (Ph−Kas) was the richest in γ-tocopherol (204.9 mg/kg). Lipid fractions had a low content of δ-tocopherol (1.2 to 3.6 mg/kg. The highest content of γ-tocotrienol (∼18 %) was determined for P. halepensis (Ph−Dc and Ph−Hn). Thirteen fatty acids were identified by GC-FID with significant variation between the investigated species. The linoleic acid was the major fatty acid followed by oleic acid and palmitic acid. The chemical differentiation among species for the composition of fatty acids and vitamin E was confirmed by PCA. Significant correlations were observed between the content of vitamin E and fatty acids and ecological parameters of P. halepensis populations.     

Generation of CTL recognizing an HLA-A*0201-restricted epitope shared by MAGE-A1, -A2, -A3, -A4, -A6, -A10, and -A12 tumor antigens: implication in a broad-spectrum tumor immunotherapy

MAGE-A1, -A2, -A3, -A4, -A6, -A10, and -A12 are expressed in a significant proportion of primary and metastatic tumors of various histological types and are targets of tumor Ag-specific CTL. Individual MAGE-A expression varies from one tumor type to the other but, overall, the large majority of tumors expresses at least one MAGE-A Ag. Therefore, targeting epitopes shared by all MAGE-A Ags would be of interest in immunotherapy against a broad spectrum of cancers. In the present study, we describe a heteroclitic MAGE-A peptide (p248V9) that induces CTL in vivo in HLA-A*0201 transgenic HHD mice and in vitro in healthy donors. These CTL are able to recognize two low HLA-A*0201 affinity peptides differing at their C-terminal position and derived from MAGE-A2, -A3, -A4, -A6, -A10, and -A12 (p248G9) and MAGE-A1 (p248D9). Interestingly, p248V9-specific CTL respond to endogenous MAGE-A1, -A2, -A3, -A4, -A6, -A10, and -A12 in an HLA-A*0201-restricted manner and recognize human HLA-A*0201(+)MAGE-A(+) tumor cells of various histological origin. Therefore, this heteroclitic peptide may be considered as a potent candidate for a broad-spectrum tumor vaccination.     

Functional consequences of mutating conserved SF2 helicase motifs in the Type III restriction endonuclease EcoP15I translocase domain

For efficient DNA hydrolysis, Type III restriction endonuclease EcoP15I interacts with two inversely oriented recognition sites in an ATP-dependent process. EcoP15I consists of two methylation (Mod) subunits and a single restriction (Res) subunit yielding a multifunctional enzyme complex able to methylate or to hydrolyse DNA. Comprehensive sequence alignments, limited proteolysis and mass spectroscopy suggested that the Res subunit is a fusion of a motor or translocase (Tr) domain of superfamily II helicases and an endonuclease domain with a catalytic PD…EXK motif. In the Tr domain, seven predicted helicase motifs (I, Ia, II–VI), a recently discovered Q-tip motif and three additional regions (IIIa, IVa, Va) conserved among Type III restriction enzymes have been identified that are predicted to be involved in DNA binding and ATP hydrolysis. Because DNA unwinding activity for EcoP15I (as for bona fide helicases) has never been found and EcoP15I ATPase rates are only low, the functional importance of the helicase motifs and regions was questionable and has never been probed systematically. Therefore, we mutated all helicase motifs and conserved regions predicted in Type III restriction enzyme EcoP15I and examined the functional consequences on EcoP15I enzyme activity and the structural integrity of the variants by CD spectroscopy. The resulting eleven enzyme variants all, except variant IVa, are properly folded showing the same secondary structure distribution as the wild-type enzyme. Classical helicase motifs I–VI are important for ATP and DNA cleavage by EcoP15I and mutations therein led to complete loss of ATPase and cleavage activity. Among the catalytically inactive enzyme variants three preserved the ability to bind ATP. In contrast, newly assigned motifs Q-tip, Ia and Va are not essential for EcoP15I activity and the corresponding enzyme variants were still catalytically active. DNA binding was only marginally reduced (2–7 fold) in all enzyme variants tested.     

Cryptococcus festucosus sp. nov. a new hymenomycetous yeast in the Holtermannia clade

Five yeast strains belonging to the genus Cryptococcus Vuillemin were isolated from steppe plants and turf collected in the Prioksko-terrasny biosphere reserve (Moscow region, Russia). Sequence analyses of the D1/D2 domains of the 26S rDNA and of the internal transcribed spacer region revealed that these yeast strains and strain CBS 8016 have almost identical sequences and belong to the Holtermannia clade of the Tremellomycetidae (Basidiomycota, Hymenomycetes). A novel species named Cryptococcus festucosus (type strain VKM Y-2930) is proposed to accommodate these strains. Physiological characteristics and mycocin sensitivity patterns distinguishing Cryptococcus festucosus from the other species of this clade are presented.     

Effect of Cry1F maize on the behavior of susceptible and resistant Spodoptera frugiperda and Ostrinia nubilalis

Understanding the behavior of pests targeted with Bacillus thuringiensis Berliner (Bt) crops is important to define resistance management strategies. Particularly the study of larval movement between plants is important to determine the feasibility of refuge configurations. Exposure to Bt maize, Zea mays L. (Poaceae), has been suggested to increase larval movement in lepidopteran species but few studies have examined the potential for resistance to interact with behavioral responses to Bt toxins. Choice and no‐choice experiments were conducted with Spodoptera frugiperda (JE Smith) (Lepidoptera: Noctuidae) and Ostrinia nubilalis (Hübner) (Lepidoptera: Crambidae) to determine whether Cry1F resistance influences neonate movement. Leaf discs of Cry1F maize and the corresponding isoline were used to characterize behavioral responses. In both experiments, the location (on or off of plant tissues) and mortality of susceptible and Cry1F resistant neonates was recorded for 5 days, but the analysis of larvae location was performed until 7 h. Our results indicated no strong difference between resistant and susceptible phenotypes in S. frugiperda and O. nubilalis, although a small percentage of susceptible neonates in both species abandoned maize tissue expressing Cry1F. However, significant behavioral differences were observed between species. Ostrinia nubilalis exhibited increased movement between leaf discs, whereas S. frugiperda selected plant tissue within the first 30 min and remained on the chosen plant regardless of the presence of Cry1F. Spodoptera frugiperda reduced larval movement may have implications to refuge configuration. This study represents the first step toward understanding the effects of Cry1F resistance on Lepidoptera larval behavior. Information regarding behavioral differences between species could aid in developing better and more flexible resistance management strategies.     

Synergistic effects of habitat configuration and land-use intensity shape the structure of bird assemblages in human-modified landscapes across three major neotropical biomes

Maximizing biodiversity persistence in heterogeneous human-modified landscapes is hindered by the complex interactions between habitat quality and configuration of native and non-native habitats. Here we examined these complex interactions considering avian diversity across 26 sampling sites, each of which comprised of three sampling points located across a gradient of disturbance: core native habitat fragment, fragment edge, and non-native adjacent matrix. The 78 sampling points were further nested within three neotropical biomes—Amazonia, Cerrado and Pantanal—in central-western Brazil. Matrix type consisted of cattle pastures in the Amazon and teak plantations in the Pantanal and Cerrado. We considered the interactive effects of (1) disturbance-context: fragment core, edge and adjacent matrix, (2) matrix type: tree plantation or cattle pastures, both subject to varying land-use intensity, and (3) native habitat configuration (fragment size, shape and isolation) on bird species richness, abundance and composition. Based on point-count surveys, we recorded 210 bird species. Bird species richness and abundance declined across the disturbance gradient, while genus composition only differed within the adjacent matrix, particularly cattle-pastures. The effect of native habitat area was positive but only detected at fragment edges. Overall bird diversity increased at sites characterized by higher availability of either relict trees within pasture landscapes or old-growth trees within teak plantation landscapes. The core of native fragments played a primary role in ensuring the persistence of bird diversity, regardless of fragment size. In contrast to pastures, tree plantations likely harbour a higher proportion of forest-dependent species while bird diversity can be further enhanced by reduced management intensity in both matrix types. Strategies to maximize avian persistence should not only include retaining native habitats, but also maximizing the size of core native habitats. Likewise, more structurally complex matrix types should be encouraged while maintaining low levels of land-use intensity.