Import of cytochrome c into mitochondria

The import of cytochrome c into mitochondria can be resolved into a number of discrete steps. Here we report on the covalent attachment of heme to apocytochrome c by the enzyme cytochrome c heme lyase in mitochondria from Neurospora crassa. A new method was developed to measure directly the linkage of heme to apocytochrome c. This method is independent of conformational changes in the protein accompanying heme attachment. Tryptic peptides of [35S]cysteine-labelled apocytochrome c, and of enzymatically formed holocytochrome c, were resolved by reverse-phase HPLC. The cysteine-containing peptide to which heme was attached eluted later than the corresponding peptide from apocytochrome c and could be quantified by counting 35S radioactivity as a measure of holocytochrome c formation. Using this procedure, the covalent attachment of heme to apocytochrome c, which is dependent on the enzyme cytochrome c heme lyase, could be measured. Activity required heme (as hemin) and could be reversibly inhibited by the analogue deuterohemin. Holocytochrome c formation was stimulated 5–10-fold by NADH > NADPH > glutathione and was independent of a potential across the inner mitochondrial membrane. NADH was not required for the binding of apocytochrome c to mitochondria and was not involved in the reduction of the cysteine thiols prior to heme attachment. Holocytochrome c formation was also dependent on a cytosolic factor that was necessary for the heme attaching step of cytochrome c import. The factor was a heat-stable, protease-insensitive, low-molecular-mass component of unknown function. Cytochrome c heme lyase appeared to be a soluble protein located in the mitochondrial intermembrane space and was distinct from the previously identified apocytochrome c binding protein having a similar location. A model is presented in which the covalent attachment of heme by cytochrome c heme lyase also plays an essential role in the import pathway of cytochrome c

Estimation of wood volume and height of olive tree plantations using airborne discrete-return LiDAR data

The aim of this study is to analyze methodologies based on airborne LiDAR (light detection and ranging) technology of low pulse density points (0.5m(-2)) for height and volume quantification of olive trees in Viver (Spain). A total of 29 circular plots, each with a radius of 20m, were sampled and their volumes and heights were obtained by dendrometric methods. For these estimations, several statistics derived from LiDAR data were calculated in each plot. Regression models were used to predict volume and height. The results showed good performance for estimating volume (R-2=0.70) and total height (R-2=0.67).The authors appreciate the financial support provided by the Spanish Ministerio de Ciencia e Innovacion (Ministry for Science & Innovation) within the framework of the project AGL2010-15334 and by the Vice-Rectorate for Research of the Universitat Politecnica de Valencia [Grant PAID-06-12-3297; SP20120534].Estornell Cremades, J.; Velázquez Martí, B.; López Cortés, I.; Salazar Hernández, DM.; Fernández-Sarría, A. (2014). Estimation of wood volume and height of olive tree plantations using airborne discrete-return LiDAR data. GIScience and Remote Sensing. 51(1):17-29. https://doi.org/10.1080/15481603.2014.883209S1729511Estornell, J., Ruiz, L. A., Velázquez-Martí, B., & Fernández-Sarría, A. (2011). Estimation of shrub biomass by airborne LiDAR data in small forest stands. Forest Ecology and Management, 262(9), 1697-1703. doi:10.1016/j.foreco.2011.07.026García, M., Riaño, D., Chuvieco, E., & Danson, F. M. (2010). Estimating biomass carbon stocks for a Mediterranean forest in central Spain using LiDAR height and intensity data. Remote Sensing of Environment, 114(4), 816-830. doi:10.1016/j.rse.2009.11.021Hyyppa, J., Kelle, O., Lehikoinen, M., & Inkinen, M. (2001). A segmentation-based method to retrieve stem volume estimates from 3-D tree height models produced by laser scanners. IEEE Transactions on Geoscience and Remote Sensing, 39(5), 969-975. doi:10.1109/36.921414Kim, Y., Yang, Z., Cohen, W. B., Pflugmacher, D., Lauver, C. L., & Vankat, J. L. (2009). Distinguishing between live and dead standing tree biomass on the North Rim of Grand Canyon National Park, USA using small-footprint lidar data. Remote Sensing of Environment, 113(11), 2499-2510. doi:10.1016/j.rse.2009.07.010Moorthy, I., Miller, J. R., Berni, J. A. J., Zarco-Tejada, P., Hu, B., & Chen, J. (2011). Field characterization of olive (Olea europaea L.) tree crown architecture using terrestrial laser scanning data. Agricultural and Forest Meteorology, 151(2), 204-214. doi:10.1016/j.agrformet.2010.10.005Næsset, E. (2004). Accuracy of forest inventory using airborne laser scanning: evaluating the first nordic full-scale operational project. Scandinavian Journal of Forest Research, 19(6), 554-557. doi:10.1080/02827580410019544Popescu, S. C. (2007). Estimating biomass of individual pine trees using airborne lidar. Biomass and Bioenergy, 31(9), 646-655. doi:10.1016/j.biombioe.2007.06.022Popescu, S. C., Wynne, R. H., & Nelson, R. F. (2002). Estimating plot-level tree heights with lidar: local filtering with a canopy-height based variable window size. Computers and Electronics in Agriculture, 37(1-3), 71-95. doi:10.1016/s0168-1699(02)00121-7Velázquez-Martí, B., Estornell, J., López-Cortés, I., & Martí-Gavilá, J. (2012). Calculation of biomass volume of citrus trees from an adapted dendrometry. Biosystems Engineering, 112(4), 285-292. doi:10.1016/j.biosystemseng.2012.04.011Velázquez-Martí, B., Fernández-González, E., Estornell, J., & Ruiz, L. A. (2010). Dendrometric and dasometric analysis of the bushy biomass in Mediterranean forests. Forest Ecology and Management, 259(5), 875-882. doi:10.1016/j.foreco.2009.11.027Velázquez-Martí, B., Fernández-González, E., López-Cortés, I., & Salazar-Hernández, D. M. (2011). Quantification of the residual biomass obtained from pruning of trees in Mediterranean olive groves. Biomass and Bioenergy, 35(7), 3208-3217. doi:10.1016/j.biombioe.2011.04.042Yu, X., Hyyppä, J., Kaartinen, H., & Maltamo, M. (2004). Automatic detection of harvested trees and determination of forest growth using airborne laser scanning. Remote Sensing of Environment, 90(4), 451-462. doi:10.1016/j.rse.2004.02.00

Identified charged hadron production in p+p collisions at sqrt(s)=200 and 62.4 GeV

Transverse momentum distributions and yields for $\pi^{\pm}$, $K^{\pm}$, $p$ and $\bar{p}$ in $p+p$ collisions at $\sqrt{s}$=200 and 62.4 GeV at midrapidity are measured by the PHENIX experiment at the Relativistic Heavy Ion Collider (RHIC). These data provide important baseline spectra for comparisons with identified particle spectra in heavy ion collisions at RHIC. We present the inverse slope parameter $T_{\rm inv}$, mean transverse momentum $$ and yield per unit rapidity $dN/dy$ at each energy, and compare them to other measurements at different $\sqrt{s}$ in $p+p$ and $p+\bar{p}$ collisions. We also present the scaling properties such as $m_T$ scaling, $x_T$ scaling on the $p_T$ spectra between different energies. To discuss the mechanism of the particle production in $p+p$ collisions, the measured spectra are compared to next-to-leading-order or next-to-leading-logarithmic perturbative quantum chromodynamics calculations.Comment: 431 authors from 62 institutions, 32 pages, 23 figures, and 18 tables. Submitted to Physical Review C. Plain text data tables for the points plotted in figures for this and previous PHENIX publications are (or will be) publicly available at http://www.phenix.bnl.gov/papers.htm

Measurement of jet-medium interactions via direct photon-hadron correlations in Au++Au and dd++Au collisions at sNN=200\sqrt{s_{_{NN}}}=200 GeV

We present direct photon-hadron correlations in 200 GeV/A Au$+$Au, $d$$+$Au and $p$$+$$p$ collisions, for direct photon $p_T$ from 5--12 GeV/$c$, collected by the PHENIX Collaboration in the years from 2006 to 2011. We observe no significant modification of jet fragmentation in $d$$+$Au collisions, indicating that cold nuclear matter effects are small or absent. Hadrons carrying a large fraction of the quark's momentum are suppressed in Au$+$Au compared to $p$$+$$p$ and $d$$+$Au. As the momentum fraction decreases, the yield of hadrons in Au$+$Au increases to an excess over the yield in $p$$+$$p$ collisions. The excess is at large angles and at low hadron $p_T$ and is most pronounced for hadrons associated with lower momentum direct photons. Comparison to theoretical calculations suggests that the hadron excess arises from medium response to energy deposited by jets.Comment: 578 authors from 80 institutions, 11 pages, 7 figures, data from 2007, 2008, 2010, and 2011. v2 is version accepted for publication in Physical Review C. Plain text data tables for the points plotted in figures for this and previous PHENIX publications are (or will be) publicly available at http://www.phenix.bnl.gov/papers.htm

LPS induces IL-10 production by human alveolar macrophages via MAPKinases- and Sp1-dependent mechanisms

<p>Abstract</p> <p>Background</p> <p>IL-10 is a cytokine mainly produced by macrophages that plays key roles in tolerance to inhaled antigens and in lung homeostasis. Its regulation in alveolar macrophages (HAM), the resident lung phagocytes, remains however unknown.</p> <p>Methods</p> <p>The present study investigated the role of intracellular signalling and transcription factors controlling the production of IL-10 in LPS-activated HAM from normal nonsmoking volunteers.</p> <p>Results</p> <p>LPS (1–1000 pg/ml) induced <it>in vitro </it>IL-10 production by HAM, both at mRNA and protein levels. LPS also activated the phosphorylation of ERK, p38 and JNK MAPkinases (immunoblots) and Sp-1 nuclear activity (EMSA). Selective inhibitors of MAPKinases (respectively PD98059, SB203580 and SP600125) and of Sp-1 signaling (mithramycin) decreased IL-10 expression in HAM. In addition, whilst not affecting IL-10 mRNA degradation, the three MAPKinase inhibitors completely abolished Sp-1 activation by LPS in HAM.</p> <p>Conclusion</p> <p>These results demonstrate for the first time that expression of IL-10 in lung macrophages stimulated by LPS depends on the concomitant activation of ERK, p38 and JNK MAPKinases, which control downstream signalling to Sp-1 transcription factor. This study further points to Sp-1 as a key signalling pathway for IL-10 expression in the lung.</p

Loss of Deacetylation Activity of Hdac6 Affects Emotional Behavior in Mice

Acetylation is mediated by acetyltransferases and deacetylases, and occurs not only on histones but also on diverse proteins. Although histone acetylation in chromatin structure and transcription has been well studied, the biological roles of non-histone acetylation remain elusive. Histone deacetylase 6 (Hdac6), a member of the histone deacetylase (HDAC) family, is a unique deacetylase that localizes to cytoplasm and functions in many cellular events by deacetylating non-histone proteins including α-tubulin, Hsp90, and cortactin. Since robust expression of Hdac6 is observed in brain, it would be expected that Hdac6-mediated reversible acetylation plays essential roles in CNS. Here we demonstrate the crucial roles of Hdac6 deacetylase activity in the expression of emotional behavior in mice. We found that Hdac6-deficient mice exhibit hyperactivity, less anxiety, and antidepressant-like behavior in behavioral tests. Moreover, administration of Hdac6-specific inhibitor replicated antidepressant-like behavior in mice. In good agreement with behavioral phenotypes of Hdac6-deficient mice, Hdac6 dominantly localizes to the dorsal and median raphe nuclei, which are involved in emotional behaviors. These findings suggest that HDAC6-mediated reversible acetylation might contribute to maintain proper neuronal activity in serotonergic neurons, and also provide a new therapeutic target for depression

Plasmacytoid Dendritic Cells Capture and Cross-Present Viral Antigens from Influenza-Virus Exposed Cells

Among the different subsets of dendritic cells (DC), plasmacytoid dendritic cells (PDC) play a unique role in secreting large amounts of type I interferons upon viral stimulation, but their efficiency as antigen-presenting cells has not been completely characterized. We show here, by flow cytometry, with human primary blood PDC and with a PDC cell line, that PDC display poor endocytic capacity for soluble or cellular antigens when compared to monocyte-derived myeloid DC. However, immature PDC efficiently take up cellular material from live influenza-exposed cells, subsequently mature and cross-present viral antigens very efficiently to specific CD8+ T cells. Therefore, during viral infection PDC not only secrete immunomodulatory cytokines, but also recognize infected cells and function as antigen cross-presenting cells to trigger the anti-viral immune response

Novel mutations in TLR genes cause hyporesponsiveness to Mycobacterium avium subsp. paratuberculosis infection

<p>Abstract</p> <p>Background</p> <p>Toll like receptors (TLR) play the central role in the recognition of pathogen associated molecular patterns (PAMPs). Mutations in the TLR1, TLR2 and TLR4 genes may change the ability to recognize PAMPs and cause altered responsiveness to the bacterial pathogens.</p> <p>Results</p> <p>The study presents association between TLR gene mutations and increased susceptibility to <it>Mycobacterium avium </it>subsp. <it>paratuberculosis </it>(MAP) infection. Novel mutations in TLR genes (TLR1- Ser150Gly and Val220Met; TLR2 – Phe670Leu) were statistically correlated with the hindrance in recognition of MAP legends. This correlation was confirmed subsequently by measuring the expression levels of cytokines (IL-4, IL-8, IL-10, IL-12 and IFN-γ) in the mutant and wild type moDCs (mocyte derived dendritic cells) after challenge with MAP cell lysate or LPS. Further <it>in silico </it>analysis of the TLR1 and TLR4 ectodomains (ECD) revealed the polymorphic nature of the central ECD and irregularities in the central LRR (leucine rich repeat) motifs.</p> <p>Conclusion</p> <p>The most critical positions that may alter the pathogen recognition ability of TLR were: the 9^thamino acid position in LRR motif (TLR1–LRR10) and 4^thresidue downstream to LRR domain (exta-LRR region of TLR4). The study describes novel mutations in the TLRs and presents their association with the MAP infection.</p

Eye bank issues: II. Preservation techniques: warm versus cold storage

Most of the tissue used for penetrating keratoplasty is issued through eye banks that store the corneoscleral button either in hypothermic storage at 2–6°C or in organ culture at 31–37°C

Regulation of Mycobacterium tuberculosis-Dependent HIV-1 Transcription Reveals a New Role for NFAT5 in the Toll-Like Receptor Pathway

Tuberculosis (TB) disease in HIV co-infected patients contributes to increased mortality by activating innate and adaptive immune signaling cascades that stimulate HIV-1 replication, leading to an increase in viral load. Here, we demonstrate that silencing of the expression of the transcription factor nuclear factor of activated T cells 5 (NFAT5) by RNA interference (RNAi) inhibits Mycobacterium tuberculosis (MTb)-stimulated HIV-1 replication in co-infected macrophages. We show that NFAT5 gene and protein expression are strongly induced by MTb, which is a Toll-like receptor (TLR) ligand, and that an intact NFAT5 binding site in the viral promoter of R5-tropic HIV-1 subtype B and subtype C molecular clones is required for efficent induction of HIV-1 replication by MTb. Furthermore, silencing by RNAi of key components of the TLR pathway in human monocytes, including the downstream signaling molecules MyD88, IRAK1, and TRAF6, significantly inhibits MTb-induced NFAT5 gene expression. Thus, the innate immune response to MTb infection induces NFAT5 gene and protein expression, and NFAT5 plays a crucial role in MTb regulation of HIV-1 replication via a direct interaction with the viral promoter. These findings also demonstrate a general role for NFAT5 in TLR- and MTb-mediated control of gene expression