Lack of mitochondrial nitric oxide production in the mouse brain

Based on our initial finding that the nitric oxide (NO) sensitive fluorochrome diaminofluorescein (DAF) was localized to mitochondria in cultured primary neurons, we investigated whether brain mitochondria produce NO through a mitochondrial NO synthase (mtNOS) enzyme. Isolated brain mitochondria were loaded with DAF and subjected to flow cytometry analysis. Neither the application of NOS inhibitors nor the genetic disruption of either NOS gene diminished the DAF-fluorescence. However, peroxynitrite scavengers reduced the mitochondrial DAF fluorescence, indicating that the DAF signal is not specific to NO. Chemiluminescence detection in the head space gas and a Clark-type NO-sensitive electrode in the solution failed to detect NO release in brain mitochondria. NOS activity in mitochondria was only 1% of the whole brain NOS activity level, which may be attributed to extramitochondrial contamination. Extensive immunoblotting and immunoprecipitation experiments failed to show the presence of endothelial, neuronal, or inducible NOS in mouse brain mitochondria using a variety of primary antibodies. Arginine, calmodulin or 2,5-ADP affinity purification protocols successfully concentrated eNOS and nNOS from full brain tissue but failed to show any signal in mitochondria. We conclude that mouse brain mitochondria do not contain NOS isoforms, nor do they produce NO through a NOS-dependent mechanism.     

Ramet demography of a nurse bromeliad in Brazilian restingas

Restingas are sandy coastal plains that stand between the sea and the Brazilian Atlantic forest mountains. The predominant restinga vegetation type in northern Rio de Janeiro, Brazil, is characterized by the formation of islands that begins with colonization by some pioneer herbs and/or woody plants. Pioneer plants are stress-resistant and nurse many other less-resistant plant species. Determining the spatiotemporal variation in the dynamics of nurse plants is essential to understand the ecological functioning of restingas as a whole. The goal of this study was to analyze the spatiotemporal variation in population dynamics of the nurse bromeliad Aechmea nudicaulis. We monitored A. nudicaulis ramets in different habitats, microhabitats, and years. We analyzed the spatiotemporal variation in demographic traits and in population growth rate. Results showed young ramet traits were more variable at the microhabitat level, and when variable, vegetative ramet traits varied at all spatiotemporal scales. Overall, λ values indicated that A. nudicaulis basically remained spatiotemporally stable as most of the λ values did not significantly differ from unity. Hence, the stability of A. nudicaulis in different microhabitats and habitats in the restinga may create several settlement opportunities for many other less-resistant species.     

Molecular phylogenetics of Limonium and related genera (Plumbaginaceae): biogeographical and systematic implications

Phylogenetic relationships within Limonium (Plumbaginaceae) are evaluated using sequence data from three plastid regions (rbcL, the trnL intron, and the trnL-trnF intergenic spacer). Sixty-six species representing the major genera of Staticoideae, including representatives of all sections and genera formerly included in Limonium, have been analyzed using four species of Plumbaginoideae as an outgroup. Analyses of each separate and combined data set yield similar results. Afrolimon is embedded in Limonium and related to L. vulgare, the type of Limonium. Limonium is split into two major clades corresponding to subgenera, but otherwise the current infrageneric classification proved to be artificial. Some groups restricted to particular areas can be recognized, and their synapomorphies are discussed. The presence of an isolated taxon in the Canary Islands is used as a calibration point for age estimates of the major events in the genus, including migrations to the Southern Hemisphere, the Canary Islands, and Asia. The rapid radiation of Limonium in the Mediterranean basin appears to coincide with the desiccation of the Mediterranean Sea in the Messinian (late Miocene).     

A role for human Sp alpha as a pattern recognition receptor

Human Sp alpha is a soluble protein belonging to group B of the scavenger receptor cysteine-rich (SRCR) superfamily for which little functional information is available. It is expressed by macrophages present in lymphoid tissues (spleen, lymph node, thymus, and bone marrow), and it binds to myelomonocytic and lymphoid cells, which suggests that it may play an important role in the regulation of the innate and adaptive immune systems. In the present study we show that recombinant human Sp alpha (rSp alpha) binds to the surface of several gram-positive and gram-negative bacterial strains. Competition studies indicated that such binding is mediated by the recognition of lipoteichoic acid (LTA) and lipopolysaccharide (LPS), respectively, through nonoverlapping sites on the Sp alpha molecule. The most conserved part of LPS (2-keto-3-deoxyoctulosonic acid and lipid A) was shown to be involved in the recognition by Sp alpha. Bacterial binding studies using the SRCR domain 1 of Sp alpha showed that this domain retains both the LPS and LTA binding activities, indicating that both bacterial interacting sites are retained in a single SRCR domain. Furthermore, rSp alpha induced aggregation of gram-positive and gram-negative bacteria strains. On the other hand, rSp alpha inhibited tumor necrosis factor-alpha secretion by human monocytes stimulated with LPS or LTA. Binding of Sp alpha to conserved components of bacterial surfaces and modulation of the monocyte response indicate that this molecule is an active constituent of the innate immune response of the host.     

Quinone-dependent delayed fluorescence from the reaction center of photosynthetic bacteria

Millisecond delayed fluorescence from the isolated reaction center of photosynthetic bacteria Rhodobacter sphaeroides was measured after single saturating flash excitation and was explained by thermal repopulation of the excited bacteriochlorophyll dimer from lower lying charge separated states. Three exponential components (fastest, fast, and slow) were found with lifetimes of 1.5, 102, and 865 ms and quantum yields of 6.4 x 10(-9), 2.2 x 10(-9), and 2.6 x 10(-9) (pH 8.0), respectively. While the two latter phases could be related to transient absorption changes, the fastest one could not. The fastest component, dominating when the primary quinone was prereduced, might be due to a small fraction of long-lived triplet states of the radical pair and/or the dimer. The fast phase observed in the absence of the secondary quinone, was sensitive to pH, temperature, and the chemical nature of the primary quinone. The standard free energy of the primary stable charge pair relative to that of the excited dimer was -910 +/- 20 meV at pH 8 and with native ubiquinone, and it showed characteristic changes upon pH and quinone replacement. The interaction energy ( approximately 50 meV) between the cluster of the protonatable groups around GluL212 and the primary semiquinone provides evidence for functional linkage between the two quinone binding pockets. An empirical relationship was found between the in situ free energy of the primary quinone and the rate of charge recombination, with practical importance in the estimation of the free energy levels from the easily available lifetime of the charge recombination. The ratio of the slow and fast components could be used to determine the pH dependence of the free energy level of the secondary stable charge pair relative to that of the excited dimer.     

Cholesterol efflux promotes acrosome reaction in goat spermatozoa

Cholesterol efflux and membrane destabilization play an important role in sperm capacitation and membrane fusion in the acrosome reaction (AR). In this study we establish the effect of cholesterol removal from spermatozoa on acrosomal responsiveness. Mature goat spermatozoa were incubated in BSA-free medium in the presence of beta-cyclodextrin (betaCD) as cholesterol acceptor. After incubation with 8 mM betaCD, 50-60% of cholesterol was released from sperm membranes with no loss in the phospholipid content, and 35% of AR was induced. However, when 30% of cholesterol was lost, this moderate cholesterol decrease was unable to initiate AR. Cholesterol desorption was very rapid, following an exponential kinetics with a half-time of around 10 min, which is in contrast with the slow sigmoidal kinetics of acrosomal responsiveness: around 2 h was required for maximal AR. Our results suggest that cholesterol efflux has a direct influence on the onset of the AR, that is, merely removing cholesterol would trigger the AR.     

Localization of disulfide bonds in the cystine knot domain of human von Willebrand factor

von Willebrand factor (VWF) is a multimeric glycoprotein that is required for normal hemostasis. After translocation into the endoplasmic reticulum, proVWF subunits dimerize through disulfide bonds between their C-terminal cystine knot-like (CK) domains. CK domains are characterized by six conserved cysteines. Disulfide bonds between cysteines 2 and 5 and between cysteines 3 and 6 define a ring that is penetrated by a disulfide bond between cysteines 1 and 4. Dimerization often is mediated by additional cysteines that differ among CK domain subfamilies. When expressed in a baculovirus system, recombinant VWF CK domains (residues 1957-2050) were secreted as dimers that were converted to monomers by selective reduction and alkylation of three unconserved cysteine residues: Cys(2008), Cys(2010), and Cys(2048). By partial reduction and alkylation, chemical and proteolytic digestion, mass spectrometry, and amino acid sequencing, the remaining intrachain disulfide bonds were characterized: Cys(1961)-Cys(2011) (), Cys(1987)-Cys(2041) (), Cys(1991)-Cys(2043) (), and Cys(1976)-Cys(2025). The mutation C2008A or C2010A prevented dimerization, whereas the mutation C2048A did not. Symmetry considerations and molecular modeling based on the structure of transforming growth factor-beta suggest that one or three of residues Cys(2008), Cys(2010), and Cys(2048) in each subunit mediate the covalent dimerization of proVWF.