Peroxynitrite efficiently mediates the interconversion of redox intermediates of myeloperoxidase

Nitric oxide-derived oxidants (e.g., peroxynitrite) are believed to participate in antimicrobial activities as part of normal host defenses but also in oxidative tissue injury in inflammatory disorders. A similar role is ascribed to the heme enzyme myeloperoxidase (MPO), the most abundant protein of polymorphonuclear leukocytes, which are the terminal phagocytosing effector cells of the innate immune system. Concomitant production of peroxynitrite and release of millimolar MPO are characteristic events during phagocytosis. In order to understand the mode of interaction between MPO and peroxynitrite, we have performed a comprehensive stopped-flow investigation of the reaction between all physiological relevant redox intermediates of MPO and peroxynitrite. Both iron(III) MPO and iron(II) MPO are rapidly converted to compound II by peroxynitrite in monophasic reactions with calculated rate constants of (6.8+/-0.1) x 10(6) M(-1)s(-1) and (1.3+/-0.2) x 10(6) M(-1)s(-1), respectively (pH 7.0 and 25 degrees C). Besides these one- and two-electron reduction reactions of peroxynitrite, which produce nitrogen dioxide and nitrite, a one-electron oxidation to the oxoperoxonitrogen radical must occur in the fast monophasic transition of compound I to compound II mediated by peroxynitrite at pH 7.0 [(7.6+/-0.1) x 10(6) M(-1)s(-1)]. In addition, peroxynitrite induced a steady-state transition from compound III to compound II with a rate of (1.0+/-0.3) x 10(4) M(-1)s(-1). Thus, the interconversion among the various oxidation states of MPO that is prompted by peroxynitrite is remarkable. Reaction mechanisms are proposed and the physiological relevance is discussed.     

A concise synthesis of 4-nitrophenyl 2-azido-2-deoxy- and 2-acetamido-2-deoxy-D-mannopyranosides

4-nitrophenyl 3,4,6-tri-O-acetyl-2-azido-2-deoxy-alpha- and beta-D-mannopyranosides were prepared from methyl 4,6-O-benzylidene-alpha-D-glucopyranoside and 1,3,4,6-tetra-O-acetyl-alpha-D-glucopyranose, respectively. Chemoselective reduction of both azides with hydrogen sulfide readily afforded 4-nitrophenyl 2-acetamido-4,6-di-O-acetyl-2-deoxy-alpha-D- and -beta-D-mannopyranosides in higher yields than reduction with triphenylphosphine or a polymer-supported triarylphosphine. Subsequent de-O-acetylation yielded 4-nitrophenyl 2-acetamido-2-deoxy-alpha-D-mannopyranoside and 4-nitrophenyl 2-acetamido-2-deoxy-beta-D-mannopyranoside in 20% and 44% overall yields, respectively.     

Genetic ablation of polysialic acid causes severe neurodevelopmental defects rescued by deletion of the neural cell adhesion molecule

Poly-alpha2,8-sialic acid (polySia) is a unique modification of the neural cell adhesion molecule, NCAM, tightly associated with neural development and plasticity. However, the vital role attributed to this carbohydrate polymer has been challenged by the mild phenotype of mice lacking polySia due to NCAM-deficiency. To dissect polySia and NCAM functions, we generated polySia-negative but NCAM-positive mice by simultaneous deletion of the two polysialyltransferase genes, St8sia-II and St8sia-IV. Beyond features shared with NCAM-null animals, a severe phenotype with specific brain wiring defects, progressive hydrocephalus, postnatal growth retardation, and precocious death was observed. These drastic defects were selectively rescued by additional deletion of NCAM, demonstrating that they originate from a gain of NCAM functions because of polySia deficiency. The data presented in this study reveal that the essential role of polySia resides in the control and coordination of NCAM interactions during mouse brain development. Moreover, this first demonstration in vivo that a highly specific glycan structure is more important than the glycoconjugate as a whole provides a novel view on the relevance of protein glycosylation for the complex process of building the vertebrate brain.     

A telomeric satellite in Drosophila virilis and its sibling species

Telomere elongation by telomerase is the most widespread mechanism among eukaryotes. However, alternative mechanisms such as homologous recombination between terminal satellite DNAs are probably used in lower dipteran insects and in some plants. Drosophila melanogaster uses the very unusual telomere elongation pathway of transposition of telomere-specific retrotransposable elements. The uniqueness of this telomere elongation mechanism raises the question of its origin. We, therefore, analyzed sequences located at telomeres of fairly distantly related Drosophila species, and in this paper we describe the characterization of complex satellite DNA sequences located at the telomeres of D. virilis and other species in the virilis group. We suggest an involvement of these DNA satellites in telomere elongation by homologous recombination similar to that found in lower dipterans. Our findings raise the possibility that telomere elongation by specific retrotransposons as found in D. melanogaster and its sibling species is a recent event in the evolution of dipteran insects.     

Thermoluminescence and fluorescence study of changes in Photosystem II photochemistry in desiccating barley leaves

An effect of desiccation (a decrease of relative water content from 97% to 10% within 35 h) on Photosystem II was studied in barley leaf segments (Hordeum vulgare L. cv. Akcent) using chlorophyll a fluorescence and thermoluminescence (TL). The O-J-I-P fluorescence induction curve revealed a decrease of F_P and a slight shift of the J step to a shorter time with no change in its height. The analysis of the fluorescence decline after a saturating light flash revealed an increased portion of slow exponential components with increasing desiccation. The TL bands obtained after excitation by continuous light were situated at about –27°C (Z_v band – recombination of P680⁺Q_A ⁻), –14 °C (A band – S₃Q_A ⁻), +12 °C (B band – S_2/3Q_B ⁻) and +45 °C (C band – TyrD⁺Q_A ⁻). The bands related to the S-states of oxygen evolving complex (A and B) were reduced by desiccation and shifted to higher and lower temperatures, respectively. In accordance with this, the band observed at about +27 °C (S₂Q_B ⁻) after excitation by 1 flash fired at –10 °C and band at about +20 °C (S_2/3Q_B ⁻) after 2 flashes decreased with increasing water deficit and shifted to lower temperatures. A new band around 5 °C appeared in both regimes of TL excitation for a relative water content of under 42% and was attributed to the Q band (S₂Q_A ⁻). It is suggested that under desiccation, an inhibition of the formation of S₂- and S₃-states in OEC occurred simultaneously with a lowering of electron transport on the acceptor side of PS II. The temperature down-shift of the TL bands obtained after the flash excitation was induced at the initial phases of water stress, indicating a decrease of the activation energy for the S_2/3Q_B ⁻recombination. This revised version was published online in June 2006 with corrections to the Cover Date.     

The influence of incompetent lip seal on the growth and development of craniofacial complex

Abnormal orofacial functions in the period of growth and development can cause morphological anomalies of the craniofacial complex. The aim of this study was to determine the correlation between open mouth posture and morphology of craniofacial complex. The shape, size and relationships of skeletal parts of craniofacial complex were determined by analysis of lateral cephalograms in the sample of 84 children--45 girls and 39 boys (aged 8.96 +/- 0.66 years). The sample was divided into two groups--lip competence and lip incompetence group. Differences in cephalometric values between observed groups were found. The values of inclination of lower central incisors (angle ILi/NB), interbasal angle (NL/NSL), angle between occlusal and mandibular plane and anterior lower facial height were significantly higher in the group with open mouth posture. It can be concluded that lip incompetence plays an important role in growth and development of craniofacial complex.     

Calpain Sensitive Regions in the N-terminal Cytoplasmic Domains of Glycine Transporters GlyT1A and GlyT1B

Glycine transporters are members of the Na⁺/Cl⁻ dependent transporter gene family and play crucial roles in regulating inhibitory as well as excitatory neurotransmission. In this report we show that calcium elevation in spinal cord synaptosomes decreases the levels of glycine transporter, GlyT1, N-terminal immunoreactivity, and that this decrease can be blocked by calpain inhibitor. Sequencing of GST fusion proteins containing the N-terminal domains of GlyT1A and B splice variants cleaved with rat recombinant calpain identified calpain cleavage sites after glycine 17 in GlyT1B and N-terminally of the first conserved arginine residue in both GlyT1A and GlyT1B. Expression in HEK293 cells revealed that truncation of the N-terminus of GlyT1 results in significant inhibition of glycine uptake. A syntaxin1A GST fusion protein was able to pull-down N-terminally deleted GlyT1, indicating that calpain cleavage does not eliminate syntaxin1A binding. These results suggest that calpain cleavage may regulate the transport activity/turnover of GlyT1 in vivo by cleaving its N-terminal domain.     

Ascorbic acid decreases neutral endopeptidase activity in cultured osteoblastic cells

Neprilysin (NEP) is a plasma membrane-bound peptidase with wide expression in kidney, lung, brain and bone. Decreased NEP activity has been linked to increased growth of some cancer cells, but it is unknown whether its activity is related to growth of cells belonging to the osteoblast lineage. We assessed NEP activity in an osteoblastic cell line, PyMS, by cleavage of N-Dansyl-d-Ala-Gly-p-nitro-Phe-Gly to Dansyl-d-Ala-Gly. NEP activity was completely blocked by 1 muM thiorphan. Most agents affecting growth of these cells (e.g. calcium, insulin-like growth factor I and dexamethasone) did not regulate NEP activity. Ascorbic acid (ASA) increased thymidine incorporation into DNA and potentiated the stimulatory effect of IGF I on DNA synthesis, an effect which was attenuated by echistatin. ASA decreased NEP activity in a dose-dependent manner, and decreased Western-detectable NEP protein in plasma membranes. ASA affects both integrin receptor-mediated signalling and the processing of regulatory peptides.     

Coordinate regulation of the mother centriole component nlp by nek2 and plk1 protein kinases

Mitotic entry requires a major reorganization of the microtubule cytoskeleton. Nlp, a centrosomal protein that binds gamma-tubulin, is a G(2)/M target of the Plk1 protein kinase. Here, we show that human Nlp and its Xenopus homologue, X-Nlp, are also phosphorylated by the cell cycle-regulated Nek2 kinase. X-Nlp is a 213-kDa mother centriole-specific protein, implicating it in microtubule anchoring. Although constant in abundance throughout the cell cycle, it is displaced from centrosomes upon mitotic entry. Overexpression of active Nek2 or Plk1 causes premature displacement of Nlp from interphase centrosomes. Active Nek2 is also capable of phosphorylating and displacing a mutant form of Nlp that lacks Plk1 phosphorylation sites. Importantly, kinase-inactive Nek2 interferes with Plk1-induced displacement of Nlp from interphase centrosomes and displacement of endogenous Nlp from mitotic spindle poles, while active Nek2 stimulates Plk1 phosphorylation of Nlp in vitro. Unlike Plk1, Nek2 does not prevent association of Nlp with gamma-tubulin. Together, these results provide the first example of a protein involved in microtubule organization that is coordinately regulated at the G(2)/M transition by two centrosomal kinases. We also propose that phosphorylation by Nek2 may prime Nlp for phosphorylation by Plk1.