High affinity interaction between a bivalve C-type lectin and a biantennary complex-type N-glycan revealed by crystallography and microcalorimetry

Codakine is an abundant 14-kDa mannose-binding C-type lectin isolated from the gills of the sea bivalve Codakia orbicularis. Binding studies using inhibition of hemagglutination indicated specificity for mannose and fucose monosaccharides. Further experiments using a glycan array demonstrated, however, a very fine specificity for N-linked biantennary complex-type glycans. An unusually high affinity was measured by titration microcalorimetry performed with a biantennary Asn-linked nonasaccharide. The crystal structure of the native lectin at 1.3A resolution revealed a new type of disulfide-bridged homodimer. Each monomer displays three intramolecular disulfide bridges and contains only one calcium ion located in the canonical binding site that is occupied by a glycerol molecule. The structure of the complex between Asn-linked nonasaccharide and codakine has been solved at 1.7A resolution. All residues could be located in the electron density map, except for the capping beta1-4-linked galactosides. The alpha1-6-linked mannose binds to calcium by coordinating the O3 and O4 hydroxyl groups. The GlcNAc moiety of the alpha1,6 arm engages in several hydrogen bonds with the protein, whereas the GlcNAc on the other antenna is stacked against Trp(108), forming an extended binding site. This is the first structural report for a bivalve lectin.     

CLIP-170 tracks growing microtubule ends by dynamically recognizing composite EB1/tubulin-binding sites

The microtubule cytoskeleton is crucial for the internal organization of eukaryotic cells. Several microtubule-associated proteins link microtubules to subcellular structures. A subclass of these proteins, the plus end–binding proteins (+TIPs), selectively binds to the growing plus ends of microtubules. Here, we reconstitute a vertebrate plus end tracking system composed of the most prominent +TIPs, end-binding protein 1 (EB1) and CLIP-170, in vitro and dissect their end-tracking mechanism. We find that EB1 autonomously recognizes specific binding sites present at growing microtubule ends. In contrast, CLIP-170 does not end-track by itself but requires EB1. CLIP-170 recognizes and turns over rapidly on composite binding sites constituted by end-accumulated EB1 and tyrosinated α-tubulin. In contrast to its fission yeast orthologue Tip1, dynamic end tracking of CLIP-170 does not require the activity of a molecular motor. Our results demonstrate evolutionary diversity of the plus end recognition mechanism of CLIP-170 family members, whereas the autonomous end-tracking mechanism of EB family members is conserved.     

CD36 N-terminal cytoplasmic domain is not required for the internalization of oxidized low-density lipoprotein

The uptake of OxLDLs (oxidized low density lipoproteins) by CD36-expressing macrophages in the arterial intima and the subsequent 'foam cell' formation represents a crucial step in the initiation and development of atherosclerotic plaques. The present study has addressed the function of the CD36 N-terminal cytoplasmic domain in the binding and internalization of OxLDL. A selection of CD36 N-terminal cytoplasmic domain mutants were generated and stably expressed in HEK-293 (human embryonic kidney) cells. The capacity of three mutants [CD36_C3/7-A (CD36-C3A/C7A), CD36_D4/R5-A (CD36-D4A/R5A) and CD36_nCPD(-) (CD36 lacking the N-terminal cytoplasmic domain)] to bind and endocytose OxLDL was then studied using immunofluorescence microscopy and quantitative fluorimetry. Each of the CD36 constructs was expressed at differing levels at the cell surface, as measured by flow cytometry and Western blotting. Following incubation with DiI (1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate)-OxLDL, cells bearing the CD36_wt (wild-type CD36), CD36_C3/7-A, CD36_D4/R5-A and CD36_nCPD(-) constructs all internalized DiI-OxLDL into endosomal structures, whereas empty-vector-transfected cells failed to do so, indicating that, unlike the C-terminal cytoplasmic domain, the N-terminal cytoplasmic domain is not essential for the endocytosis of OxLDL. In conclusion, the uptake of OxLDL by CD36 is not reliant on the presence of the CD36 N-terminal cytoplasmic domain. However, the N-terminal cytoplasmic domain may conceivably be implicated in the maturation of CD36.     

BDNF-TrkB signaling interacts with the GABAergic system to inhibit rhythmic swallowing in the rat

Brain-derived neurotrophic factor (BDNF) acts as an anorexigenic factor in the dorsal vagal complex (DVC) of the adult rat brain stem. The DVC contains the premotoneurons controlling swallowing, a motor component of feeding behavior. Although rats with transected midbrain do not seek out food, they are able to swallow and to ingest food. Because BDNF and tropomyosin-related kinase B (TrkB) receptors are expressed in the DVC, this study hypothesized that BDNF could modify the activity of premotoneurons involved in swallowing. Repetitive electrical stimulation of the superior laryngeal nerve (SLN) induces rhythmic swallowing that can be recorded with electromyographic electrodes inserted in sublingual muscles. We show that a microinjection of BDNF in the swallowing network induced a rapid, transient, and dose-dependant inhibition of rhythmic swallowing. This BDNF effect appeared to be mediated via TrkB activation, since it no longer occurred when TrkB receptors were antagonized by K-252a. Interestingly, swallowing was inhibited when subthreshold doses of BDNF and GABA were coinjected, suggesting a synergistic interaction between these two signaling substances. Moreover, BDNF no longer had an inhibitory effect on swallowing when coinjected with bicuculline, a GABA(A) receptor antagonist. This blockade of BDNF inhibitory effect on swallowing was reversible, since it reappeared when BDNF was injected 15 min after bicuculline. Finally, we show that stimulation of SLN induced a decrease in BDNF protein within the DVC. Together, our results strongly suggest that BDNF inhibits swallowing via modulation of the GABAergic signaling within the central pattern generator of swallowing.     

Detection of quantitative trait loci for carcass composition traits in pigs

A quantitative trait locus (QTL) analysis of carcass composition data from a three-generation experimental cross between Meishan (MS) and Large White (LW) pig breeds is presented. A total of 488 F2 males issued from six F1 boars and 23 F1 sows, the progeny of six LW boars and six MS sows, were slaughtered at approximately 80 kg live weight and were submitted to a standardised cutting of the carcass. Fifteen traits, i.e. dressing percentage, loin, ham, shoulder, belly, backfat, leaf fat, feet and head weights, two backfat thickness and one muscle depth measurements, ham + loin and back + leaf fat percentages and estimated carcass lean content were analysed. Animals were typed for a total of 137 markers covering the entire porcine genome. Analyses were performed using a line-cross (LC) regression method where founder lines were assumed to be fixed for different QTL alleles and a half/full sib (HFS) maximum likelihood method where allele substitution effects were estimated within each half-/full-sib family. Additional analyses were performed to search for multiple linked QTL and imprinting effects. Significant gene effects were evidenced for both leanness and fatness traits in the telomeric regions of SSC 1q and SSC 2p, on SSC 4, SSC 7 and SSC X. Additional significant QTL were identified for ham weight on SSC 5, for head weight on SSC 1 and SSC 7, for feet weight on SSC 7 and for dressing percentage on SSC X. LW alleles were associated with a higher lean content and a lower fat content of the carcass, except for the fatness trait on SSC 7. Suggestive evidence of linked QTL on SSC 7 and of imprinting effects on SSC 6, SSC 7, SSC 9 and SSC 17 were also obtained.     

Flight Phases in the Song of Skylarks: Impact on Acoustic Parameters and Coding Strategy

Skylarks inhabit open fields and perform an aerial song display which serves as a territorial signal. The particularly long and elaborate structure of this song flight raises questions about the impact of physical and energetic constraints acting on a communication signal. Song produced during the three distinct phases of the flight - ascending, level and descending phase could be subject to different constraints, serve different functions and encode different types of information. We compared song parameters during the ascending and the level phases. We found that the structure of the song varied with the phase of the flight. In particular, song had a higher tempo when skylarks were ascending which might be related to higher oxygen and energetic demands. We also explored which phase of the song flight might encode individuality. Earlier studies reported that skylarks reduced their territorial response to established neighbours if the neighbour song was broadcasted from the correct adjacent boundary, but reacted aggressively if the neighbour songs were broadcasted from an incorrect boundary (mimicking a displaced neighbour). Such differential response provides some evidence for individual recognition. Here, we exposed subjects to playback stimuli of neighbour song in which we had replaced either the song produced during the level or the ascending phase by the relevant song of the neighbour from the incorrect border. Singing response was higher towards stimuli in which the ‘level phase song’ was replaced, indicating that skylarks could be able to recognise their neighbours based on song of this phase. Thus, individuality seems to be primarily coded in the level phase of the flight song.     

Palaeoreas,Majoreas, and Stryfnotherium gen. nov. (Mammalia: Artiodactyla: Bovidae) from the Late Miocene of Greece

A new comparative study of the two crania, LGPUT RZO-03 and LGPUT RZO-68 attributed to Palaeoreas zouavei Bouvrain, 1980 from the Late Miocene of Axios Valley, Greece reveals great morphological and metric differences between them, which require the exclusion of LGPUT RZO-68 from this species and even from Palaeoreas Gaudry, 1861. This decision leaves unsupported many diagnostic features of P. zouavei applied by previous authors and along with recent data from Bulgaria put in doubt the validity of P. zouavei as a whole. As a result, the genus Palaeoreas is re-defined as monotypic and the cranial morphometric changes of P. lindermayeri (Wagner, 1848) are discussed on a temporal basis. An updated taxonomic analysis confirms the presence of P. lindermayeri in the Late Turolian mammal assemblage of Dytiko-1 (Axios Valley, Greece) but rejects its occurrence at Dytiko-2. Instead, a re-examination of old cranial specimens and a study of new ones suggest the presence in both sites of Majoreas Kostopoulos, 2004 a Late Miocene taxon previously mostly known from the eastern banks of the Aegean Sea. A reassessment of the LGPUT RZO-68 cranium, and its comparison with Palaeoreas and Criotherium Forsyth-Major, 1891, suggests that it deserves taxonomic distinction at genus and species levels: Stryfnotherium exophthalmon.     

Determination of acyclovir and its metabolite 9-carboxymethoxymethylguanine in serum and urine using solid-phase extraction and high-performance liquid chromatography

A reversed-phase ion-pair high-performance liquid chromatography method for the determination of acyclovir and its metabolite 9-carboxymethoxymethylguanine is described. The samples are purified by reversed-phase solid-phase extraction. The components are separated on a C₁₈ column with a mobile phase containing 18% acetonitrile, 5 mM dodecyl sulphate and 30 mM phosphate buffer, pH 2.1, and measured by fluorescence detection using an excitation wavelength of 285 nm and an emission wavelenght of 380 nm. Detection limits are 0.12 μM (plasma)) and 0.60 μM (urine) for acyclovir, and 0.26 μM (plasma) and 1.3 μM (urine) for metabolite. Correlation coefficients that were better than 0.998 were obtained normally. This analytical method, which enables simultaneous measurement of parent compound and metabolite, has been used in kinetics studies and for therapeutic drug monitoring in different patient groups with variable degrees of renal dysfunction.     

GROWTH STIMULATION OF ALEXANDRIUM TAMARENSE (DINOPHYCEAE) BY HUMIC SUBSTANCES FROM THE MANICOUAGAN RIVER (EASTERN CANADA)1

In the St. Lawrence Estuary, annual recurrent blooms of the toxic dinoflagellate Alexandrium tamarense L. Balech are associated with brackish waters. Riverine inputs are suspected to favor bloom development by increasing water column stability and/or by providing growth stimulants such as humic substances (HS). A 17‐day culture experiment was conducted to evaluate the importance of HS as growth factors for A. tamarense. Nonaxenic cultures were exposed to four HS extracts from three different sources: humic and fulvic acids isolated from the Manicouagan River, Quebec, Canada; humic acids from the Suwannee River, Georgia, United States; and a desalted alkaline soil extract. For each extract, four concentrations were tested as supplements to the artificial Keller medium, a nitrate‐rich algal culture medium. Additions of HS from all sources significantly enhanced the overall growth rates relative to the controls. Concentrations of HS, estimated by UV spectrophotometry, remained constant throughout the exponential growth phase, suggesting that the HS were acting mainly as growth promoters during our experiment. Dose–response curves indicated that HS could increase the growth rate of A. tamarense even at low concentrations, such as those encountered in the St. Lawrence Estuary. Our results support the hypothesis that HS from the Manicouagan River plume can stimulate the development of toxic dinoflagellate blooms.     

Determinants of Human Immunodeficiency Virus Type 1 Envelope Glycoprotein Activation by Soluble CD4 and Monoclonal Antibodies

Infection by some human immunodeficiency virus type 1 (HIV-1) isolates is enhanced by the binding of subneutralizing concentrations of soluble receptor, soluble CD4 (sCD4), or monoclonal antibodies directed against the viral envelope glycoproteins. In this work, we studied the abilities of different antibodies to mediate activation of the envelope glycoproteins of a primary HIV-1 isolate, YU2, and identified the regions of gp120 envelope glycoprotein contributing to activation. Binding of antibodies to a variety of epitopes on gp120, including the CD4 binding site, the third variable (V3) loop, and CD4-induced epitopes, enhanced the entry of viruses containing YU2 envelope glycoproteins. Fab fragments of antibodies directed against either the CD4 binding site or V3 loop also activated YU2 virus infection. The activation phenotype was conferred on the envelope glycoproteins of a laboratory-adapted HIV-1 isolate (HXBc2) by replacing the gp120 V3 loop or V1/V2 and V3 loops with those of the YU2 virus. Infection by the YU2 virus in the presence of activating antibodies remained inhibitable by macrophage inhibitory protein 1β, indicating dependence on the CCR5 coreceptor on the target cells. Thus, antibody enhancement of YU2 entry involves neither Fc receptor binding nor envelope glycoprotein cross-linking, is determined by the same variable loops that dictate enhancement by sCD4, and probably proceeds by a process fundamentally similar to the receptor-activated virus entry pathway.