Structural basis of the broadly neutralizing anti-interferon-α antibody rontalizumab

Interferons-alpha (IFN-α) are the expressed gene products comprising thirteen type I interferons with protein pairwise sequence similarities in the 77–96% range. Three other widely expressed human type I interferons, IFN-β, IFN-κ and IFN-ω have sequences 29–33%, 29–32% and 56–60% similar to the IFN-αs, respectively. Type I interferons act on immune cells by producing subtly different immune-modulatory effects upon binding to the extracellular domains of a heterodimeric cell-surface receptor composed of IFNAR1 and IFNAR2, most notably anti-viral effects. IFN-α has been used to treat infection by hepatitis-virus type C (HCV) and a correlation between hyperactivity of IFN-α-induced signaling and systemic lupus erythematosis (SLE), or lupus, has been noted. Anti-IFN-α antibodies including rontalizumab have been under clinical study for the treatment of lupus. To better understand the rontalizumab mechanism of action and specificity, we determined the X-ray crystal structure of the Fab fragment of rontalizumab bound to human IFN-α2 at 3Å resolution and find substantial overlap of the antibody and IFNA2 epitopes on IFN-α2.     

hnRNP Q regulates Cdc42-mediated neuronal morphogenesis

The RNA-binding protein hnRNP Q has been implicated in neuronal mRNA metabolism. Here, we show that knockdown of hnRNP Q increased neurite complexity in cultured rat cortical neurons and induced filopodium formation in mouse neuroblastoma cells. Reexpression of hnRNP Q1 in hnRNP Q-depleted cells abrogated the morphological changes of neurites, indicating a specific role for hnRNP Q1 in neuronal morphogenesis. A search for mRNA targets of hnRNP Q1 identified functionally coherent sets of mRNAs encoding factors involved in cellular signaling or cytoskeletal regulation and determined its preferred binding sequences. We demonstrated that hnRNP Q1 bound to a set of identified mRNAs encoding the components of the actin nucleation-promoting Cdc42/N-WASP/Arp2/3 complex and was in part colocalized with Cdc42 mRNA in granules. Using subcellular fractionation and immunofluorescence, we showed that knockdown of hnRNP Q reduced the level of some of those mRNAs in neurites and redistributed their encoded proteins from neurite tips to soma to different extents. Overexpression of dominant negative mutants of Cdc42 or N-WASP compromised hnRNP Q depletion-induced neurite complexity. Together, our results suggest that hnRNP Q1 may participate in localization of mRNAs encoding Cdc42 signaling factors in neurites, and thereby may regulate actin dynamics and control neuronal morphogenesis.     

Coated vesicles participate in the receptor-mediated endocytosis of insulin

We have purified coated vesicles from rat liver by differential ultracentrifugation. Electron micrographs of these preparations reveal only the polyhedral structures typical of coated vesicles. SDS PAGE of the coated vesicle preparation followed by Coomassie Blue staining of proteins reveals a protein composition also typical of coated vesicles. We determined that these rat liver coated vesicles possess a latent insulin binding capability. That is, little if any specific binding of 125I-insulin to coated vesicles is observed in the absence of detergent. However, coated vesicles treated with the detergent octyl glucoside exhibit a substantial specific 125I-insulin binding capacity. We visualized the insulin binding structure of coated vesicles by cross-linking 125I-insulin to detergent-solubilized coated vesicles using the bifunctional reagent disuccinimidyl suberate followed by electrophoresis and autoradiography. The receptor structure thus identified is identical to that of the high-affinity insulin receptor present in a variety of tissues. We isolated liver coated vesicles from rats which had received injections of 125I-insulin in the hepatic portal vein. We found that insulin administered in this fashion was rapidly and specifically taken up by liver coated vesicles. Taken together, these data are compatible with a functional role for coated vesicles in the receptor-mediated endocytosis of insulin.     

Fluorine-18 isotope labeling for positron emission tomography imaging. Direct evidence for DBPR211 as a peripherally restricted CB1 inverse agonist

The [¹⁸F] isotope-labelled CB1 inverse agonist 3 was elaborated and synthesized for positron emission tomography scanning studies. After immediate purification and calibration with its unlabeled counterpart, compound 3 was intravenously injected in mice and revealed that its distribution percentage in brain over 90-min scans among five region of interests, including brain, liver, heart, thigh muscle and kidney was lower than 1%, thus providing direct evidence to justify itself as a peripherally restricted CB1 antagonist.     

浙江省檫木林生境与生态位研究

植被生境是群落乃至整个生态系统的物质基础,生态位宽度、生态位重叠、种间关系在群落生态学研究领域也具有重要地位,因此在植被生境基础之上展开的生态位、种间关系研究对了解生态系统功能具有重要意义。主成分分析表明:土壤养分和海拔因子对檫木生长分布起着重要的限制性作用;通过重要值对环境因子趋势拟合得出檫木在浙江省的典型生境为:海拔400—800 m,坡度20°—40°,阴坡半阴坡,土壤厚度大且肥沃的区域;在出现檫木的189个监测样地中,共出现的乔木树种有149种,主要优势树种(以重要值排序)为杉木Cunninghamia lanceolata、马尾松Pinus massoniana Lamb.、木荷Schima superba、枫香Liquidambar formosana Hance、青冈Cyclobalanopsis glauca (Thunb) Oerst、苦槠Castanopsis sclerophylla、石栎Lithocarpus glaber(Thunb.) Nakai、檫木Sassafras tzumu (Hemsl.) Hemsl.等。生态位宽度大小依次为杉木、檫木、马尾松、木荷、枫香、青冈、苦槠、板栗Castanea mollissima等,可见杉木、檫木、马尾松、木荷对外部环境有较强的适应能力,在各个资源位中出现较多,对资源的利用优势比较明显,檫木的生态位宽度达到了61.65,仅次于杉木的89.64,甚至大于马尾松的57.28,也说明檫木能在研究区拥有较好的群落地位;檫木与野樱桃Cerasus serrula (Franch.) Yüet L的生态位重叠值高达0.9以上,和浙江楠Phoebe chekiangensisC. B. Shang、构树Broussonetia papyrifera、鸡爪槭Acer palmatum Thunb.的生态位重叠值分别达0.831、0.785、0.531,说明檫木和野樱桃、浙江楠、构树、鸡爪槭对生境要求比较相近,在生态资源充足的情况下可尝试混交造林;通过监测样地中11个优势树种的种间联结分析,显示檫木与杉木、马尾松、木荷的种间具有显著的正联结关系,与枫香、苦槠和青冈也存在正联结关系,可见在研究区内檫木能够与这些树种互相吸引。     

Real-Time Determination of Structural Changes of Sodium Caseinate-Stabilized Emulsions Containing Pectin Using High Resolution Ultrasonic Spectroscopy

The interactions that lead to structure transitions in oil-in-water emulsions were investigated using high-resolution ultrasonic spectroscopy. High methoxyl pectin (HMP) was added to the emulsions at various concentrations and the dynamics of aggregation induced by changes in pH were observed. Two independent ultrasonic parameters, velocity and attenuation, were measured as a function of time or pH. At pH 6.8, both velocity and attenuation of sound changed as a function of HMP concentration. During acidification, caused by the addition of glucono-δ-lactone, there were small changes in the overall ultrasonic velocity, but it was possible to relate these changes to the structural changes in the emulsion. The values of ultrasonic attenuation decreased at high pH with increasing amount of HMP, indicating changes in the flocculation state of the oil droplets caused by depletion forces. During acidification at pH 5.4, emulsions containing HMP showed a steep increase in the ultrasonic attenuation, and this pH corresponds to the pH of association of HMP with the casein-covered oil droplets. The adsorption of HMP onto the interface causes a rearrangement of the oil droplets, and the emulsions containing sufficient amounts of HMP no longer gel at acid pH. This is well described by the ultrasonic attenuation changes in the various emulsions. This research demonstrated for the first time that ultrasonic spectroscopy can be employed for in situ monitoring and analysis of acid-induced destabilization of food emulsions.     

The effects of foreign transmembrane domains on the biosynthesis of the influenza virus hemagglutinin

Eleven chimeric proteins were created in which the transmembrane, the cytoplasmic, or both topological domains of the influenza virus hemagglutinin (HA) were replaced with those from five other glycoproteins. All of the chimeric HAs reached the cell surface but appeared to differ in the degree to which they were stably folded. Comparisons of the rates of folding, passage into the Golgi, and arrival at the plasma membrane of wild-type HA and the chimeric proteins suggest that formation of a stable HA trimer is not an absolute requirement for export from the endoplasmic reticulum. In addition, there appear to be at least two steps at which the rate of transport can be altered during exocytosis, one occurring before and the other after the trimming of oligosaccharides by Golgi mannosidases. Certain of the chimeras differed from HA in their ability to pass through each of these steps. Replacement of the HA transmembrane domain with the analogous sequences from other proteins affected folding and transport of the chimeric HAs in ways that suggest that the HA transmembrane sequences form a specific structure in the membrane that differs from that formed by analogous sequences from the other proteins.     

EGF-induced tyrosine phosphorylation of Endofin is dependent on PI3K activity and proper localization to endosomes

In our previous study, Endofin was validated to be a novel tyrosine phosphorylation target downstream of EGFR. Here, we attempted to map the signaling events associated with Endofin following activation of EGFR with EGF. Tyrosine phosphorylation of endogenous Endofin peaked around 15 min and was modulated within 30 min of EGF treatment. Phosphatidylinositol 3-kinase (PI3K) activity and FYVE domain-mediated localization of Endofin to EEA1-marked endosomes were shown to be necessary for the tyrosine phosphorylation of Endofin. Tyrosine 515 was mapped to be a major phosphorylation site on Endofin but disruption of phosphorylation at Y515 neither affected Endofin's localization nor its co-localization with EGFR in the endosomes. Instead, abrogation of Y515 phosphorylation and mislocalization of Endofin were found to enhance the amplitude of the MAPK cascade, suggesting a possible role of Endofin in the modulation of MAPK pathway. Our study has identified a novel signaling cascade involving EGFR, PI3K, Endofin and MAPK in the EGFR signaling network.     

The insulin receptor protein kinase. Physicochemical requirements for activity

We determined that the rate of insulin-stimulated autophosphorylation of the insulin receptor is independent of receptor concentration and thus proceeds via an intramolecular process. This result is consistent with the possibility that ligand-dependent autophosphorylation may be a means by which cells can distinguish occupied from unoccupied receptors. We employed dithiothreitol to dissociate tetrameric receptor into alpha beta halves in order to further elucidate the structural requirements for the receptor-mediated kinase activity. Dithiothreitol had a complex biphasic effect on insulin-stimulated receptor kinase activity. Marked stimulation of kinase activity was observed at 1-2 mM dithiothreitol when the receptor was predominantly tetrameric and kinase activity diminished when dimeric alpha beta receptor halves predominate (greater than 2 mM dithiothreitol). N-Ethylmaleimide inhibits insulin-stimulated receptor kinase activity. We suggest that the tetrameric holoreceptor is the most active kinase structure and this structure requires for maximal activity, a reduced sulfhydryl group at or near the active site. We treated receptor preparations with elastase to generate receptor proteolytically "nicked" in the beta subunit. This treatment completely abolishes insulin-dependent autophosphorylation and histone phosphorylation with essentially no effects on insulin binding as determined by affinity labeling of the receptor alpha subunit. We suggest such treatment functionally uncouples insulin binding from insulin-stimulated receptor kinase activity. The possible physiological significance of these findings is discussed.     

A polymorphic satellite sequence maps to the pericentric region of the bovine Y chromosome

Exploiting a serendipitously observed bovine male-specific signal, generated by the mouse pSP64.2.5EI minisatellite probe, we have cloned a bovine (Bos taurus) Y-specific sequence: btDYZ-1. This sequence is composed of 60 tandem repetitions of a motif consisting of two parts: a 40-bp-long unit, showing a mean divergence of 27% between repeats, separated from the next repeat by a TG-rich stretch varying in length between 12 and 63 bp. The number of copies of this repeated motif has been estimated at 6 X 10(4) per male genome. As a consequence, the corresponding satellite, DYZ-1, might represent approximately 1/20 of the bovine Y chromosome. btDYZ-1 has been mapped by in situ hybridization to the pericentric region of the Y chromosome. It is characterized by a substantial genetic polymorphism and has been shown to be conserved within the Bos and Bison genera of the Bovinae subfamily. This sequence is being used to develop a sexing procedure for bovine preimplantation embryos based on the polymerase chain reaction.