Visualization of the uptake of high-density lipoprotein by rat aortic endothelial cells and smooth muscle cells in vitro

High-density lipoproteins (HDL) were conjugated to Fluorescein 1,1-dioctadecyl 3,3,3,3-tetramethylindocarbocyanine perchlorate (DiI) or colloidal gold for the investigation of ultrastructural aspects of binding and uptake of HDL by cholesterol-loaded cultured endothelial and smooth muscle cells from rat aorta. When cells were incubated for 2h at 4°C, HDL–DiI and HDL–gold conjugates were seen only on the cell surface. When cells were returned to incubation at 37°C for 5min, HDL–DiI appeared in the cytoplasm and colocalized with the fluorescent cholesteryl ester tag BODIPY-FL-C₁₂. HDL–gold conjugates appeared in the plasmalemmal invaginations and plasmalemmal vesicles. After incubation for 15min, most of the HDL–gold conjugates reappeared on the cell surface. After incubation for 30min, only a few conjugates were observed and they localized in lysosomal-like bodies. Quantitative data indicated that when the cholesterol-loaded cells were incubated at 4°C for 2h, the numbers of HDL–gold associated in clusters on the endothelial cell surface was 1.18 clusters/m. When cells were returned to incubation at 37°C for 5min, this value decreased to 0.7, increased again to 1.13 at 15min, and decreased to 0.29 at 30min. The numbers of clusters in the plasmalemmal invaginations were 0.06 clusters/m at 4°C for 2h, increased to 0.34 at 37°C for 5min and decreased gradually to 0.19 and 0.04 at 15 and 30min, respectively. The incidence of clusters in the plasmalemmal vesicles per non-nuclear cytoplasm was 0.01 clusters/m² at 4°C for 2h, increased significantly to 1.08 at 37°C for 5min, and decreased to 0.43 and 0.14 at 15 and 30min, respectively. This work supports that the plasmalemmal invaginations and plasmalemmal vesicles are linked to the HDL uptake in cholesterol-loaded aortic endothelial cells and smooth muscle cells.     

Mutagenesis of D80-82 and G83 residues in West Nile Virus NS2B: Effects on NS2B-NS3 activity and viral replication

Flaviviral NS2B is a required cofactor for NS3 serine protease activity and plays an important role in promoting functional NS2B-NS3 protease configuration and maintaining critical interactions with protease catalysis substrates. The residues D⁸⁰DDG in West Nile virus (WNV) NS2B are important for protease activity. To investigate the effects of D⁸⁰DDG in NS2B on protease activity and viral replication, the negatively charged region D⁸⁰DD and the conserved residue G83 of NS2B were mutated (D⁸⁰DD/E⁸⁰EE, D⁸⁰DD/K⁸⁰KK, D⁸⁰DD/A⁸⁰AA, G83F, G83S, G83D, G83K, and G83A), and NS3 D75A was designated as the negative control. The effects of the mutations on NS2B-NS3 activity, viral translation, and viral RNA replication were analyzed using kinetic analysis of site-directed enzymes and a transient replicon assay. All substitutions resulted in significantly decreased enzyme activity and blocked RNA replication. The negative charge of D⁸⁰DD is not important for maintaining NS2B function, but side chain changes in G83 have dramatic effects on protease activity and RNA replication. These results demonstrate that NS2B is important for viral replication and that D⁸⁰DD and G83 substitutions prevent replication; they will be useful for understanding the relationship between NS2B and NS3.     

Factors influencing parasitism of Trichogramma dendrolimi on eggs of the Asian corn borer, Ostrinia furnacalis

Laboratory studies were made to determine the capacity of Trichogramma dendrolimi to parasitize eggs of Ostrinia furnacalis, as affected by the rearing host species, substrate of host eggs, host age, original locality of host populations, and cold storage of host eggs. Wasps reared from eggs of Antheraea pernyi showed parasitic capacity on eggs of O. furnacalis on average twice as high as that of the wasps reared from eggs of Corcyra cephalonica. When the age of O. furnacalis eggs at 26 °C increased from 0–6 h to 18–24 h, the proportion of wasps that successfully parasitized host eggs, the number of host eggs parasitized, and the rate of parasitization all decreased by >50%. The number of O. furnacalis eggs parasitized per female T. dendrolimi increased with the number of host eggs available, and reached 22.9 in a 24 h period. However, the parasitic capacity of female T. dendrolimi on eggs of O. furnacalis laid on plant leaves was similar to that of O. furnacalis eggs laid on wax paper. Levels of parasitism of O. furnacalis eggs from two widely separated localities, i.e. Changchun (43.50° N, 125.20° E) and Hangzhou (30.18° N, 120.07° E), were similar. Cold storage of O. furnacalis eggs at 4 °C for 5 days did not affect parasitization. Results obtained in this study indicate that although O. furnacalis is a less preferred and less suitable host than many other hosts, such as Dendrolimus punctatus, Actias selene ningpoane, Philosamia cynthia, A. pernyi, C. cephalonica, within the host-species range of T. dendrolimi, the parasitoid has the potential to achieve 50–60% or even higher rates of parasitization of O. furnacalis eggs in corn fields under suitable conditions, and could be used in the biological control of the pest.     

Comparative studies of products obtained from solvolysis liquefaction of oil palm empty fruit bunch fibres using different solvents

Solvolysis of oil palm empty fruit bunches (EFB) fibres using different solvents (acetone, ethylene glycol (EG), ethanol, water and toluene) were carried out using an autoclave at 275°C for 60 min. The solvent efficiency in term of conversion yield was found to be: EG>water>ethanol>acetone>toluene. The liquid products and residue obtained were analyzed using Fourier transform infrared spectroscopy (FTIR) and gas chromatography/mass selectivity. The obtained results showed that the chemical properties of the oil product were significantly affected by the type of solvent used for the solvolysis process. The higher heating value (HHV) of oil products obtained using ethanol is ～29.42 MJ/kg, which is the highest among the oil products produced using different solvents. Water, ethanol and toluene yield major phenolic compounds. While EG favors the formation of alcohol compounds and acetone yields ketone and aldehyde compounds.     

Cytoplasmic targeting of mutant poly(A)-binding protein nuclear 1 suppresses protein aggregation and toxicity in oculopharyngeal muscular dystrophy

Oculopharyngeal muscular dystrophy (OPMD) is an adult-onset disorder characterized by progressive eyelid drooping, swallowing difficulties and proximal limb weakness. The autosomal dominant form of this disease is caused by a polyalanine expansion from 10 to 12-17 residues, located at the N-terminus of the poly(A)-binding protein nuclear 1 (PABPN1). A distinct pathological hallmark of OPMD is the presence of filamentous intranuclear aggregates in patients' skeletal muscle cells. Wildtype PABPN1 protein is expressed ubiquitously and was shown to be mostly concentrated in discrete nuclear domains called 'speckles'. Using an established cell- culture model, we show that most mutant PABPN1- positive (alanine expanded form) intranuclear aggregates are structures distinct from intranuclear speckles. In contrast, the promyelocytic leukaemia protein, a major component of nuclear bodies, strongly colocalized to intranuclear aggregates of mutant PABPN1. Wildtype PABPN1 can freely shuttle between the nucleus and cytoplasm. We determined whether the nuclear environment is necessary for mutant PABPN1 inclusion formation and cellular toxicity. This was achieved by inactivating the mutant PABPN1 nuclear localization signal and by generating full-length mutant PABPN1 fused to a strong nuclear export sequence. A green fluorescence protein tag inserted at the N-terminus of both wildtype PABPN1 (ala10) and mutant PABPN1 (ala17) proteins allowed us to visualize their subcellular localization. Targeting mutant PABPN1 to the cytoplasm resulted in a significant suppression of both intranuclear aggregates formation and cellular toxicity, two histological consequences of OPMD. Our results indicate that the nuclear localization of mutant PABPN1 is crucial to OPMD pathogenesis.     

Epidermal growth factor receptor and notch pathways participate in the tumor suppressor function of gamma-secretase

Gamma-secretase, a unique aspartyl protease, is required for the regulated intramembrane proteolysis of Notch and APP, pathways that are implicated, respectively, in the pathogenesis of cancer and Alzheimer disease. However, the mechanism whereby reduction of gamma-secretase causes tumors such as squamous cell carcinoma (SCC) remains poorly understood. Here, we demonstrate that gamma-secretase functions in epithelia as a tumor suppressor in an enzyme activity-dependent manner. Notch signaling is down-regulated and epidermal growth factor receptor (EGFR) is activated in SCC caused by genetic reduction of gamma-secretase. Moreover, the level of EGFR is inversely correlated with the level of gamma-secretase in fibroblasts, suggesting that the up-regulation of EGFR stimulates hyperproliferation in epithelia of mice with genetic reduction of gamma-secretase. Supporting this notion is our finding that the proliferative response of fibroblasts lacking gamma-secretase activity is more sensitive when challenged by either EGF or an inhibitor of EGFR as ompared with wild type cells. Interestingly, the up-regulation of EGFR is independent of Notch signaling, suggesting that the EGFR pathway functions in parallel with Notch in the tumorigenesis of SCC. Collectively, our results establish a novel mechanism linking the EGFR pathway to the tumor suppressor role of gamma-secretase and that mice with genetic reduction of gamma-secretase represent an excellent rodent model for clarifying pathogenesis of SCC and for testing therapeutic strategy to ameliorate this type of human cancer.     

Osteopontin increases the expression of β1, 4-Galactosyltransferase-I and promotes adhesion in human RL95-2 cells

Beta1, 4-Galactosyltransferase-I (β1, 4-GalT-I), which transfers galactose from UDP-Gal to N-acetylglucosamine and N-acetylglucosamine-terminated oligosaccharides of N- and O-linked glycans in a β(1-4) linkage, plays a critical role in cell adhesion, sperm-egg recognition, neurite growth, and tumor cell migration and invasion. Our previously experiments also show that β1, 4-GalT-I was up-regulated by estrogens and some important cytokines of embryo implantation especially Interleukin-1 (IL-1), TGF-α and Leukemia Inhibitory Factor (LIF) in endometrial cells. In the receptive phase human uterus, osteopontin (OPN) is the most highly up-regulated extracellular matrix/adhesion molecule/cytokine. In this study, we demonstrated the correlated expression of OPN and β1, 4-GalT-I in endometrium during early pregnancy, and recombinant human OPN (rhOPN) protein induced the β1, 4-GalT-I up-regulation in RL95-2 cells. Inhibition of MEK/ERK, PI3K/AKT and NF-κB suppressed rhOPN-induced β1, 4-GalT-I expression. In addition, rhOPN promoted the adhesion of blastocysts cells in vitro in β1, 4-GalT-I-dependent manner. Moreover, the adhesion is greatly inhibited when β1, 4-GalT-I was blocked with the specific antibody. Taken together, our data suggest that β1, 4-GalT-I provides a mechanism to bridge embryo to endometrium during implantation.     

The Effect of Attractive Interactions and Macromolecular Crowding on Crystallins Association

In living systems proteins are typically found in crowded environments where their effective interactions strongly depend on the surrounding medium. Yet, their association and dissociation needs to be robustly controlled in order to enable biological function. Uncontrolled protein aggregation often causes disease. For instance, cataract is caused by the clustering of lens proteins, i.e., crystallins, resulting in enhanced light scattering and impaired vision or blindness. To investigate the molecular origins of cataract formation and to design efficient treatments, a better understanding of crystallin association in macromolecular crowded environment is needed. Here we present a theoretical study of simple coarse grained colloidal models to characterize the general features of how the association equilibrium of proteins depends on the magnitude of intermolecular attraction. By comparing the analytic results to the available experimental data on the osmotic pressure in crystallin solutions, we identify the effective parameters regimes applicable to crystallins. Moreover, the combination of two models allows us to predict that the number of binding sites on crystallin is small, i.e. one to three per protein, which is different from previous estimates. We further observe that the crowding factor is sensitive to the size asymmetry between the reactants and crowding agents, the shape of the protein clusters, and to small variations of intermolecular attraction. Our work may provide general guidelines on how to steer the protein interactions in order to control their association.     

渤海东部海域秋季底层游泳动物种类组成及群落多样性

根据2014年9月在渤海东部海域(37°40'—38°20'N、120°00'—121°20'E)进行的游泳动物底拖网调查数据,应用相对重要性指数、生物多样性指数、群落结构多元统计分析等方法对该海域游泳动物种类组成及群落多样性特征进行了分析。结果表明:调查海域共捕获游泳动物54种,以头足类枪乌贼(Loliolus spp.)为绝对优势种;游泳动物资源密度为28.82kg/h和5166尾/h。游泳动物种类Margalef丰富度指数(D)的变化范围为1.99—3.67,Shannon-Wiener多样性指数(H')变化范围为0.89—2.28,Pielou均匀度指数(J')变化范围为0.29—0.69。群落结构多元统计分析表明,渤海东部海域游泳动物群落结构以60%的相似性可划分为3个组群:长岛群岛海域组群(A组群)、龙口海域组群(B组群)和蓬莱海域组群(C组群);ANOSIM分析表明,组群之间差异极显著,两两之间差异亦极显著。受增殖放流影响,C组群资源密度和种类多样性均保持较高的水平,而A、B组群人为活动频繁,过度捕捞严重,对海域生态环境和渔业资源整体结构的破坏较大。