Biosynthesis of the cell surface sialomucin complex of ascites 13762 rat mammary adenocarcinoma cells from a high molecular weight precursor

Cell surfaces of metastatic 13762 ascites rat mammary adenocarcinoma cells are covered with a sialomucin complex composed of the high Mr sialomucin ASGP-1 (approximately 600,000) and a concanavalin A-binding, integral membrane glycoprotein ASGP-2 (120,000). Antibodies prepared against ASGP-2 and deglycosylated ASGP-1 react on immunoblots of ascites cells or their isolated microvilli with the Mr = 120,000 species and the high Mr sialomucin, respectively. No cross-reactivity was observed. Under complex dissociating conditions, anti-ASGP-2 immunoprecipitated primarily components of Mr = 120,000 and about 400,000 from lysates of cells labeled for 1 h with mannose, glucosamine, and threonine. Under similar conditions, anti-ASGP-1 immunoprecipitated the Mr = 400,000 component and a second major labeled component of about 330,000. Pulse-chase labeling with 35S-labeled amino acids followed by immunoprecipitation with anti-ASGP-2 indicated a precursor-product relationship for the Mr = 400,000 component, designated pSMC-1 (precursor, sialomucin complex), and ASGP-2. Similar pulse-chase analyses of threonine-labeled cells using anti-ASGP-1 showed equivalent amounts of immunoprecipitated pSMC-1 and pSMC-2, both of which disappeared with kinetics similar to those observed for pSMC-1 immunoprecipitated with anti-ASGP-2. A precursor-product relationship of both pSMC-1 and pSMC-2 to ASGP-1 was suggested by combined precipitations with anti-ASGP-1 and peanut agglutinin, which precipitates ASGP-1 specifically. Immunoblot and lectin blot analyses indicated that pSMC-1 and pSMC-2 from the immunoprecipitates bind anti-ASGP-2, anti-ASGP-1, and concanavalin A. Moreover, these three components can also be labeled with mannose; the mannose was removed from 30-min pulse-labeled anti-ASGP-2 immunoprecipitates by incubation with endo-beta-N-acetylglucosaminidase H, indicating the presence of only high mannose N-linked oligosaccharides in pSMC-1. One-dimensional peptide maps of 35S-labeled pSMC-1 and Mr = 120,000 ASGP-2 showed several corresponding bands. These results indicate that both ASGP-1 and ASGP-2 can be synthesized from a common high Mr precursor. We propose that complex is formed from pSMC-1 by proteolytic cleavage to yield Mr = 120,000 ASGP-2 plus the precursor to ASGP-1 early in the transit pathway from the endoplasmic reticulum to the cell surface.     

Incorporation of the transmembrane hydrophobic domain of glycophorin into small unilamellar phospholipid vesicles Ion Flux studies

Human erythrocyte glycophorin is one of the best characterized integral membrane proteins. Reconstitution of the membrane-spanning hydrophobic segment of glycophorin (the tryptic insoluble peptide released when glycophorin is treated with trypsin) with liposomes results in the production of freeze-fracture intrabilayer particles of 80 Å diameter (Segrest, J.P., Gulik-Krzywicki, T. and Sardet, C. (1974) Proc. Natl. Acad. Sci. U.S.A. 71, 3294–3298), with particles appearing at or above a tryptic insoluble peptide concentration of 4 mmol per mol phosphatidylcholine. In the present study, increasing concentrations of tryptic insoluble peptide were added to sonicated small unilamellar egg phosphatidylcholine vesicles and the rate of efflux of ²²Na⁺ was examined by rapid (30 s) gel filtration on Sephadex G-50. Below a concentation of 3–5 mmol tryptic insoluble peptide/mol phosphatidylcholine, ²²Na⁺ efflux occurs at a constant slow rate at given tryptic insoluble peptide concentrations. Above a concentration of 3–5 mM, the rate of efflux is biphasic at given tryptic insoluble peptide concentrations, exhibiting both an initial fast and a subsequent slow component. On the basis of graphic and computer curve-fitting analysis, with increasing tryptic insoluble peptide concentration, the rate of the slow component reaches a plateau at a tryptic insoluble peptide concentration of 3–5 mM and remains essentially constant until much higher concentrations are reached; the fast component increases linearly with increasing tryptic insoluble peptide concentration well beyond 5 mM. The most consistent interpretation of this data is as follows. The slow ²²Na⁺ efflux component is due to perturbations of small unilamellar vesicle integrity by tryptic insoluble peptide monomers. At a tryptic insoluble peptide concentration of 3–5 mmol/mol, a critical concentration is reached following which there is intrabilayer tryptic insoluble peptide self-association. The fast ²²Na⁺ efflux component is due to the increasing presence of tryptic insoluble peptide self-associated multimers the 80-Å particles seen by freeze-fracture electron microscopy) which results in a significantly larger bilayer defect than do tryptic insoluble peptide monomers. The failure of complete saturation of efflux by the fast component is ascribed to the presence of two populations of small unilamellar vesicles, some of which contain tryptic insoluble peptide multimers and some of which do not.Addition of cholesterol to the tryptic insoluble peptide/phosphatidylcholine vesicles decreases the rate of ²²Na⁺ efflux by inhibiting primarily the fast component. Freeze-fracture electron microscopy indicates that the presence of cholesterol has no effect on the size, number or distribution of 80-Å intra-bilayer particles in the tryptic insoluble peptide/phosphatidylcholine vesicles. These results are consistent with a mechanism to explain the fast Na⁺ efflux component involving protein-lipid boundary perturbations.Efflux of ⁴⁵Ca²⁺ from phosphatidylcholine vesicles is also enhanced by incorporation of tryptic insoluble peptide, but only if divalent cations (Ca²⁺ or Mg²⁺) are present in the external bathing media as well as inside the sonicated vesicles. If monovalent Na⁺ only is present in the bathing media no ⁴⁵Ca²⁺ efflux is seen. Under conditions where ⁴⁵Ca²⁺ efflux is seen, both a fast and a slow component are present, although both appear lower than corresponding rate constants for ²²Na⁺ efflux. These results suggest a coordinated mechanism for ion efflux induced by tryptic insoluble peptide and, together with the ²²Na⁺ efflux studies, may have mechanistic implications for the transbilayer phospholipid exchange (flip-flop) suggesed to be induced at glycophorin/phospholipid interfaces (de Kruiff, B., van Zoelen, E.J.J. and van Deenen, L.L.M. (1978) Biochim. Biophys. Acta 509, 537–542).     

A global invasion by the thrip,Frankliniella occidentalis: Current virus vector status and its management

Western flower thrip, Frankliniella occidentalis (Pergande), is among the most economically important agricultural pests globally, attacking a wide range of vegetable and horticultural crops. In addition to causing extensive crop damage, the species is notorious for vectoring destructive plant viruses, mainly belonging to the genera Orthotospovirus, Ilarvirus, Alphacarmovirus and Machlomovirus. Once infected by orthotospoviruses, thrips can remain virulent throughout their lifespan and continue transmitting viruses to host plants when and wherever they feed. These irruptive viral outbreaks in crops will permanently disrupt functional integrated pest management systems, and typically require a remedial treatment involving insecticides, contributing to further development of insecticide resistance. To mitigate against this continuing cycle, the most effective management is early and comprehensive surveillance of the pest species and recognition of plant viruses in the field. This review provides information on the pest status of F. occidentalis, discusses the current global status of the viruses vectored by this thrip species, examines the mechanisms involved in transmitting virus‐induced diseases by thrips, and reviews different management strategies, highlighting the potential management tactics developed for various cropping systems. The early surveillance and the utilization of potential methods for control of both F. occidentalis and viruses are proposed.     

Physiological functions of Wilms’ tumor 1-associating protein and its role in tumourigenesis

The Wilms’ tumor-associated gene WT1 encodes a tumor suppressor gene, which is implicated in renal differentiation and development of adult urogenital system. Wilms’ tumor 1-associating protein (WTAP) is initially identified as a nuclear protein that specifically interacts with WT1 in both in vitro and in vivo assays. WTAP is ubiquitously expressed in different tissues and various growth periods, and its expression is involved in cell cycle, RNA splicing and stabilization, N6-methyladenosine RNA modification, cell proliferation, and apoptosis as well as embryonic development. In the present review, we aimed to summarize the functions of WTAP in various physiological and pathological processes, in particular with regard to the current knowledge about the role of WTAP in tumorigenesis of different cancers.     

Seasonal dynamics of soil fauna in the subalpine and alpine forests of west Sichuan at different altitudes

The climate (especially temperature) often plays an important role in the structure, function as well as composition of soil organisms in different latitudes and altitudes. As one of the essential components of soil ecosystem, soil faunal community not only lays their roles as soil engineer in material cycling and energy flow, but also acts as the sensitive bio-indicator to environmental change. However, little information has been available on the responses of soil faunal community to the changed environment at different altitudes and seasons. In order to understand the seasonal dynamics of soil faunal diversity under different forests with varying altitudes, three fir (Abies faxoniana) forests were selected covering a 600 m vertical transition zone. The primary fir forest at 3600 m (A1) of altitude, mixed fir and birch forest at 3300 m (A2) of altitude, and secondary fir forest at 3000 m (A3) of altitude are representative forests in the subalpine and alpine region of west Sichuan. A 2 years study was conducted in the three subalpine and alpine forests from May in 2009 until October in 2010. Soil samples were collected in both the soil organic layer and mineral soil layer. Soil macro-fauna were picked up by hand in the fields. Meso/micro-fauna and damp living fauna were separated and collected from the soil samples by Baermann and Tullgren methods in laboratory, respectively. A total of 74,827 individuals were collected in the 2 years, belonging to seven phyla, 16 classes, 31 orders and 125 families by preliminary identification. Similar dominant groups were detected in different forests at different altitudes, consisting of Spirostreptida, Formicidae, Staphylinidae, Hesperinidae, Onychiuridae, Isotomidae, Oribatuloidae, Alicoragiidae, Secernentea, and Adenophorea. In contrast, the ordinary species of macro-fauna and the ratios of Acarina to Collembolan were obviously different. For instance, the ordinary species were dominated by Cydmaenidae and Mycetophilidae at the A1, Scaphidiidae and Helicinidae at the A2, and Lumbricida and Agelenidae at the A3, respectively. Both the individual density and the number of soil faunal groups were significantly higher in soil organic layer than those in mineral soil layer. The density and group of macro-, meso- and micro-fauna in different forests showed the order as A2 > A1 > A3, but the density of damp living fauna showed the order as A1 > A2 > A3. The functional groups of macro-fauna were mainly dominated by saprozoic. The highest density and group of macro-fauna was observed in August, while the highest value of meso/micro-fauna was detected in October. In addition, the Jacard similarity indices showed that the composition and structure of soil fauna were similar in the different forests varied with altitudes, but the Shannon–Wiener indices were significantly different. The highest values of Shannon–Wiener indices were observed in October at both the A1 and A3, and in August at the A2. The results suggested that soil faunal community kept a high diversity in the subalpine and alpine forests of west Sichuan, and their structures were significantly affected by the variation of altitudes, which provided important scientific evidences for understanding the ecological processes in the subalpine and alpine coniferous forests.     

A modified cornish pasty method for ex ovo culture of the chick embryo development

Chick embryos grown in ex ovo culture by the modified Cornish pasty method reported in Nagai, Lin and Sheng in this issue.     

Dissecting the molecular mechanism of drosophila odorant receptors through activity modeling and comparative analysis

To gain insight into the molecular mechanism of odorant receptors (ORs) in Drosophila species, we developed a Quantitative Structure Activity Relationship (QSAR) model that predicts experimentally measured electrophysiological activities between 24 D. melanogaster ORs and 108 odorants. Although the model is limited by the tested odorants,analyzing the model allowed dissection of specific topological and chemical properties necessary for an odorant to elicit excitatory or inhibitory receptor response. Linear odorants with five to eight nonhydrogen atoms at the main chain and hydrogen‐bond acceptor and/or hydrogen‐bond donor at its ends were found to stimulate strong excitatory response. A comparative sequence analysis of 90 ORs in 15 orthologous groups identified 15 putative specificity‐determining residues (SDRs) and 15 globally conserved residues that we postulate as functionally key residues. Mapping to a model of secondary structure resulted in 14 out of 30 key residues locating to the transmembrane (TM) domains. Twelve residues, including six SDRs and six conserved residues, are located at the extracellular halves of the TM domains. Combining the evidence from the QSAR modeling and the comparative sequence analysis, we hypothesize that the Drosophila ORs accept odorants into a binding pocket located on the extracellular halves of its TM domains. The QSAR modeling suggests that the binding pocket is around 15 Å in depth and about 6 Å in width. Twelve mainly polar or charged key residues, both SDRs and conserved, are located inthis pocket and postulated to distinguish docked odorants via primarily geometry fitting and hydrogen‐bond interaction. Proteins 2010. © 2009 Wiley‐Liss, Inc.     

Colonization of endophyte Acremonium sp. D212 in Panax notoginseng and rice mediated by auxin and jasmonic acid

Endophytic fungi can be beneficial to plant growth. However, the molecular mechanisms underlying colonization of Acremonium spp. remain unclear.In this study, a novel endophytic Acremonium strain was isolated from the buds of Panax notoginseng and named Acremonium sp. D212. The Acremonium sp. D212 could colonize the roots of P. notoginseng,enhance the resistance of P. notoginseng to root rot disease, and promote root growth and saponin biosynthesis in P. notoginseng. Acremonium sp. D212 could secrete indole-3-acetic acid(IAA) and jasmonic acid(JA), and inoculation with the fungus increased the endogenous levels of IAA and JA in P. notoginseng. Colonization of the Acremonium sp. D212 in the roots of the rice line Nipponbare was dependent on the concentration of methyl jasmonate(Me JA)(2–15 μmol/L) and 1-naphthalenacetic acid(NAA)(10–20 μmol/L). Moreover, the roots of the JA signaling-defective coi1-18 mutant were colonized by Acremonium sp. D212 to a lesser degree than those of the wild-type Nipponbare and mi R393 boverexpressing lines, and the colonization was rescued by Me JA but not by NAA. It suggests that the cross-talk between JA signaling and the auxin biosynthetic pathway plays a crucial role in the colonization of Acremonium sp. D212 in host plants.