Variation in Efficacy of Isolates of the Fungus ARF Against the Soybean Cyst Nematode Heterodera glycines

An unnamed fungus, designated ARF, that parasitizes eggs and sedentary stages of cyst nematodes is a potential biological control agent of Heterodera glycines. The objectives of this study were to determine whether ARF isolates differ in their ability to suppress nematode numbers in soil and to compare the efficacy of ARF in heat-treated and native soil. The effectiveness of 11 ARF isolates was compared by introducing homogenized mycelium into heat-treated soil. Soybean seedlings were transplanted into pots containing fungus-infested soil and inoculated with H. glycines. After 30 or 60 days, the number of nematodes and the percentage of parasitized eggs were determined. Three isolates (907, 908, and TN14), which were previously reported to be weak egg parasites in vitro, consistently suppressed nematode numbers by 50% to 100%. Of the isolates previously reported to be aggressive egg parasites, four (903, BG2, MS3, and TN12) reduced nematode numbers by 56% to 69% in at least one experimental trial, but the other four had no effect on nematode numbers. When the efficacy of isolate TN14 was tested in heat-treated and native soil, nematode suppression was greater in the heat-treated soil in only one of two trials. In both soil treatments, nematode numbers were reduced by more than 60%. We conclude that virulence toward nematode eggs in vitro is a poor indicator of effectiveness of an ARF isolate in soil, and that the presence of soil microbes may reduce, but does not completely inhibit, activity of isolate TN14.     

ACAP4 protein cooperates with Grb2 protein to orchestrate epidermal growth factor-stimulated integrin β1 recycling in cell migration

ARF6 GTPase is an important regulator of membrane trafficking and actin-based cytoskeleton dynamics active at the leading edge of migrating cells. The integrin family heterodimeric transmembrane proteins serve as major receptors for extracellular matrix proteins, which play essential roles in cell adhesion and migration. Our recent proteomic analyses of ARF6 effectors have identified a novel ARF6 GTPase-activating protein, ACAP4, essential for EGF-induced cell migration. However, molecular mechanisms underlying ACAP4-mediated cell migration have remained elusive. Here, we show that ACAP4 regulates integrin β1 dynamics during EGF-stimulated cell migration by interaction with Grb2. Our biochemical study shows that EGF stimulation induces phosphorylation of tyrosine 733, which enables ACAP4 to bind Grb2. This interaction of ACAP4 with Grb2 regulates integrin β1 recycling to the plasma membrane. Importantly, knockdown of ACAP4 by siRNA or overexpression of ACAP4 decreased recycling of integrin β1 to the plasma membrane and reduced integrin-mediated cell migration. Taken together, these results suggest a novel function for ACAP4 in the regulation of cell migration through controlling integrin β1 dynamics.     

Glycogen synthase kinase-3 acts upstream of ADP-ribosylation factor 6 and Rac1 to regulate epithelial cell migration

Cell sheet movement during epithelial wound closure is a complex process involving collective cell migration. We have found that glycogen synthase kinase-3 (GSK-3) activity is required for membrane protrusion and crawling of cells at the wound edge and those behind it in wounded Madin-Darby canine kidney (MDCK) epithelial cell monolayers. RNA interference-based silencing of GSK-3alpha and GSK-3beta expression also results in slowed cell sheet migration, with the effect being more pronounced with knockdown of GSK-3beta. Both GSK-3alpha and GSK-3beta are in activated states during the most active phase of cell migration. In addition to having a positive control or permissive, rather than negative, function in MDCK cell migration, GSK-3 appears to act upstream of the small GTPases ADP-ribosylation factor 6 (ARF6) and Rac1. Expression of constitutively active ARF6 restores a protrusive, migratory phenotype in cells treated with GSK-3 inhibitors. It does not, however, restore to normal levels the directional polarization of cells behind the wound edge toward the wound area, implying the existence of a separate ARF6-independent branch of the GSK-3 pathway that regulates proper wound-directed polarization of these cells. Finally, inhibition of GSK-3 also strongly reduces activation of Rac1 and cell scatter in response to hepatocyte growth factor/scatter factor, which triggers dispersal and migration of cells in monolayer culture as fibroblast-like individual cells, a mode of epithelial cell motility distinct from the collective migration of wound closure.     

A set of Arabidopsis thaliana miRNAs involve shoot regeneration in vitro

Plant miRNAs, the critical regulator of gene expression, involve many development processes in vivo. However, the roles of miRNAs in plant cell proliferation and redifferntiation in vitro remain unknown. To determine better the molecular mechanism of these processes, we have recently reported that a set of miRNAs with different expression patterns between cells of totipotent and non-totipotent Arabidopsis calli. Some of these were specifically up- or downregulated during callus formation or shoot regeneration, and other development. Among them, miR160, and one of its target genes, ARF10, regulated Arabidopsis in vitro shoot regeneration via WUS, CLV3 and CUC1/2. The miR160-overexpressing, 35S transgenic lines, exhibited reduced shoot regeneration efficiency. The mARF10, a miR160-resistant form of ARF10, showed a high level of shoot regeneration ability. In the transgenic, expression of the above shoot meristem-specific genes was elevated, which is consistent with the improved shoot regeneration. In contrast, the ARF10 deficient knockout mutant produced fewer regenerated shoot. However, overexpressors of ARF10 were only marginally more efficient than the wild type with the respect to shoot regeneration. Our observation strongly supports that proper shoot regeneration from in vitro cultured cells requires the miR160-directed negative influence of ARF10. The enhanced expression of ARF10 is likely to have contributed to the improved regeneration ability.     

The OSBP-related proteins: a novel protein family involved in vesicle transport,cellular lipid metabolism,and cell signalling

Proteins/genes showing high sequence homology to the mammalian oxysterol binding protein (OSBP) have been identified in a variety of eukaryotic organisms from yeast to man. The unifying feature of the gene products denoted as OSBP-related proteins (ORPs) is the presence of an OSBP-type ligand binding (LB) domain. The LB domains of OSBP and its closest homologue bind oxysterols, while data on certain other family members suggest interaction with phospholipids. Many ORPs also have a pleckstrin homology (PH) domain in the amino-terminal region. The PH domains of the family members studied in detail are known to interact with membrane phosphoinositides and play an important role in the intracellular targeting of the proteins. It is plausible that the ORPs constitute a regulatory apparatus that senses the status of specific lipid ligands in membranes, using the PH and/or LB domains, and mediates information to yet poorly known downstream machineries. Functional studies carried out on the ORP proteins in different organisms indicate roles of the gene family in diverse cellular processes including control of lipid metabolism, regulation of vesicle transport, and cell signalling events.     

Effect of cell cycle inhibitor p19ARF on senescence of human diploid cell

To investigate the effect of cell cycle inhibitor p19ARF on replicative senescence of human diploid cell, recombinant p19ARF eukaryotic expression vector was constructed and p19ARF gene was transfected into human diploid fibroblasts (WI-38 cells) by liposome-mediated transfection for overexpression. Then, the effects of p19ARF on replicative senescence of WI-38 cells were observed. The results re- vealed that, compared with control cells, the WI-38 cells in which p19ARF gene was introduced showed significant up-regulation of p53 and p21 expression level, decrease of cell generation by 10 12 generations, decline of cell growth rate with cell cycle being arrested at G1 phase, increase of positive rate of senescent marker SA-β-gal staining, and decrease of mitochondrial membrane potential. The morphology of the transfected fibroblasts presented the characteristics changes similar to senescent cells. These results indicated that high expression of p19ARF may promote the senescent process of human diploid cells.     

Mating type differentiation in tetrahymena thermophila: Characterization of the delayed refeeding effect and its implications concerning intranuclear coordination

Mating type determination in Tetrahymena thermophila involves developmentally programmed, heritable alterations of the macronucleus, localized to the mtd locus. This determination can be predictably controlled by the environmental conditions during macronuclear development, eg, temperature and time of refeeding. In this article we have further characterized the effects of delayed refeeding on mating type determination, as revealed by the frequency of mating types among the progeny of a cross. Our results show that 1) the magnitude of this starvation effect decreases with temperature of conjugation and becomes undetectable at 18°C; 2) starvation during the interval 14 to 22 hr (after conjugation is induced at 30°C) is a necessary and sufficient condition for the induction of starvation effects; 3) relative mating type frequencies vary monotonically with nutrient concentration present during this critical period; and 4) sister macronuclei, developing under starvation conditions in the same cytoplasm, differentiate majority mating types characteristic of early or late refeeding; sister macronuclei show no apparent correlation with each other. On the basis of our observations on early and late refed cells, we propose that the composition of the newly developed macronucleus is the outcome of two key events: 1) mating type determination at the mtd locus and 2) differential molecular cloning of generally one or two autonomously replicating fragments (ARFs) of the macronuclear DNA bearing the mtd locus.     

氧化应激对Hsp90α、ARF1 细胞内定位和相互作用的影响

目的:探讨氧化应激对热休克蛋白90α(Hsp90α)与ADP-核糖基化因子1(ARF1)细胞内定位、相互作用的影响。方法:应用500μM H2O2处理HepG2细胞,建立氧化应激模型,MTT比色法检测细胞活力,Western blotting检测Hsp90α和ARF1水平,细胞免疫荧光法、免疫共沉淀检测上述蛋白在氧化应激下的分布、共定位变化和相互作用。结果:MTT比色法结果提示,随氧化应激时间延长,细胞存活力降低;Western blotting结果显示,氧化应激可提高胞内Hsp90α和ARF1蛋白水平;免疫共沉淀结果显示,随氧化应激作用时间延长,Hsp90α与ARF1相互结合增多;细胞免疫荧光结果显示,随氧化应激作用时间延长,Hsp90α与ARF1荧光强度增强,并趋于沿胞膜分布。结论:提示氧化应激影响Hsp90α和ARF1的水平、胞内分布及相互作用。