Ecto-Protein Kinase and Surface Protein Phosphorylation in PC12 Cells: Interactions with Nerve Growth Factor

Abstract: The phosphorylation of surface proteins by ectoprotein kinase has been proposed to play a role in mechanisms underlying neuronal differentiation and their responsiveness to nerve growth factor (NGF). PC 12 clones represent an optimal model for investigating the mode of action of NGF in a homogeneous cell population. In the present study we obtained evidence that PC12 cells possess ectoprotein kinase and characterized the endogenous phosphorylation of its surface protein substrates. PC12 cells maintained in a chemically defined medium exhibited phosphorylation of proteins by [γ-³²P]ATP added to the medium at time points preceding the intracellular phosphorylation of proteins in cells labeled with ³²P_i. This activity was abolished by adding apyrase or trypsin to the medium but was not sensitive to addition of an excess of unlabeled P_i. As also expected from ecto-protein kinase activity, PC12 cells catalyzed the phosphorylation of an exogenous protein substrate added to the medium, dephospho-α-casein, and this activity competed with the endogenous phosphorylation for extracellular ATP. Based on these criteria, three protein components migrating in sodium dodecyl sulfate gels with apparent molecular weights of 105K, 39K, and 20K were identified as exclusive substrates of ecto-protein kinase in PC12 cells. Of the phosphate incorporated into these proteins from extracellular ATP, 75–87% was found in phosphothreonine. The phosphorylation of the 39K protein by ecto-protein kinase did not require Mg²⁺, implicating this activity in the previously demonstrated regulation of Ca²⁺-dependent, high-affinity norepinephrine uptake in PC12 cells by extracellular ATP. The protein kinase inhibitor K-252a inhibited both intra- and extracellular protein phosphorylation in intact PC12 cells. Its hydrophilic analogue K-252b, had only minimal effects on intracellular protein phosphorylation but readily inhibited the phosphorylation of specific substrates of ecto-protein kinase in PC12 cells incubated with extracellular ATP, suggesting the involvement of ecto-protein kinase in the reported inhibition of NGF-induced neurite extension by K-252b. Preincubation of PC12 cells with 50 ng/ml of NGF for 5 min stimulated the activity of ecto-protein kinase toward all its endogenous substrates. Exposure of PC12 cells to the same NGF concentration for 3 days revealed another substrate of ecto-protein kinase, a 53K protein, whose surface phosphorylation is expressed only after NGF-induced neuronal differentiation. In the concentration range (10–100 μM) at which 6-thioguanine blocked NGF-promoted neurite outgrowth in PC12 cells, 6-thioguanine effectively inhibited the phosphorylation of specific proteins by ecto-protein kinase. This study provides the basis for continued investigation of the involvement of ecto-protein kinase and its surface protein substrates in neuronal differentiation, neuritogenesis, and synaptogenesis.     

Spring dispersal ofSitona lineatus: The use of aggregation pheromone traps for monitoring

The spring dispersal ofSitona lineatus L. (Coleoptera; Curculionidae) was investigated on a Danish farm.S. lineatus dispersed by flight in the early spring on sunny, calm days with temperatures above ca. 15°C. Two thirds of the population ofS. lineatus dispersed from perennial leguminous crops (clover and lucerne) in the first period of flight activity. The next dispersal did not occur until one month later despite several intermediate flight activity periods. The first period of dispersal occurred before the germination of the spring sown summer host crop,Vicia faba L. The field bean crop was infested in three later invasions during a period of more than three weeks. The aggregation pheromone, 4-methyl-3,5-heptanedione, had a significant effect on captures of both males and females in cone traps placed on the ground. There was no effect of the pheromone on captures in yellow sticky traps placed 1.5 m above ground. The pheromone effect is discussed in relation to behavioural observations. Both types of traps may be used in a survey system for monitoring spring dispersal ofS. lineatus and optimal timing of insecticide spraying. However, the pheromone cone traps were highly specific whereas all kinds of flying insects were caught in the yellow sticky traps, thus making the latter traps less suitable for monitoring.     

Immunolocalization of tenascin and cellular fibronectins in diverse glomerulopathies

Frozen samples of minimal change glomerulopathy (MCG), and of membranous, segmental and diffuse lupus glomerulonephritis (MGN, SGN, DLGN) were studied to assess the distribution of tenascin (Ten), and the extradomains A and B (EDA-and EDB-) and oncofetal (Onc-) isoforms of cellular fibronectin (cFn). Cryosections were immunostained by the ABC method with specific monoclonal antibodies. In MCG, mesangial Ten and EDA-cFn reactions were increased. In MGN, mesangial Ten and EDA-cFn staining was enhanced except in segmental scars; convincing reactions were seen in cases with membranous transformation; spikes stained strongly. In SGN, variably intense staining for Ten and all cFn isoforms was seen in glomerular necrosis, proliferation and crescents; parietal epithelium EDA-cFn staining was noted. In DLGN, strong and extensive mesangial Ten and EDA-cFn staining was seen as were focal EDB-and Onc-cFn reactions. Parietal cells with and without crescents stained variably with all Mabs. Obsolete glomeruli were unreactive save for rare periglomerular Ten rims. Interstitial inflammation and fibrosis in MGN, SGN and DLGN had moderate to strong Ten and EDA-cFn staining with rare traces of EDB-and Onc-cFn. We conclude that enhanced Ten and EDA-cFn is a potentially reversible response to glomerular injury whereas the expression of EDB-and Onc-cFn apparently result from necrosis and/or cellular proliferation which lead to scarring. And, while mesangial cells are the major source of these molecules, epithelial cells might also partake in their synthesis.     

Production of pectin lyase by Penicillium griseoroseum as a function of the inoculum and culture conditions

Optimum activity of an extracellular pectin lyase produced by Penicillium griseoroseum in submerged culture was after 120 h using 0.1% (w/v) citrus pectin as substrate. Sucrose at 0.1% (w/v) stimulated enzyme production and citrus pectin gave the highest activity of enzyme per unit growth.     

Localisation of acidic and basic fibroblast growth factors during mouse palate development and their effects on mouse palate mesenchyme cells in vitro

Summary The distribution of acidic and basic fibroblast growth factors (aFGF, bFGF) was mapped during mouse embryonic palate development. Generally, they localised most intensely in the basement membrane and epithelia rather than the mesenchyme. Localisation was predominantly restricted to the palatal nasal, and medial edge epithelia. Staining was particularly intense in the medial edge epithelia at the time of mid-line epithelial seam formation. Intense staining persisted in the epithelia of the degenerating seam and later in the oral and nasal epithelial triangles. Mouse embryonic palate mesenchyme (MEPM) cells cultured in vitro on a variety of substrata (on plastic, on the surface of a collagen gel and within a collagen gel) responded to treatment with aFGF or bFGF. These responses were modulated by the culture substratum. The FGFs stimulated MEPM cell proliferation on plastic and on collagen, but inhibited cell growth in collagen. The FGFs had little effect on protein production when cells were cultured on plastic, but caused a large reduction in on-collagen and incollagen cultures. This reduction was greater in collagenous than non-collagenous proteins. Generally, treatment with FGFs stimulated the production of glycosaminoglycans (GAGs), particularly hyaluronan (HA) and dermatan sulphate (DS). In addition, the size class of HA was shifted to a higher molecular weight form. These data indicate that aFGF and bFGF may play a role in modulating mesenchymal cell matrix biosynthesis, so facilitating palatal epithelial seam degeneration. Correspondence to: M.W.J. Ferguson     

Human pluripotent stem cell-derived extracellular vesicles: From now to the future

Extracellular vesicles (EVs) are nanometric particles that enclose cell-derived bioactive molecules in a lipid bilayer and serve as intercellular communication tools. Accordingly, in various biological contexts, EVs are reported to engage in immune modulation, senescence, and cell proliferation and differentiation. Therefore, EVs could be key elements for potential off-the-shelf cell-free therapy. Little has been studied regarding EVs derived from human pluripotent stem cells (hPSC-EVs), even though hPSCs offer good opportunities for induction of tissue regeneration and unlimited proliferative ability. In this review article, we provide an overview of studies using hPSC-EVs, focusing on identifying the conditions in which the cells are cultivated for the isolation of EVs, how they are characterized, and applications already demonstrated. The topics reported in this article highlight the incipient status of the studies in the field and the significance of hPSC-EVs’ prospective applications as PSC-derived cell-free therapy products.     

Dispersal patterns and chromatic response of Scaphoideus titanus Ball (Homoptera Cicadellidae), vector of the phytoplasma agent of grapevine flavescence dorée

Abstract 1 Scaphoideus titanus Ball, a nearctic leafhopper introduced into Europe in the 1950s, is known to be the vector of the phytoplasma agent of flavescence dorée (FD), a persistent disease of grapevine. Knowledge of its dispersal patterns is thus very important to prevent disease outbreaks. 2 Yellow sticky traps were used to study the seasonal flight activity of S. titanus, its vertical flight, its movement outside the vineyard and the influence of plant density. Sticky traps of different colours (yellow, red, blue, and white) were also compared. The behaviour of males and females was tested for all those conditions. 3 Abundance was greater in normal than in low plant density conditions, and a positive relationship was found between number of plants per square metre and presence of S. titanus. Leafhoppers did not appear capable of spreading significantly outside a vineyard. Few individuals were trapped above the canopy. Red sticky traps caught more individuals than white, yellow or blue, with the latter showing a poor attractiveness. Sex ratio was almost always male biased. 4 Scaphoideus titanus is monophagous and appears incapable of great dispersal away from its host plant, and females are less likely to fly than males. Further studies on the influence of different factors on the behaviour of this leafhopper are suggested.     

粘质赛氏菌胞外蛋白酶的提取、纯化及部分性质研究

经过硫酸铵３０％～５０％分级沉淀、二步柱层析可获聚丙烯酰胺凝胶电泳均一的粘质赛氏菌胞外蛋白酶制品,收率可达５３％,并制备了酶的结晶,该酶以ＳｅｐｈａｄｅｘＧ１００柱层析及ＳＤＳ－ＰＡＧＥ测得分子量约为８１０００,该酶的最适ｐＨ为７．０,最适温度为４５℃,Ｚｎ２＋、Ｍｎ２＋、Ｆｅ２＋、Ｃｕ２＋、Ｃｏ２＋等重金属离子不同程度地抑制酶活性。     

Laminin and Neuropeptide Y Are Increased by Synapsin Transfection in Cultured NG108-15 Neuroblastoma/Glioma Hybrid Cells

Abstract: We have investigated the presence and expression of laminin and neuropeptide Y (NPY) in several NG108-15 cell lines transfected with synapsin Ib, IIa, or IIb. The content of laminin, a basal membrane glycoprotein that promotes adhesion and induces neurite outgrowth and neuronal differentiation, was increased in all transfected cell lines examined. In cells that were chemically differentiated with prostaglandin E₁ plus 3-isobutyl-1-methylxanthine, laminin levels were increased even further. The content of NPY, suggested to be a neurotransmitter/neuromodulator in peripheral sympathetic neurons as well as in central neurons, was also increased in all transfected cell lines examined. Immunohistochemical analysis combined with confocal laser microscopy showed that NPY staining was granular and very often enriched in neuritic varicosities. The distribution and the staining pattern of NPY were consistent with storage of NPY in large dense-cored vesicles. The results indicate that, in differentiated neurons, the synapsins increase the levels of a neuropeptide transmitter stored in large dense-cored vesicles and of an extracellular matrix protein associated with neuronal maturation.     

Intrasynaptosomal Free Mg2+ Concentration Measured with the Fluorescent Indicator Mag-Fura-2: Modulation by Na+ Gradient and by Extrasynaptosomal ATP

Abstract: Synaptosomes can be loaded with mag-fura-2 without significant perturbation of their ATP content by incubation for 10 min at 37°C with 10 µM mag-fura-2 acetoxymethyl ester in Hanks'-HEPES buffer (pH 7.45). The intrasynaptosomal free Mg²⁺ concentration ([Mg²⁺]_i) was found to be dependent on external Mg²⁺ concentration, increasing from 0.8 to 1.25 mM when the concentration of Mg²⁺ in the incubation medium increased from 1 to 8 mM. Dissipation of the Na⁺ gradient across the plasma membrane of synaptosomes by treatment with the Na⁺ ionophore monensin (0.2 mM) or with veratridine (0.2 mM) and ouabain (0.6 mM) produced a moderate increase of [Mg²⁺]_i, from 1.0 to 1.2–1.3 mM in an incubation medium containing 5 mM Mg²⁺. Plasma membrane depolarization by incubation of synaptosomes in a medium containing 68 mM KCl and 68 mM NaCl had no effect on [Mg²⁺]_i. Reversal of the Na⁺ gradient by incubation of synaptosomes in a medium in which external Na⁺ was replaced by choline increased [Mg²⁺]_i up to 1.6 and 2.2 mM for extrasynaptosomal Mg²⁺ concentrations of 1 and 8 mM, respectively. We conclude that a Na⁺/Mg²⁺ exchange operates in the plasma membrane of synaptosomes. In the presence of Mg²⁺ in the incubation medium, extrasynaptosomal ATP, but not ADP or adenosine, increased [Mg²⁺]_i from 1.1 ± 0.1 up to 1.6 ± 0.1 mM. The nonhydrolyzable ATP analogue adenosine 5′-(βγ-imido)triphosphate antagonized the effect of ATP, but had no effect by itself on [Mg²⁺]_i. It is concluded that Mg²⁺ transport across the plasma membrane of synaptosomes is modulated by the activity of an ecto-ATPase or an ecto-protein kinase.