Subcellular compartmentalization has become an important theme in cell signaling such as spatial regulation of Ras by RasGRP1 and MEK/ERK by Sef. Here, we report spatial regulation of Raf kinase by RKTG (Raf kinase trapping to Golgi). RKTG is a seven-transmembrane protein localized at the Golgi apparatus. RKTG expression inhibits EGF-stimulated ERK and RSK phosphorylation, blocks NGF-mediated PC12 cell differentiation, and antagonizes Ras-and Raf-1-stimulated Elk-1 transactivation. Through interaction with Raf-1, RKTG changes the localization of Raf-1 from cytoplasm to the Golgi apparatus, blocks EGF-stimulated Raf-1 membrane translocation, and reduces the interaction of Raf-1 with Ras and MEK1. In RKTG-null mice, the basal ERK phosphorylation level is increased in the brain and liver. In RKTG-deleted mouse embryonic fibroblasts, EGF-induced ERK phosphorylation is enhanced. Collectively, our results reveal a paradigm of spatial regulation of Raf kinase by RKTG via sequestrating Raf-1 to the Golgi apparatus and thereby inhibiting the ERK signaling pathway.R af kinase relays the signals from Ras to MEK (MAPK and ERK kinase) and ERK/MAPK (1, 2). This pathway regulates many fundamental cellular functions, including cell proliferation, apoptosis, differentiation, motility, and metabolism, and is implicated in many human diseases including cancer (3). In the Ras/Raf/MEK/ ERK signaling cascade, several players within this pathway are exquisitely regulated by subcellular compartmentalization (4, 5). Kinase suppressor of Ras (KSR) is a scaffold protein that coordinates the assembly of multiprotein MAPK complex in plasma membrane (6). ␤-Arrestin could also function as a scaffold protein to target the MAPK protein complex to early endosome (7). Different lipid anchors are able to shuttle Ras between different membrane compartments to modulate subcellular Ras signaling (8-10). In T lymphocyte, Ras could be activated in situ on the Golgi apparatus via the Ras exchange factor RasGRP1, which is activated by phospholipase C (11). Compartmentalized signaling of Ras/ MAPK in T lymphocyte on either plasma membrane or Golgi apparatus leads to distinct output of ERK activation and thereby determines the threshold of thymic selection (12). MEK could be targeted to endosome by a scaffold protein MP1 through adaptor p14 (13). It was also found that MEK/ERK can be recruited to the Golgi by Sef, and that such spatial regulation blocks the Ras signaling to the nucleus but not to the cytosol (14). However, how Raf is regulated in a spatial manner has not been characterized.The activity of Raf kinase is mainly regulated by phosphorylation events and scaffold proteins (1, 2). In addition, Raf could be negatively regulated by interaction with other proteins. Raf kinase inhibitor protein (RKIP) is able to interact with Raf-1, MEK, and ERK (15). RKIP blocks Raf-1 signaling to MEK by competitively disrupting the interaction between Raf-1 and MEK because both Raf-1 and MEK bind to overlapping sites in RKIP (15, 16). After stimulation of G protein...

The oxidation potentials of 19 nitrogen bases (abbreviated as B: six primary amines, five secondary amines, two tertiary amines, three anilines, pyridine, quinuclidine, and 1,4-diazabicyclo[2,2,2]octane), i.e., E(ox)(B) values in dimethyl sulfoxide (DMSO) and/or acetonitrile (AN), have been measured. Combination of these E(ox)(B) values with the acidity values of the corresponding acids (pK(HB)(+)) in DMSO and/or AN using the equation: BDE(HB)(+) = 1.37pK(HB)(+) + 23.1 E(ox)(B) + C (C equals 59.5 kcal/mol in AN and 73.3 kcal/mol in DMSO) gave estimates of solution phase homolytic bond dissociation energies of H-B(+) bonds. Gas-phase BDE values of H-B(+) bonds were estimated from updated proton affinities (PA) and adiabatic ionization potentials (aIP) using the equation, BDE(HB(+))(g) = PA + aIP - 314 kcal/mol. The BDE(HB)(+) values estimated in AN were found to be 5-11 kcal/mol higher than the corresponding gas phase BDE(HB(+))(g) values. These bond-strengthening effects in solution are interpreted as being due to the greater solvation energy of the HB(+) cation than that of the B(+*) radical cation.

The homolytic bond dissociation energies (BDEs) of the O−H bonds in DMSO solution for (a) phenol and a number of its derivatives, (b) three oximes, (c) three alcohols, (d) three hydroxylamines, and (e) two hydroxamic acids have been estimated by eq 1:  BDEHA = 1.37pK HA + 23.1E ox(A-) + 73.3 kcal/mol. For most of these hydroxylic acids, the BDEs of the O−H bonds estimated by eq 1 are within ±2 kcal/mol of the literature values in nonpolar solvents or in the gas phase. There is no reason to believe, therefore, that these BDEs are “seriously in error because of failure to correct for solvent effects” as has been claimed on the basis that BDEs in highly polar solvents estimated for the O−H bond in phenol by photoacoustic calorimetry must be so corrected.

The etiology and epidemiology of Pythium root rot in hydroponically-grown crops are reviewed with emphasis on knowledge and concepts considered important for managing the disease in commercial greenhouses. Pythium root rot continually threatens the productivity of numerous kinds of crops in hydroponic systems around the world including cucumber, tomato, sweet pepper, spinach, lettuce, nasturtium, arugula, rose, and chrysanthemum. Principal causal agents include Pythium aphanidermatum, Pythium dissotocum, members of Pythium group F, and Pythium ultimum var. ultimum. Perspectives are given of sources of initial inoculum of Pythium spp. in hydroponic systems, of infection and colonization of roots by the pathogens, symptom development and inoculum production in host roots, and inoculum dispersal in nutrient solutions. Recent findings that a specific elicitor produced by P. aphanidermatum may trigger necrosis (browning) of the roots and the transition from biotrophic to necrotrophic infection are considered. Effects on root rot epidemics of host factors (disease susceptibility, phenological growth stage, root exudates and phenolic substances), the root environment (rooting media, concentrations of dissolved oxygen and phenolic substances in the nutrient solution, microbial communities and temperature) and human interferences (cropping practices and control measures) are reviewed. Recent findings on predisposition of roots to Pythium attack by environmental stress factors are highlighted. The commonly minor impact on epidemics of measures to disinfest nutrient solution as it recirculates outside the crop is contrasted with the impact of treatments that suppress Pythium in the roots and root zone of the crop. New discoveries that infection of roots by P. aphanidermatum markedly slows the increase in leaf area and whole-plant carbon gain without significant effect on the efficiency of photosynthesis per unit area of leaf are noted. The platform of knowledge and understanding of the etiology and epidemiology of root rot, and its effects on the physiology of the whole plant, are discussed in relation to new research directions and development of better practices to manage the disease in hydroponic crops. Focus is on methods and technologies for tracking Pythium and root rot, and on developing, integrating, and optimizing treatments to suppress the pathogen in the root zone and progress of root rot.
A etiologia e a epidemiologia da podridão radicular causada por Pythium spp. em cultivo hidropônico são revisadas com ênfase em conhecimentos e conceitos considerados importantes para o manejo de doenças em estufas comerciais. A podridão radicular causada por Pythium continuamente ameaça a produtividade de diversas culturas em sistemas hidropônicos, incluindo pepino, tomate, pimentão, espinafre, alface, capuchinha, rúcula, rosa, e crisântemo. Os principais agentes causais incluem Pythium aphanidermatum, Pythium dissotocum, espécies de Pythium do grupo F e Pythium ultimum var. ultimum. São apresentadas e discutidas as princip...

Netrin-1 is a critical molecule for axonal pathfinding during embryo development, and because of its structural homology to the endothelial mitogens, it may share its effects on vascular network formation. Using an adeno-associated viral netrin-1 vector (AAV-NT-1) gene transfer, we demonstrated that netrin-1 was able to stimulate the proliferation and migration of human cerebral endothelial cells (HCECs) and human aortic smooth muscle cells (HASMCs) compared with the control (P < 0.05), and could also promote HCEC tube formation on matrigel (P < 0.05) in vitro. Moreover, netrin-1 hyperstimulation could promote focal neovascularization (P < 0.05) in the adult brain in vivo. Unlike VEGF-induced microvessel increase, netrin-1-induced newly formed vessels showed an artery-like phenotype, with an intact endothelial cell monolayer surrounded by multiple cell layers, including smooth muscle cells and an astrocyte-connected outer layer. Our findings suggest that netrin-1 plays an important role in promoting blood vessel formation in the adult rodent central nervous system, and could have broad implication in cerebrovascular development and remodeling.

Background and Purpose-Exogenous delivery of vascular endothelial growth factor gene (VEGF) may provide a useful approach to the treatment of brain ischemia. We investigated the use of a hypoxia-responsive element to control VEGF expression given for neuroprotection. Methods-Three groups (nϭ36) of mice received AAVH9-VEGF, AAVH9-lacZ, or saline injection. Five days after gene transfer, the mice underwent 45 minutes of transient middle cerebral artery occlusion (tMCAO) followed by 1 to 7 days of reperfusion. Infarct volume was determined using cresyl violet staining; neuronal injury was examined using TUNEL, cleaved caspase-3, and fluoro-Jade B staining. Results-Hypoxia-inducible factor-1 (HIF-1) was overexpressed after tMCAO in the ischemic hemisphere in the brain.Expression of lacZ, mediated by AAV-lacZ, was seen before and after tMCAO; however, AAVH9-lacZ-mediated lacZ expression was detected only after tMCAO.

Maintaining nitric oxide (NO) homeostasis is essential for normal plant physiological processes. However, very little is known about the mechanisms of NO modulation in plants. Here, we report a unique mechanism for the catabolism of NO based on the reaction with the plant hormone cytokinin. We screened for NO-insensitive mutants in Arabidopsis and isolated two allelic lines, cnu1-1 and 1-2 (continuous NO-unstressed 1), that were identified as the previously reported altered meristem program 1 (amp1) and as having elevated levels of cytokinins. A double mutant of cnu1-2 and nitric oxide overexpression 1 (nox1) reduced the severity of the phenotypes ascribed to excess NO levels as did treating the nox1 line with trans-zeatin, the predominant form of cytokinin in Arabidopsis. We further showed that peroxinitrite, an active NO derivative, can react with zeatin in vitro, which together with the results in vivo suggests that cytokinins suppress the action of NO most likely through direct interaction between them, leading to the reduction of endogenous NO levels. These results provide insights into NO signaling and regulation of its bioactivity in plants.is one of the most widespread signaling molecules in living organisms (1, 2). In plants, NO is involved in the regulation of numerous physiological processes during growth and development and is also an important modulator of disease resistance (2-4). Several laboratories discovered that NO is produced not only from nitrate/nitrite but also from L-arginine (L-Arg), which is the main substrate for NO synthesis in animals (4-6). NO is also a widespread atmospheric pollutant. Therefore, this gas not only is a pivotal player in signal transduction but also has the potential to exert significant deleterious effects by being a pollutant. As an inevitable result, increased NO levels in the atmosphere can influence multiple NO-regulated processes in organisms. Despite the wealth of information gathered from analyses of NO functioning in plants, the molecular processes underlying NO effects in plants are still largely unknown.NO differs from other signaling molecules by being reactive, lipophilic, and volatile. In fact, chemically, NO is a free radical, and such a reactive molecule is unlikely to interact specifically with a single specific receptor (3). In animals, NO appears to act through the chemical modification of targets. NO can bind to transition metals of metalloproteins (metal nitrosylation). It also can bind covalently to cysteine (S-nitrosylation) and tyrosine (tyrosine nitration) residues (3,7,8). Such specific protein modifications are emerging as key mechanistic intermediates for NO signal transduction. In plant cells, NO has also been found to regulate the activity of various target proteins through S-or metal-nitrosylation and probably through tyrosine nitration as well (9-13).Furthermore, it has been shown that NO takes part in different phytohormone signaling pathways, frequently under the control of hormonal stimuli. For instance, NO functions in auxin-induc...

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