Irreversible oxidative inactivation of protein kinase C by photosensitive inhibitor calphostin C.

Isolated protein kinase C (PKC) was irreversibly inactivated by a brief (min) incubation with calphostin C in the presence of light. This inactivation required Ca2+ either in a millimolar range in the absence of lipid activators or in a submicromolar range in the presence of lipid activators. In addition, an oxygen atmosphere was required suggesting the involvement of oxidation(s) in this inactivation process. Furthermore, PKC inactivation might involve a site-specific oxidative modification of the enzyme at the Ca(2+)-induced hydrophobic region. Physical quenchers of singlet oxygen such as lycopene, beta-carotene, and alpha-tocopherol all reduced the calphostin C-induced inactivation of PKC. In intact cells treated with calphostin C, the inactivation of PKC was rapid in the membrane fraction compared to cytosol. This intracellular PKC inactivation was also found to be irreversible. Therefore, calphostin C can bring prolonged effects for several hours in cells treated for a short time. Taken together these results suggest that the calphostin C-mediated inactivation of PKC involves a site-specific and a 'cage' type oxidative modification of PKC.     

Revisiting the use of Iprodione and Trichoderma in the integrated management of onion white rot

The biocontrol properties of Trichoderma species are well documented, but their effectiveness in antagonism of the problematic Sclerotium cepivorum, the causal agent of white rot in Allium species, appears limited with reports of significant control only relating to deliberately-mutated strains of Trichoderma. Our previous studies have indicated the possibility of using selected naturally-occurring strains of the antagonist in the suppression of other diseases; now in vitro and controlled environment in vivo studies have indicated that a degree of control of Onion White Rot is possible, and that the selected antagonist strains can be used in integrated treatments with Iprodione to good effect. The possible value of such treatments is considered in light of other approaches to the suppression of this continuing problem.     

A spatiotemporal characterization of the effect of p53 phosphorylation on its interaction with MDM2

The interaction of p53 and MDM2 is modulated by the phosphorylation of p53. This mechanism is key to activating p53, yet its molecular determinants are not fully understood. To study the spatiotemporal characteristics of this molecular process we carried out Brownian dynamics simulations of the interactions of the MDM2 protein with a p53 peptide in its wild type state and when phosphorylated at Thr18 (pThr18) and Ser20 (pSer20). We found that p53 phosphorylation results in concerted changes in the topology of the interaction landscape in the diffusively bound encounter complex domain. These changes hinder phosphorylated p53 peptides from binding to MDM2 well before reaching the binding site. The underlying mechanism appears to involve shift of the peptide away from the vicinity of the MDM2 protein, peptide reorientation, and reduction in peptide residence time relative to wild-type p53 peptide. pThr18 and pSr20 p53 peptides experience reduction in residence times by factors of 13.6 and 37.5 respectively relative to the wild-type p53 peptide, indicating a greater role for Ser20 phosphorylation in abrogating p53 MDM2 interactions. These detailed insights into the effect of phosphorylation on molecular interactions are not available from conventional experimental and theoretical approaches and open up new avenues that incorporate molecular interaction dynamics, for stabilizing p53 against MDM2, which is a major focus of anticancer drug lead development.     

Polyphenols from green tea prevent antineuritogenic action of Nogo‐A via 67‐kDa laminin receptor and hydrogen peroxide

Axonal regeneration after injury to the CNS is hampered by myelin‐derived inhibitors, such as Nogo‐A. Natural products, such as green tea, which are neuroprotective and safe for long‐term therapy, would complement ongoing various pharmacological approaches. In this study, using nerve growth factor‐differentiated neuronal‐like Neuroscreen‐1 cells, we show that extremely low concentrations of unfractionated green tea polyphenol mixture (GTPP) and its active ingredient, epigallocatechin‐3‐gallate (EGCG), prevent both the neurite outgrowth‐inhibiting activity and growth cone‐collapsing activity of Nogo‐66 (C‐terminal domain of Nogo‐A). Furthermore, a synergistic interaction was observed among GTPP constituents. This preventive effect was dependent on 67‐kDa laminin receptor (67LR) to which EGCG binds with high affinity. The antioxidants N‐acetylcysteine and cell‐permeable catalase abolished this preventive effect of GTPP and EGCG, suggesting the involvement of sublethal levels of H₂O₂ in this process. Accordingly, exogenous sublethal concentrations of H₂O₂, added as a bolus dose (5 μM) or more effectively through a steady‐state generation (1–2 μM), mimicked GTPP in counteracting the action of Nogo‐66. Exogenous H₂O₂ mediated this action by bypassing the requirement of 67LR. Taken together, these results show for the first time that GTPP and EGCG, acting through 67LR and elevating intracellular sublethal levels of H₂O₂, inhibit the antineuritogenic action of Nogo‐A.

     

Laminin-1 induces endocytosis of 67KDa laminin receptor and protects Neuroscreen-1 cells against death induced by serum withdrawal

Although the function of laminin in the basement membrane is known, the function of soluble “neuronal” laminin is unknown. Since laminin is neuroprotective, we determined whether the soluble laminin-1 induces signaling for neuroprotection via its 67KDa laminin-1 receptor (67LR). Treatment of Neuroscreen-1 (NS-1) cells with laminin-1 or YIGSR peptide, which corresponds to a sequence in laminin-1 β1 chain that binds to 67LR, induced a decrease in the cell-surface expression of 67LR and caused its internalization. Furthermore, intracellular cAMP-elevating agents, dibutyryl-cAMP, forskolin, and rolipram, also induced this internalization. Both soluble laminin-1 and YIGSR induced a sustained elevation of intracellular cAMP under defined conditions, suggesting a causal role of cAMP in the endocytosis of 67LR. This endocytosis was not observed in cells deficient in protein kinase A (PKA) nor in cells treated with either SQ 22536, an inhibitor for adenylyl cyclase, or ESI-09, an inhibitor for the exchange protein directly activated by cAMP (Epac). In addition, when internalization occurred in NS-1 cells, 67LR and adenylyl cyclase were localized in early endosomes. Under conditions in which endocytosis had occurred, both laminin-1 and YIGSR protected NS-1 cells from cell death induced by serum withdrawal. However, under conditions in which endocytosis did not occur, neither laminin-1 nor YIGSR protected these cells. Conceivably, the binding of laminin-1 to 67LR causes initial signaling through PKA and Epac, which causes the internalization of 67LR, along with signaling enzymes, such as adenylyl cyclase, into early endosomes. This causes sustained signaling for protection against cell death induced by serum withdrawal.     

Ground wētā in vines of the Awatere Valley,Marlborough: biology,density and distribution

The endemic New Zealand ground wētā (Hemiandrus sp. ‘promontorius’) has a Naturally Uncommon conservation status. This is because of the paucity of information on its density and distribution. Here, the biology, density and distribution of a population of this wētā found in and around vineyards in the Awatere Valley, Marlborough was studied. Wētā density was assessed in vineyards, paddocks and shrublands in this valley. Soil moisture, penetration resistance, pH and organic matter were recorded at locations with and without wētā. Wētā density in vineyards was significantly higher than in either paddocks or shrub habitats. In vineyards, the density of this insect was significantly higher under-vines than in the inter-rows. Higher numbers of this wētā were found in moist soils that required lower force to burrow. Females laid an average of 55 eggs between March and April, which hatched in September. These findings highlight the intersection between agriculture and conservation.     

Capitalizing on multi-element interactions through bal-anced nutrition——A pathway to improve nitrogen use efficiency in China,India and North America

A viable option for increasing nitrogen (N) use efficiency and mitigation of negative impacts of N on the environment is to capitalize on multi-element interactions through implementation of nutrient management programs that provide balanced nutrition. Numerous studies have demonstrated the immediate efficacy of this approach in the developing regions like China and India as well as developed countries in North America. Based on 241 site-years of experiments in these countries, the first-year N recovery efficiency (RE) for the conventional or check treatments averaged 21% while the balanced treatments averaged 54% RE, for an average increase of 33% in RE due to balanced nutrition. Effective policies to promote adoption are most likely those that enable site-specific approaches to nutrient management decisions rather than sweeping, nation-wide incentives supporting one nutrient over another. Local farmers, advisers and officials need to be empowered with tools and information to help them define necessary changes in practices to create more balanced nutrient management.     

Excess of de novo deleterious mutations in genes associated with glutamatergic systems in nonsyndromic intellectual disability

Little is known about the genetics of nonsyndromic intellectual disability (NSID). We hypothesized that de novo mutations (DNMs) in synaptic genes explain an important fraction of sporadic NSID cases. In order to investigate this possibility, we sequenced 197 genes encoding glutamate receptors and a large subset of their known interacting proteins in 95 sporadic cases of NSID. We found 11 DNMs, including ten potentially deleterious mutations (three nonsense, two splicing, one frameshift, four missense) and one neutral mutation (silent) in eight different genes. Calculation of point-substitution DNM rates per functional and neutral site showed significant excess of functional DNMs compared to neutral ones. De novo truncating and/or splicing mutations in SYNGAP1, STXBP1, and SHANK3 were found in six patients and are likely to be pathogenic. De novo missense mutations were found in KIF1A, GRIN1, CACNG2, and EPB41L1. Functional studies showed that all these missense mutations affect protein function in cell culture systems, suggesting that they may be pathogenic. Sequencing these four genes in 50 additional sporadic cases of NSID identified a second DNM in GRIN1 (c.1679_1681dup/p.Ser560dup). This mutation also affects protein function, consistent with structural predictions. None of these mutations or any other DNMs were identified in these genes in 285 healthy controls. This study highlights the importance of the glutamate receptor complexes in NSID and further supports the role of DNMs in this disorder.     

Nonphorbol tumor promoters okadaic acid and calyculin-A induce membrane translocation of protein kinase C.

The cell-permeable inhibitors of type 1 and 2A protein phosphatases, okadaic acid and calyculin-A, induced a redistribution of protein kinase C (PKC) activity and immunoreactivity (40 to 60%) from cytosol to membrane in some cell types. Calyculin-A was 100-fold more potent than okadaic acid and required only 5 to 10 nM concentrations to induce this PKC translocation. The concentration of these agents required to induce the redistribution of PKC correlated with the potency of these agents to inhibit both type 1 and 2A protein phosphatases. There was a lag period of 15 to 30 min before the onset of PKC translocation, as this process might have been induced by indirect cellular events triggered by inhibitions of protein phosphatases (1 and 2A). Taken together these results suggest that although the okadaic acid class of tumor promoters and phorbol ester-related agents bind to two different cellular receptors having counteracting enzymic activities, they share a common mechanism of action, namely the induction of cytosol to membrane translocation of PKC.