Analysis of the Arabidopsis cell suspension phosphoproteome in response to short-term low temperature and abscisic acid treatment

To shed light on the early protein phosphorylation events involved in plant cell signaling in response to environmental stresses, we studied changes in the phosphorylation status of the Arabidopsis cell suspension proteome after short-term low temperature and abscisic acid (ABA) treatment. We used radioactive pulse-labeling of Arabidopsis cell suspension cultures and two-dimensional (2-D) gel electrophoresis to identify proteins that are differentially phosphorylated in response to these treatments. Changes in the phosphorylation levels of several proteins were detected in response to short-term (5 min or less) cold (4°C) and chilling (12°C) stress and ABA treatment, and we observed that some of these changes were common between these treatments. In addition, we used Pro-Q Diamond phosphoprotein gel stain to study the steady-state protein phosphorylation status under the same treatments. We demonstrated that Pro-Q Diamond effectively stained phosphorylated proteins, however, the overall Pro-Q Diamond 2-D gel staining pattern of proteins extracted from low-temperature and ABA-treated cells was not consistent with the gel patterns obtained by in vivo radioactive labeling of phosphoproteins. These in vivo pulsed-labeling experiments demonstrate that the Arabidopsis phosphoproteome is dynamic in response to short-term low temperature and ABA treatment, and thus represents a strategy for the identification of signaling proteins that could be utilized in the production of chilling or freeze tolerant crop varieties.     

Spatial and temporal regulation of RACK1 function and N-methyl-D-aspartate receptor activity through WD40 motif-mediated dimerization

Efficient signaling requires accurate spatial and temporal compartmentalization of proteins. RACK1 is a scaffolding protein that fulfils this role through interaction of binding partners with one of its seven WD40 domains. We recently identified the kinase Fyn and the NR2B subunit of the N-methyl-D-Aspartate receptor (NMDAR) as binding partners of RACK1. Scaffolding of Fyn near its substrate NR2B by RACK1 inhibits Fyn phosphorylation of NR2B and thereby negatively regulates channel function. We found that Fyn and NR2B share the same binding site on RACK1; however, their binding to RACK1 is not mutually exclusive (Yaka, R., Thornton, C., Vagts, A. J., Phamluong, K., Bonci, A., and Ron, D. (2002) Proc. Natl. Acad. Sci. U. S. A. 99, 5710-5715). We therefore tested the hypothesis that RACK1 forms a homodimer that allows the simultaneous binding of Fyn and NR2B. We found that RACK1 binds to itself both in vitro and in the brain. Deletion analyses identified a RACK1-RACK1 dimer-binding site within the 4th WD40 repeat, and application of the 4th WD40 repeat or a peptide derivative to hippocampal slices inhibited NMDAR activity. We further found that in hippocampal slices, both RACK1 and NR2B associated with another WD40 protein, the beta-subunit of G protein (Gbeta), previously shown to heterodimerize with RACK1 in vitro (Dell, E. J., Connor, J., Chen, S., Stebbins, E. G., Skiba, N. P., Mochly-Rosen, D., and Hamm, H. E. (2002) J. Biol. Chem. 277, 49888-49895). However, activation of the pituitary adenylate cyclase polypeptide (1-38) G protein-coupled receptor, previously found to induce the dissociation of RACK1 from the NMDAR complex (Yaka, R., He, D. Y., Phamluong, K., and Ron, D. (2003) J. Biol. Chem. 278, 9630-9638), attenuated the association of Gbeta with RACK1 and NR2B. Based on these results, we propose that WD40-mediated homo- and heterodimerization of RACK1 mediate the formation of a transient signaling complex that includes the NMDAR, a G protein and Fyn.     

Post-ischemic activation of caspase-3 in the rat hippocampus: evidence of an axonal and dendritic localisation

The relationship between caspase-3 activation and delayed neuronal death after ischemia was examined. Expression of caspase-3 was evaluated by colorimetric assay, immunoblotting and by immunohistochemistry. Apoptosis was characterised by terminal desoxynucleotidyl transferase-mediated uridine 5'-triphosphate-biotin nick end-labelling. Immunohistochemistry showed caspase-3 activation in the whole hippocampus as early as 30 min after ischemia with exclusive localisation in fiber systems, especially in the perforant path and mossy fibers, Schaffer-collaterals, as well as apical and basal dendrites of pyramidal cells. One day post-ischemia, the 18 kDa cleavage product of caspase-3 (p18) was seen in all cell compartments (nucleus, cytosol and dendrites) throughout the entire subfields and the dentate gyrus with high distribution in mossy fibers. Two days post-ischemia, p18 kDa was only seen in the nuclei and cytosol of hippocampal cells without specific regional differences among hippocampal subfields. A significant number of apoptotic cells appeared only in the CA1 pyramidal cells at 2-3 days post-ischemia. Our data provides the first evidence that caspase-3 activation was detectable in the trisynaptic pathway fiber bundles which probably correspond to perforant path, alvear path and collaterals of Schaffer, and that activation of caspase-3 led to execution of apoptotic cell death program in selectively vulnerable areas, but not in the resistant area of the hippocampus.     

Novaluron (Rimon), a novel IGR: potency and cross-resistance

The potency of novaluron on laboratory susceptible and field strains of S. littoralis resembles that of chlorfluazuron and both compounds are about 20-fold more potent than teflubenzuron. No appreciable resistance to novaluron or chlorfluazuron was observed in a field strain of Spodoptera littoralis collected from cucumber field in the central part of Israel. On the other hand, the field strain showed a mild resistance of about 4-fold to teflubenzuron as compared to the laboratory susceptible strain. A very resistant colony of Bemisia tabaci to pyriproxyfen (1,200- to 2,000-fold) showed no appreciable cross-resistance to novaluron. Two field colonies of B. tabaci pressurized with acetamiprid or thiamethoxam for 22 generations, resulting in a 30- to 50-fold resistance to acetamiprid and thiamethoxam, has no cross-resistance to novaluron. The above results are of special interest, indicating a possible alternation between novaluron, pyriproxyfen, and neonicotinoids in insecticide-resistance management programs aiming at preventing resistance development to these novel groups of insecticides against important pests such as whitefly and lepidopteran species.     

H-Ras modulates N-methyl-D-aspartate receptor function via inhibition of Src tyrosine kinase activity

Tyrosine phosphorylation of the NR2A and NR2B subunits of the N-methyl-d-aspartate (NMDA) receptor by Src protein-tyrosine kinases modulates receptor channel activity and is necessary for the induction of long term potentiation (LTP). Deletion of H-Ras increases both NR2 tyrosine phosphorylation and NMDA receptor-mediated hippocampal LTP. Here we investigated whether H-Ras regulates phosphorylation and function of the NMDA receptor via Src family protein-tyrosine kinases. We identified Src as a novel H-Ras binding partner. H-Ras bound to Src but not Fyn both in vitro and in brain via the Src kinase domain. Cotransfection of H-Ras and Src inhibited Src activity and decreased NR2A tyrosine phosphorylation. Treatment of rat brain slices with Tat-H-Ras depleted NR2A from the synaptic membrane, decreased endogenous Src activity and NR2A phosphorylation, and decreased the magnitude of hippocampal LTP. No change was observed for NR2B. We suggest that H-Ras negatively regulates Src phosphorylation of NR2A and retention of NR2A into the synaptic membrane leading to inhibition of NMDA receptor function. This mechanism is specific for Src and NR2A and has implications for studies in which regulation of NMDA receptor-mediated LTP is important, such as synaptic plasticity, learning, and memory and addiction.     

Fibril reinforced poroelastic model predicts specifically mechanical behavior of normal,proteoglycan depleted and collagen degraded articular cartilage

Degradation of collagen network and proteoglycan (PG) macromolecules are signs of articular cartilage degeneration. These changes impair cartilage mechanical function. Effects of collagen degradation and PG depletion on the time-dependent mechanical behavior of cartilage are different. In this study, numerical analyses, which take the compression-tension nonlinearity of the tissue into account, were carried out using a fibril reinforced poroelastic finite element model. The study aimed at improving our understanding of the stress-relaxation behavior of normal and degenerated cartilage in unconfined compression. PG and collagen degradations were simulated by decreasing the Young's modulus of the drained porous (nonfibrillar) matrix and the fibril network, respectively. Numerical analyses were compared to results from experimental tests with chondroitinase ABC (PG depletion) or collagenase (collagen degradation) digested samples. Fibril reinforced poroelastic model predicted the experimental behavior of cartilage after chondroitinase ABC digestion by a major decrease of the drained porous matrix modulus (-64+/-28%) and a minor decrease of the fibril network modulus (-11+/-9%). After collagenase digestion, in contrast, the numerical analyses predicted the experimental behavior of cartilage by a major decrease of the fibril network modulus (-69+/-5%) and a decrease of the drained porous matrix modulus (-44+/-18%). The reduction of the drained porous matrix modulus after collagenase digestion was consistent with the microscopically observed secondary PG loss from the tissue. The present results indicate that the fibril reinforced poroelastic model is able to predict specifically characteristic alterations in the stress-relaxation behavior of cartilage after enzymatic modifications of the tissue. We conclude that the compression-tension nonlinearity of the tissue is needed to capture realistically the mechanical behavior of normal and degenerated articular cartilage.     

Functional evolution in the ancestral lineage of vertebrates or when genomic complexity was wagging its morphological tail

Early vertebrate evolution is characterized by a significant increase of organismal complexity over a relatively short time span. We present quantitative evidence for a high rate of increase in morphological complexity during early vertebrate evolution. Possible molecular evolutionary mechanisms that underlie this increase in complexity fall into a small number of categories, one of which is gene duplication and subsequent structural or regulatory neofunctionalization. We discuss analyses of two gene families whose regulatory and structural evolution shed light on the connection between gene duplication and increases in organismal complexity.     

Effect of atorvastatin on plasma apoE metabolism in patients with combined hyperlipidemia

Atorvastatin, a synthetic HMG-CoA reductase inhibitor used for the treatment of hyperlipidemia and the prevention of coronary artery disease, significantly lowers plasma cholesterol and low-density lipoprotein cholesterol (LDL-C) levels. It also reduces total plasma triglyceride and apoE concentrations. In view of the direct involvement of apoE in the pathogenesis of atherosclerosis, we have investigated the effect of atorvastatin treatment (40 mg/day) on in vivo rates of plasma apoE production and catabolism in six patients with combined hyperlipidemia using a primed constant infusion of deuterated leucine. Atorvastatin treatment resulted in a significant decrease (i.e., 30-37%) in levels of total triglyceride, cholesterol, LDL-C, and apoB in all six patients. Total plasma apoE concentration was reduced from 7.4 +/- 0.9 to 4.3 +/- 0.2 mg/dl (-38 +/- 8%, P < 0.05), predominantly due to a decrease in VLDL apoE (3.4 +/- 0.8 vs. 1.7 +/- 0.2 mg/dl; -42 +/- 11%) and IDL/LDL apoE (1.9 +/- 0.3 vs. 0.8 +/- 0.1 mg/dl; -57 +/- 6%). Total plasma lipoprotein apoE transport (i.e., production) was significantly reduced from 4.67 +/- 0.39 to 3.04 +/- 0.51 mg/kg/day (-34 +/- 10%, P < 0.05) and VLDL apoE transport was reduced from 3.82 +/- 0.67 to 2.26 +/- 0.42 mg/kg/day (-36 +/- 10%, P = 0.057). Plasma and VLDL apoE residence times and HDL apoE kinetic parameters were not significantly affected by drug treatment. Percentage decreases in VLDL apoE concentration and VLDL apoE production were significantly correlated with drug-induced reductions in VLDL triglyceride concentration (r = 0.99, P < 0.001; r = 0.88, P < 0.05, respectively, n = 6). Our results demonstrate that atorvastatin causes a pronounced decrease in total plasma and VLDL apoE concentrations and a significant decrease in plasma and VLDL apoE rates of production in patients with combined hyperlipidemia.