Glycogen synthase kinase 3 beta induces caspase-cleaved tau aggregation in situ

Tau is a substrate of caspases, and caspase-cleaved tau has been detected in Alzheimer's disease brain but not in control brain. Furthermore, in vitro studies have revealed that caspase-cleaved tau is more fibrillogenic than full-length tau. Considering these previous findings, the purpose of this study was to determine how the caspase cleavage of tau affected tau function and aggregation in a cell model system. The effects of glycogen synthase kinase 3 beta (GSK3 beta), a well established tau kinase, on these processes also were examined. Tau or tau that had been truncated at Asp-421 to mimic caspase cleavage (Tau-D421) was transfected into cells with or without GSK3 beta, and phosphorylation, microtubule binding, and tau aggregation were examined. Tau-D421 was not as efficiently phosphorylated by GSK3 beta as full-length tau. Tau-D421 efficiently bound microtubules, and in contrast to the full-length tau, co-expression with GSK3 beta did not result in a reduction in the ability of Tau-D421 to bind microtubules. In the absence of GSK3 beta, neither Tau-D421 nor full-length tau formed Sarkosyl-insoluble inclusions. However, in the presence of GSK3 beta, Tau-D421, but not full-length tau, was present in the Sarkosyl-insoluble fraction and formed thioflavin-S-positive inclusions in the cell. Nonetheless, co-expression of GSK3 beta and Tau-D421 did not result in an enhancement of cell death. These data suggest that a combination of phosphorylation events and caspase activation contribute to the tau oligomerization process in Alzheimer's disease, with GSK3 beta-mediated tau phosphorylation preceding caspase cleavage.     

Regulation of the dual specificity protein phosphatase, DsPTP1, through interactions with calmodulin

Reversible phosphorylation is a key mechanism for the control of intercellular events in eukaryotic cells. In animal cells, Ca2+/CaM-dependent protein phosphorylation and dephosphorylation are implicated in the regulation of a number of cellular processes. However, little is known on the functions of Ca2+/CaM-dependent protein kinases and phosphatases in Ca2+ signaling in plants. From an Arabidopsis expression library, we isolated cDNA encoding a dual specificity protein phosphatase 1, which is capable of hydrolyzing both phosphoserine/threonine and phosphotyrosine residues of the substrates. Using a gel overlay assay, we identified two Ca2+-dependent CaM binding domains (CaMBDI in the N terminus and CaMBDII in the C terminus). Specific binding of CaM to two CaMBD was confirmed by site-directed mutagenesis, a gel mobility shift assay, and a competition assay using a Ca2+/CaM-dependent enzyme. At increasing concentrations of CaM, the biochemical activity of dual specificity protein phosphatase 1 on the p-nitrophenyl phosphate (pNPP) substrate was increased, whereas activity on the phosphotyrosine of myelin basic protein (MBP) was inhibited. Our results collectively indicate that calmodulin differentially regulates the activity of protein phosphatase, dependent on the substrate. Based on these findings, we propose that the Ca2+ signaling pathway is mediated by CaM cross-talks with a protein phosphorylation signal pathway in plants via protein dephosphorylation.     

Phospholipase C delta-type consists of three isozymes: bovine PLCdelta2 is a homologue of human/mouse PLCdelta4

To date, 12 phospholipase C (PLC) isozymes have been identified in mammals, and they are divided into five classes, beta-, gamma-, delta-, epsilon-, and zeta-type. PLCdelta-type is reported to be composed of four isozymes, PLCdelta1-delta4. Here we report that a screening for mouse PLCdelta2 from a BAC library with primers that amplify a specific region of bovine PLCdelta2 resulted in isolation of one clone containing the mouse PLCdelta4 gene. Furthermore, a database search revealed that there is only one gene corresponding to PLCdelta2 and PLCdelta4 in the mouse and human genomes, indicating that bovine PLCdelta2 is a homologue of human and mouse PLCdelta4. However, PLCdelta2 Western blot analysis with a widely used commercial anti-PLCdelta2 antibody showed an expression pattern distinct from that of PLCdelta4 in wild-type mice. In addition, an 80-kDa band, which was recognized by antibody against PLCdelta2, was smaller than an 85-kDa band detected by anti-PLCdelta4 antibody, and the 80-kDa band was detectable in lysates of brain, testis, and spleen from PLCdelta4-deficient mice. We also found that immunoprecipitates from brain lysates with this PLCdelta2 antibody contained no PLC activity. From these data, we conclude that bovine PLCdelta2 is a homologue of human and mouse PLCdelta4, and that three isozymes (delta1, delta3, and delta4) exist in the PLCdelta family.     

Under-sulfation by PAPS synthetase inhibition modulates the expression of ECM molecules during chondrogenesis

Sulfation of proteoglycans is an important post-translational modification in chondrocytes. We previously found that 3'-phosphoadenosine 5'-phosphosulfate (PAPS) synthetase-2 levels increased more than 10-fold during mesenchymal cell chondrogenesis. Given that PAPS is the sole sulfur donor, and is produced only by PAPS synthetase in all cells, increased expression of PAPS synthetase-2 should be a prerequisite for increased sulfation activity of chondrocytes. We found that sodium chlorate, a specific inhibitor of PAPS synthetase, inhibited proteoglycan sulfation during chondrogenesis. In contrast, sodium chlorate unexpectedly induced early expression of type II collagen and increased the number of cartilage nodules during chondrogenesis. Inhibition of sulfation also accelerated the down-regulation of N-cadherin and fibronectin during chondrogenesis. These findings suggest that sulfation has an important regulatory role in coordinating the timely expression of extracellular matrix molecules during chondrogenesis, and that under-sulfation may cause the breakdown of this coordination, leading to premature chondrogenesis.     

Cynaropicrin, a sesquiterpene lactone, as a new strong regulator of CD29 and CD98 functions

Cynaropicrin is a sesquiterpene lactone displaying immunomodulatory effects on the production of cytokine and nitric oxide from macrophages/monocytes. In this study we have examined inhibitory effect of cynaropicrin on activation of major adhesion molecules [CD29 (beta1 integrins), CD43, and CD98] on the cells assessed by U937 (promonocytic cells) homotypic aggregation. Cynaropicrin potently blocked CD29 (beta1 integrins)- and CD98-induced homotypic aggregation with IC(50) values of 3.46 and 2.98 microM, respectively, without displaying cytotoxicity. Similarly, flow cytometric analysis exhibited that cynaropicrin down-regulated strikingly surface level of CD29 and CD147, a functional regulator of CD98, but not CD43. More importantly, cynaropicrin inhibition was linked to blockade of extracellular signal-related kinase (ERK) activation and distinct from other enzyme inhibitors including rottlerin, propranolol, forskolin, and chloroquine, but not cytochalasin B. Therefore, our finding is the first demonstration that cynaropicrin may be a potent functional regulator of CD29 and CD98 via interrupting ERK activation which may be linked to cytoskeleton rearrangement, suggesting further application to CD29- and CD98-mediated diseases such as virus-induced chronic inflammation, and invasion, migration, and metastasis of leukocyte cancer cells.     

Proteome analysis by bio-active ceramic water in rat liver: contribution to antioxidant enzyme expression, SOD I

The purpose of this study was to investigate the protective effect of bio-active ceramic water on rat liver. Male Wistar rats were divided into 4 groups of 15 animals each. Groups 1 and 2 were fed bio-active ceramic water and tap water for 4 months, respectively. Groups 3 and 4 were treated with the same condition for 12 months. The changes of protein expression of these four groups were investigated using two-dimensional gel electrophoresis and matrix-assisted laser desorption/ionization-time of flight mass spectrometry. Eleven proteins were significantly up-regulated in bio-active ceramic water treated rat liver including aldehyde dehydrogenase I and II, albumin, fructose-1,6-bisphosphatase, and superoxide dismutase I (SOD I). The most highly expressed protein, SOD I with up-regulated enzyme activity, was confirmed by immunoblots as a major antioxidant capable of detoxifying normally generated reactive oxygen species. These data suggest that modified protein expression of the liver contributes to enhance liver function.     

Identification of vasopressin-induced genes in AQP2-transfected MDCK cells by suppression subtractive hybridization

Arginine vasopressin (AVP) plays a major role in the modulation of water reabsorption in mammalian kidney. In addition to short-term regulation of aquaporin 2 (AQP2) trafficking, AVP also has long-term effects to regulate the expression of AQP2 in renal collecting duct. However, the detailed mechanism of the long-term effects of AVP in kidney remains to be elucidated. We have searched for genes induced by AVP using the polymerase chain reaction-based suppression subtractive hybridization technique in AVP-responsive AQP2-transfected MDCK cells. We found that the expression of the genes such as VIP17/MAL, annexin II, stimulatory GTP binding protein, tubulin, and mitochondrial ATP synthase was induced by AVP treatment for 4h. These results suggest that AVP might induce the expression of several genes related to the apical targeting of newly synthesized AQP2 as well as that of AQP2 for the long-term modification of water permeability in renal collecting duct.     

Rac and protein kinase C-delta regulate ERKs and cytosolic phospholipase A2 in FcepsilonRI signaling to cysteinyl leukotriene synthesis in mast cells

Although cysteinyl leukotrienes (cysLTs) are known to be principal inflammatory lipid mediators released from IgE-stimulated mast cells, the signaling mechanisms involved in the synthesis of cysLTs remain largely unknown. In the present study, therefore, we investigated the signaling pathway by which IgE induces cysLTs synthesis after binding to its high affinity receptor (FcepsilonRI) in RBL-2H3 mast cells. We found that IgE-induced cysLT synthesis is completely abolished in RBL-2H3(Rac-N17) cells, a stable cell line expressing Rac(N17), a dominant negative Rac1 mutant; conversely, synthesis was enhanced in cells expressing Rac(V12), a constitutively active Rac1 mutant, suggesting that Rac1 is a key mediator of IgE signaling to cysLT synthesis. Further analysis aimed at identifying mediators downstream of Rac1 revealed that pretreating cells with a protein kinase C-delta (PKC-delta) inhibitor or infection with an adenoviral vector harboring a dominant negative PKC-delta mutant significantly attenuates IgE-induced ERKs phosphorylation, cytosolic phospholipase A(2) phosphorylation/translocation, and cysLT synthesis. In addition, the expression of Rac(N17) blocked PKC-delta translocation and impaired the phosphorylation of ERKs and cytosolic phospholipase A(2) otherwise elicited by IgE stimulation. Taken together these results suggest that PKC-delta also plays a critical mediatory role in the IgE signaling pathway leading to cysLT synthesis, acting downstream of Rac1. Finally, the physiological significance of PKC-delta in the IgE signaling pathway was demonstrated in an Ag (OVA)-challenged in vivo mouse model, in which induced levels of cysLTs and airway responsiveness in lung airways were significantly diminished by prior i.p. injection of a PKC-delta inhibitor.