Role of protein phosphatase 2A in mGluR5-regulated MEK/ERK phosphorylation in neurons

The regulation of protein phosphorylation requires coordinated interaction between protein kinases and protein phosphatases (PPs). Recent evidence has shown that the Galphaq-protein-coupled metabotropic glutamate receptor (mGluR) 5 up-regulates phosphorylation of MAPK/ERK1/2. However, signaling mechanisms linking mGluR5 to ERK are poorly understood. In this study, roles of a major serine/threonine PP, PP2A, in this event were evaluated in cultured neurons. We found that the PP1/2A inhibitors okadaic acid and calyculin A mimicked the effect of the mGluR5 agonists (RS)-3,5-dihydroxyphenylglycine and (RS)-2-chloro-5-hydroxyphenylglycine in facilitating phosphorylation of ERK1/2 and its upstream kinase, MEK1/2, in a PP2A-dependent but not PP1-dependent manner. Co-administration of either inhibitor with an mGluR5 agonist produced additive phosphorylation of ERK1/2. Enzymatic assays showed a basal level of phosphatase activity of PP2A under normal conditions, and activation of mGluR5 selectively inhibited PP2A, but not PP1, activity. In addition, a physical association of the cytoplasmic C terminus of mGluR5 with PP2A was observed, and ligand activation of mGluR5 reduced mGluR5-PP2A binding. Additional mechanistic studies revealed that mGluR5 activation increased tyrosine (Tyr307) phosphorylation of PP2A, which was dependent on activation of a p60c-Src family tyrosine kinase, but not the epidermal growth factor receptor tyrosine kinase and resulted in dissociation of PP2A from mGluR5 and reduced PP2A activity. Together, we have identified a novel, mGluR5-triggered signaling mechanism involving use- and Src-dependent inactivation of PP2A, which contributes to mGluR5 activation of MEK1/2 and ERK1/2.     

Sphingosine 1-phosphate receptors regulate chemokine-driven transendothelial migration of lymph node but not splenic T cells

Chemokines and chemokine receptors are required for T cell trafficking and migration. Recent evidence shows that sphingosine 1-phosphate (S1P) and S1PRs are also important for some aspects of T cell migration, but how these two important receptor-ligand systems are integrated and coregulated is not known. In this study, we have investigated CCL19-CCR7 and CXCL12-CXCR4-driven migration of both splenic and peripheral lymph node (PLN) nonactivated and naive T cells, and used both S1P and the S1PR ligand, FTY720, to probe these interactions. The results demonstrate that splenic T cell migration to CCL19 or CXCL12 is enhanced by, but does not require, S1PR stimulation. In contrast, PLN T cell migration to CXCL12, but not CCL19, requires both chemokine and S1PR stimulation, and the requirement for dual receptor stimulation is particularly important for steps involving transendothelial migration. The results also demonstrate that: 1) splenic and PLN nonactivated and naive T cells use different molecular migration mechanisms; 2) CCR7 and CXCR4 stimulation engage different migration mechanisms; and 3) S1P and FTY720 have distinct S1PR agonist and antagonist properties. The results have important implications for understanding naive T cell entry into and egress from peripheral lymphoid organs, and we present a model for how S1P and chemokine receptor signaling may be integrated within a T cell.     

In vivo correction of complement regulatory protein deficiency with an inhibitor targeting the red blood cell membrane

Because of the complement system's involvement in many human diseases and potential complications associated with its systemic blockade, site-specific regulation of this effector system is an attractive concept. We report on further developments of such an approach using a single-chain Ab fragment as a vehicle to deliver complement regulatory proteins to a defined cell type. In a model system in which RBCs deficient in complement receptor 1-related gene/protein y (Crry) are rapidly cleared after injection into wild-type animals by a complement-dependent mechanism, we selectively reconstituted these cells with N- and C-terminally targeted recombinant forms of Crry. Transfusion of Crry-coated knockout RBCs into C57BL/6 mice extended their in vivo half-life from <5 min to approximately 2 days. Maintenance of protective levels of Crry (by a combined treatment of donor and recipient RBCs) led to nearly normal RBC survival. Uniform in vitro and in vivo coating of the RBCs and the more efficient complement inhibitory capacity of C-terminally tagged Crry were other interesting features of this experimental system. These results suggest the possibility of using the single-chain Ab fragment-mediated targeting concept of complement regulatory proteins to restrict complement inhibition to the site of its excessive activation.     

Maximal efficiency of coupling between ATP hydrolysis and translocation of polypeptides mediated by SecB requires two protomers of SecA

SecA is the ATPase that provides energy for translocation of precursor polypeptides through the SecYEG translocon in Escherichia coli during protein export. We showed previously that when SecA receives the precursor from SecB, the ternary complex is fully active only when two protomers of SecA are bound. Here we used variants of SecA and of SecB that populate complexes containing two protomers of SecA to different degrees to examine both the hydrolysis of ATP and the translocation of polypeptides. We conclude that the low activity of the complexes with only one protomer is the result of a low efficiency of coupling between ATP hydrolysis and translocation.     

Oxidative Stress Decreases Phosphatidylinositol 4,5-Bisphosphate Levels by Deactivating Phosphatidylinositol- 4-phosphate 5-Kinase �� in a Syk-dependent Manner

Phosphatidylinositol 4,5-bisphosphate (PIP₂) has many essential functions and its homeostasis is highly regulated. We previously found that hypertonic stress increases PIP₂ by selectively activating the β isoform of the type I phosphatidylinositol phosphate 5-kinase (PIP5Kβ) through Ser/Thr dephosphorylation and promoting its translocation to the plasma membrane. Here we report that hydrogen peroxide (H₂O₂) also induces PIP5Kβ Ser/Thr dephosphorylation, but it has the opposite effect on PIP₂ homeostasis, PIP5Kβ function, and the actin cytoskeleton. Brief H₂O₂ treatments decrease cellular PIP₂ in a PIP5Kβ-dependent manner. PIP5Kβ is tyrosine phosphorylated, dissociates from the plasma membrane, and has decreased lipid kinase activity. In contrast, the other two PIP5K isoforms are not inhibited by H₂O₂. We identified spleen tyrosine kinase (Syk), which is activated by oxidants, as a candidate PIP5Kβ kinase in this pathway, and mapped the oxidant-sensitive tyrosine phosphorylation site to residue 105. The PIP5KβY105E phosphomimetic is catalytically inactive and cytosolic, whereas the Y105F non-phosphorylatable mutant has higher intrinsic lipid kinase activity and is much more membrane associated than wild type PIP5Kβ. These results suggest that during oxidative stress, as modeled by H₂O₂ treatment, Syk-dependent tyrosine phosphorylation of PIP5Kβ is the dominant post-translational modification that is responsible for the decrease in cellular PIP₂.Oxygen-derived free radicals are by-products of metabolic reactions in eukaryotic cells. Reactive oxygen species (ROS)⁴ act as endogenous signaling molecules (1). However, excessive ROS production leads to deleterious effects on cellular homeostasis by inducing DNA damage, lipid/protein oxidation, and ultimately apoptosis or necrosis. Acute and chronic oxidative stress have been implicated in the pathophysiology of shock and sepsis associated with traumatic injuries such as massive thermal burn (2–4), Alzheimer disease, diabetes mellitus, and atherosclerosis (5–7).Phosphatidylinositol 4,5-bisphosphate (PIP₂) has emerged as an integral component of the stress response. This is concordant with its essential role in the regulation of the actin cytoskeleton, endocytosis, exocytosis, plasma membrane (PM) scaffolding, and ion channels/transporter (8). PIP₂ is also essential for InsP₃-mediated Ca²⁺ generation, protein kinase C activation, and PIP₃ generation (9, 10). PIP₂ synthesis is depressed in the heart sarcolemma during oxidative stress, suggesting that PIP₂ depletion may contribute to cardiac dysfunctions (11). Recently, Divecha and colleagues (12) reported that prolonged (many hours) treatment of HeLa cells with hydrogen peroxide (H₂O₂) induces apoptosis by depleting PIP₂. Apoptosis can be attenuated by overexpression of a type I phosphatidylinositol-4-phosphate 5-kinase (PIP5Kβ). We found using isoform-specific PIP5K knockdown by RNA interference (RNAi) that PIP5Kβ synthesizes a large fraction of the ambient PIP₂ pool in HeLa cells (13). Hypertonicity is another type of stress that increases PIP₂ and may be protective against cell injury (14, 15) by activating PIP5Kβ through Ser/Thr dephosphorylation (16). This effect is specific for PIP5Kβ, because depletion of the other two PIP5K isoforms (α and γ) individually does not substantially abrogate the hypertonicity induced PIP₂ increase.In the present study, we used H₂O₂ to model oxidative stress in tissue culture cells, and examined the effect on PIP₂ homeostasis and PIP5Kβ function. We found that a brief H₂O₂ treatment decreases cellular PIP₂ and inactivates PIP5Kβ through tyrosine phosphorylation. We identified spleen tyrosine kinase (Syk) as a candidate kinase in this pathway. Syk is a member of the Syk/Zap-70 nonreceptor tyrosine kinase family that is abundant in hematopoietic cells (17) but is also found in nonhematopoietic lineages (18), including HeLa and COS cells (19, 20).     

Segregation analysis of esophageal cancer in a moderately high-incidence area of northern China

In order to explore the mode of inheritance of esophageal cancer in a moderately high-incidence area of northern China, we conducted a pedigree survey on 225 patients affected by esophageal cancer in Yangquan, Shanxi Province. Segregation analysis was performed using the REGTL program of S.A.G.E. The results showed that Mendelian autosomal recessive inheritance of a major gene that influences susceptibility to esophageal cancer provided the best fit to the data. In the best-fitting recessive model, the frequency of the disease allele was.2039. There was a significant sex effect on susceptibility to the disease. The maximum cumulative probability of esophageal cancer among males with the AA genotype was 100%, but, among females, it was 63.5%. The mean age at onset for both men and women was 62 years. The age-dependent penetrances for males with the AA genotype by the ages of 60 and 80 years were 41.6% and 95.2%, respectively, whereas, for females, they were 26.4% and 60.5%, respectively. Incorporating environmental risk factors-such as cigarette smoking, pipe smoking, alcohol drinking, eating hot food, and eating pickled vegetables-into the models did not provide significant improvement of the fit of the models to these data. The results suggest a major locus underlying susceptibility to esophageal cancer with sex-specific penetrance.     

Cloning and characterization of a human cDNA ACAD10 mapped to chromosome 12q24.1

Mitochondrial fatty acid -oxidation is an important energy resource for many mammal tissues. Acyl-CoA dehydrogenases (ACADs) are a family of flavoproteins that are involved in the -oxidation of the fatty acyl-CoA derivatives. Deficiency of these ACADs can cause metabolic disorders including muscle fatigue, hypoglycaemia, hepatic lipidosis and so on. By large scale sequencing, we identified a cDNA sequence of 3960 base pairs with a typical acyl-CoA dehydrogenase function domain. RT-PCR result shows that it is widely expressed in human tissues, especially high in liver, kidney, pancreas and spleen. It is hypothesized that this is a novel member of ACADs family. Abbreviations: ACADs – acyl-CoA dehydrogenases, FAD – flavinadenine dinucleotide, SCAD – short-chain acyl-CoA dehydrogenase,MCAD – medium-chain acyl-CoA dehydrogenase, LCAD – long-chain acyl-CoAdehydrogenase, VLCAD – very long- chain acyl-CoA dehydrogenase, IVD –isocalery-CoA dehydrogenase, SBCAD – short/branched chain acyl-CoAdehydrogenase, GCD – glutaryl- CoA dehydrogenase, ETF – electron transferflavoprotein, ACAD8 – acyl-CoA dehydrogenase 8, ACAD9 – acyl-CoAdehydrogenase 9, ACAD10 – acyl-CoA dehydrogenase 10.     

Relationship Between Cuticular Waxes and Storage Quality Parameters of Korla Pear Under Different Storage Methods

Cuticular wax is an important factor that affects storage quality of fruits and vegetables. Previous studies have shown that cuticular wax of pears changes significantly during storage, whereas there are few studies on the effects of different storage methods on the wax changes and the relationship with storage quality. Cuticular wax of Korla pear stored using different methods, was measured to analyze its total wax content, chemical compositions and their relationship with storage quality. At the end of storage, the highest cuticular wax content was observed in controlled atmosphere (CA) storage and the lowest in room temperature storage. The substances of the primary components with higher contents were nonacosane, (E, E)-ɑ-farnesene, dodecan-1-ol, 1,1-dimethoxynonane, nonanal, palmitic acid, and oleic acid. Total wax content, olefins and fatty acids were most significantly with the storage quality, followed by alkanes and esters. Moreover, total wax content, wax composition and weight loss were closely related to postharvest senescence. Overall, an understanding of variations in the cuticular wax under different storage methods could provide theoretical basis for further study on the storage and preservation technology of pears.