Purification and characterization of a cytosolic, 42-kDa and Ca2+-dependent phospholipase A2 from bovine red blood cells: its involvement in Ca2+-dependent release of arachidonic acid from mammalian red blood cells

It has become evident that a Ca(2+)-dependent release of arachidonic acid (AA) and subsequent formation of bioactive lipid mediators such as prostaglandins and leukotrienes in red blood cells (RBCs) can modify physiological functions of neighboring RBCs and platelets. Here we identified a novel type of cytosolic PLA(2) in bovine and human RBCs and purified it to apparent homogeneity with a 14,000-fold purification. The purified enzyme, termed rPLA(2), has a molecular mass of 42 kDa and reveals biochemical properties similar to group IV cPLA(2), but shows different profiles from cPLA(2) in several column chromatographies. Moreover, rPLA(2) did not react with any of anti-cPLA(2) and anti-sPLA(2) antibodies and was identified as an unknown protein in matrix-assisted laser desorption/ionization time-of-flight mass spectrometric analysis. Divalent metal ions tested exhibited similar effects between rPLA(2) and cPLA(2), whereas mercurials inhibited cPLA(2) but had no effect on rPLA(2). Antibody against the 42-kDa protein not only precipitated the rPLA(2) activity, but also reacted with the 42-kDa protein from bovine and human RBCs in immunoblot analysis. The 42-kDa protein band was selectively detected in murine fetal liver cells known as a type of progenitor cells of RBCs. It was found that EA4, a derivative of quinone newly developed as an inhibitor for rPLA(2), inhibited a Ca(2+) ionophore-induced AA release from human and bovine RBCs, indicating that this enzyme is responsible for the Ca(2+)-dependent AA release from mammalian RBCs. Finally, erythroid progenitor cell assay utilizing diaminobenzidine staining of hemoglobinized fetal liver cells showed that rPLA(2) detectable in erythroid cells was down-regulated when differentiated to non-erythroid cells. Together, our results suggest that the 42-kDa rPLA(2) identified as a novel form of Ca(2+)-dependent PLA(2) may play an important role in hemostasis, thrombosis, and/or erythropoiesis through the Ca(2+)-dependent release of AA.     

The role of AiiA, a quorum-quenching enzyme from Bacillus thuringiensis, on the rhizosphere competence

Bacteria sense their population density and coordinate the expression of target genes, including virulence factors in Gram-negative bacteria, by the N-acylhomoserine lactones (AHLs)-dependent quorum-sensing (QS) mechanism. In contrast, several soil bacteria are able to interfere with QS by enzymatic degradation of AHLs, referred to as quorum quenching. A potent AHL-degrading enzyme, AiiA, of Bacillus thuringiensis has been reported to effectively attenuate the virulence of bacteria by quorum quenching. However, little is known about the role of AiiA in B. thuringiensis itself. In the present study, an aiiA-defective mutant was generated to investigate the role of AiiA in rhizosphere competence in the root system of pepper. The aiiA mutant showed no detectable AHL-degrading activity and was less effective for suppression of soft-rot symptom caused by Erwinia carotovora on the potato slice. On the pepper root, the survival rate of the aiiA mutant significantly decreased over time compared with that of wild type. Interestingly, viable cell count analysis revealed that the bacterial number and composition of E. carotovora were not different between treatments of wild type and the aiiA mutant, although root application of the aiiA mutant in pepper failed to protect the plant from root rot. These results provide evidence that AiiA can play an important role in rhizosphere competentce of B. thuringiensis and bacterial quorum quenching to Gram-negative bacteria without changing bacterial number or composition.     

Comparative analysis of the secretory proteome of human adipose stromal vascular fraction cells during adipogenesis

Adipogenesis is a complex process that is accompanied by a number of molecular events. In this study, a proteomic approach was adopted to identify secretory factors associated with adipogenesis. A label‐free shotgun proteomic strategy was implemented to analyze proteins secreted by human adipose stromal vascular fraction cells and differentiated adipocytes. A total of 474 proteins were finally identified and classified according to quantitative changes and statistical significances. Briefly, 177 proteins were significantly upregulated during adipogenesis (Class I), whereas 60 proteins were significantly downregulated (Class II). Changes in the expressions of several proteins were confirmed by quantitative RT‐PCR and immunoblotting. One obvious finding based on proteomic data was that the amounts of several extracellular modulators of Wnt and transforming growth factor‐β (TGF‐β) signaling changed during adipogenesis. The expressions of secreted frizzled‐related proteins, dickkopf‐related proteins, and latent TGF‐β‐binding proteins were found to be altered during adipogenesis, which suggests that they participate in the fine regulation of Wnt and TGF‐β signaling. This study provides useful tools and important clues regarding the roles of secretory factors during adipogenic differentiation, and provides information related to obesity and obesity‐related metabolic diseases.     

An antioxidant system required for host protection against gut infection in Drosophila

A fundamental question that applies to all organisms is how barrier epithelia efficiently manage continuous contact with microorganisms. Here, we show that in Drosophila an extracellular immune-regulated catalase (IRC) mediates a key host defense system that is needed during host-microbe interaction in the gastrointestinal tract. Strikingly, adult flies with severely reduced IRC expression show high mortality rates even after simple ingestion of microbe-contaminated foods. However, despite the central role that the NF-kappaB pathway plays in eliciting antimicrobial responses, NF-kappaB pathway mutant flies are totally resistant to such infections. These results imply that homeostasis of redox balance by IRC is one of the most critical factors affecting host survival during continuous host-microbe interaction in the gastrointestinal tract.     

Interaction of profilin‐1 and F‐actin via a β‐arrestin‐1/JNK signaling pathway involved in prostaglandin E2‐induced human mesenchymal stem cells migration and proliferation

Although many previous reports have examined the function of prostaglandin E₂ (PGE₂) in the migration and proliferation of various cell types, the role of the actin cytoskeleton in human mesenchymal stem cells (hMSCs) migration and proliferation has not been reported. The present study examined the involvement of profilin‐1 (Pfn‐1) and filamentous‐actin (F‐actin) in PGE₂‐induced hMSC migration and proliferation and its related signal pathways. PGE₂ (10^?6 M) increased both cell migration and proliferation, and also increased E‐type prostaglandin receptor 2 (EP2) mRNA expression, β‐arrestin‐1 phosphorylation, and c‐Jun N‐terminal kinase (JNK) phosphorylation. Small interfering RNA (siRNA)‐mediated knockdown of β‐arrestin‐1 and JNK (‐1, ‐2, ‐3) inhibited PGE₂‐induced growth of hMSCs. PGE₂ also activated Pfn‐1, which was blocked by JNK siRNA, and induced F‐actin level and organization. Downregulation of Pfn‐1 by siRNA decreased the level and organization of F‐actin. In addition, specific siRNA for TRIO and F‐actin‐binding protein (TRIOBP) reduced the PGE₂‐induced increase in hMSC migration and proliferation. Together, these experimental data demonstrate that PGE₂ partially stimulates hMSCs migration and proliferation by interaction of Pfn‐1 and F‐actin via EP2 receptor‐dependent β‐arrestin‐1/JNK signaling pathways. J. Cell. Physiol. 226: 559–571, 2011. © 2010 Wiley‐Liss, Inc.     

Tyrosine phosphorylation enhances RAD52-mediated annealing by modulating its DNA binding

RAD52 protein has an important role in homology-directed DNA repair by mediating RAD51 nucleoprotein filament formation on single-stranded DNA (ssDNA) protected by replication protein-A (RPA) and annealing of RPA-coated ssDNA. In human, cellular response to DNA damage includes phosphorylation of RAD52 by c-ABL kinase at tyrosine 104. To address how this phosphorylation modulates RAD52 function, we used an amber suppressor technology to substitute tyrosine 104 with chemically stable phosphotyrosine analogue (p-Carboxymethyl-L-phenylalanine, pCMF). The RAD52(Y104pCMF) retained ssDNA-binding activity characteristic of unmodified RAD52 but showed lower affinity for double-stranded DNA (dsDNA) binding. Single-molecule analyses revealed that RAD52(Y104pCMF) specifically targets and wraps ssDNA. While RAD52(Y104pCMF) is confined to ssDNA region, unmodified RAD52 readily diffuses into dsDNA region. The Y104pCMF substitution also increased the ssDNA annealing rate and allowed overcoming the inhibitory effect of dsDNA. We propose that phosphorylation at Y104 enhances ssDNA annealing activity of RAD52 by attenuating dsDNA binding. Implications of phosphorylation-mediated activation of RAD52 annealing activity are discussed.     

Chromosome aberrations in workers of nuclear-power plants

The frequencies of chromosome aberrations in 135 workers from nuclear-power plants were compared with those in 135 age-matched controls. A total of 135,000 cells was scored. The frequencies of dicentric chromosome were 1.67 × 10⁻³ in the exposed group and 0.49 × 10⁻³ in the control group and those of chromosome-type deletion were 3.33 × 10⁻³ and 1.10 × 10⁻³, respectively. The frequencies of all types of chromosome aberrations in the exposed subjects were higher than those in the control group, but no significant trend of dose-dependent increase was observed when only the exposed group were considered. Poisson regression analysis, with both exposed and control included, showed that there was a significant association of chromosome aberration with radiation dose and the duration of work, but not with age, smoking habit and alcohol intake. It was also found that recent exposure to radiation, within the last 5 years, had contributed more to the observed chromosome aberration than earlier exposure.     

Direct involvement of the small GTPase Rac in activation of the superoxide-producing NADPH oxidase Nox1

Activation of the non-phagocytic superoxide-producing NADPH oxidase Nox1, complexed with p22(phox) at the membrane, requires its regulatory soluble proteins Noxo1 and Noxa1. However, the role of the small GTPase Rac remained to be clarified. Here we show that Rac directly participates in Nox1 activation via interacting with Noxa1. Electropermeabilized HeLa cells, ectopically expressing Nox1, Noxo1, and Noxa1, produce superoxide in a GTP-dependent manner, which is abrogated by expression of a mutant Noxa1(R103E), defective in Rac binding. Superoxide production in Nox1-expressing HeLa and Caco-2 cells is decreased by depletion or sequestration of Rac; on the other hand, it is enhanced by expression of the constitutively active Rac1(Q61L), but not by that of a mutant Rac1 with the A27K substitution, deficient in binding to Noxa1. We also demonstrate that Nox1 activation requires membrane recruitment of Noxa1, which is normally mediated via Noxa1 binding to Noxo1, a protein tethered to the Nox1 partner p22(phox): the Noxa1-Noxo1 and Noxo1-p22(phox) interactions are both essential for Nox1 activity. Rac likely facilitates the membrane localization of Noxa1: although Noxa1(W436R), defective in Noxo1 binding, neither associates with the membrane nor activates Nox1, the effects of the W436R substitution are restored by expression of Rac1(Q61L). The Rac-Noxa1 interaction also serves at a step different from the Noxa1 localization, because the binding-defective Noxa1(R103E), albeit targeted to the membrane, does not support superoxide production by Nox1. Furthermore, a mutant Noxa1 carrying the substitution of Ala for Val-205 in the activation domain, which is expected to undergo a conformational change upon Rac binding, fully localizes to the membrane but fails to activate Nox1.     

Tonsil-derived mesenchymal stem cells alleviate concanavalin A-induced acute liver injury

Acute liver failure, the fatal deterioration of liver function, is the most common indication for emergency liver transplantation, and drug-induced liver injury and viral hepatitis are frequent in young adults. Stem cell therapy has come into the limelight as a potential therapeutic approach for various diseases, including liver failure and cirrhosis. In this study, we investigated therapeutic effects of tonsil-derived mesenchymal stem cells (T-MSCs) in concanavalin A (ConA)- and acetaminophen-induced acute liver injury. ConA-induced hepatitis resembles viral and immune-mediated hepatic injury, and acetaminophen overdose is the most frequent cause of acute liver failure in the United States and Europe. Intravenous administration of T-MSCs significantly reduced ConA-induced hepatic toxicity, but not acetaminophen-induced liver injury, affirming the immunoregulatory capacity of T-MSCs. T-MSCs were successfully recruited to damaged liver and suppressed inflammatory cytokine secretion. T-MSCs expressed high levels of galectin-1 and -3, and galectin-1 knockdown which partially diminished interleukin-2 and tumor necrosis factor α secretion from cultured T-cells. Galectin-1 knockdown in T-MSCs also reversed the protective effect of T-MSCs on ConA-induced hepatitis. These results suggest that galectin-1 plays an important role in immunoregulation of T-MSCs, which contributes to their protective effect in immune-mediated hepatitis. Further, suppression of T-cell activation by frozen and thawed T-MSCs implies great potential of T-MSC banking for clinical utilization in immune-mediated disease.