p38 MAPK and Ca2+ contribute to hydrogen peroxide-induced increase of permeability in vascular endothelial cells but ERK does not

To examine the involvement of p38 mitogen-activated protein kinase (p38 MAPK) and extra-cellular signal-regulated kinase (ERK) in the oxidative stress-induced increase of permeability in endothelial cells, the effects of a p38 MAPK inhibitor (SB203580) and ERK inhibitor (PD90859) on the H₂O₂-induced increase of permeability in bovine pulmonary artery endothelial cells (BPAEC) were investigated using a two-compartment system partitioned by a semi-permeable filter. H₂O₂ at 1 mM caused an increase of the permeation rate of fluorescein isothiocyanate (FITC)-labeled dextran 40 through BPAEC monolayers. SB203580 inhibited the H₂O₂-induced increase of permeability but PD98059 did not, though activation (phosphorylation) of both p38 MAPK and ERK was observed in H₂O₂-treated cells in Western blot analysis. An H₂O₂-induced increase of the intracellular Ca²⁺ concentration ([Ca²⁺]_i) was also observed and an intracellular Ca²⁺ chelator (BAPTA-AM) significantly inhibited the H₂O₂-induced increase of permeability. However, it showed no inhibitory effects on the H₂O₂-induced phosphorylation of p38 MAPK and ERK. The H₂O₂-induced increase of [Ca²⁺]_i was not influenced by SB203580 and PD98059. These results indicate that the activation of p38 MAPK and the increase of [Ca²⁺]_i are essential for the H₂O₂-induced increase of endothelial permeability and that ERK is not.     

An Activator Stimulating the Hydrolysis of Phospholipids by Phospholipase B from Torulaspora delbrueckii

An activator stimulating the enzymatic hydrolysis of phospholipids was purified to a homogeneous state from autolyzed Torulaspora delbrueckii cell washings. Autolyzed cell washings were extracted with chloroform and ethanol, and the activator was purified about 130-fold by sequential column chromatographies on DEAE-Sephacel, Sephacryl S-300, and TSK gel G 3000 SW (high performance liquid chromatography, HPLC). The molecular weight of the activator was about 175,000 as estimated by gel filtration on HPLC. However, the purified activator gave two protein bands corresponding to molecular weights from 102,000 to 129,000 and from 71,000 to 88,000, respectively, on SDS- polyacrylamide gel electrophoresis, when stained with silver stain reagent and periodic acid-Shiff (PAS) reagent. The activator was sensitive to heat treatment at 70°C for lOmin. The purified activator had no enzymatic activity, but stimulated the hydrolysis of phospholipids by water-soluble and membrane-bound phospholipases B if the substrates were pre-incubated with the activator. No stimulation of hydrolysis by the enzyme was observed when the activator was pre-incubated with the enzyme. The hydrolytic rate of phosphatidylcholine by the enzyme at acidic pH (pH 2.6) depended upon the amount of activator added. On the other hand, the hydrolytic rate at alkaline pH (pH 7.6) was stimulated greatly by more than 0.04 nmol of the activator.     

Plasmacytoid dendritic cell dysfunction caused by heat stress is improved by administration of Lactococcus lactis strain plasma in mice

ABSTRACT

Plasmacytoid dendritic cells (pDCs) are crucial in anti-viral immunity, acting as regulators in both adaptive and innate immunity. In this study, brief heat stress caused a decrease in splenic pDC activity in mice. Administration of Lactococcus lactis strain Plasma (LC-Plasma) significantly suppressed the decrease in pDC activity and IFN-α production.

Abbreviations: LC-Plasma: Lactococcus lactis strain Plasma; LAB: lactic acid bacteria; pDC: plasmacytoid dendritic cell; IFN: interferons; mDC: myeloid dendritic cells     

Direct Immunochemiluminescent Assay for proBNP and Total BNP in Human Plasma proBNP and Total BNP Levels in Normal and Heart Failure

Background

Recent studies have shown that in addition to brain (or B-type) natriuretic peptide (BNP) and the N-terminal proBNP fragment, levels of intact proBNP are also increased in heart failure. Moreover, present BNP immunoassays also measure proBNP, as the anti-BNP antibody cross-reacts with proBNP. It is important to know the exact levels of proBNP in heart failure, because elevation of the low-activity proBNP may be associated with the development of heart failure.

Methodology/Principal Findings

We therefore established a two-step immunochemiluminescent assay for total BNP (BNP+proBNP) and proBNP using monoclonal antibodies and glycosylated proBNP as a standard. The assay enables measurement of plasma total BNP and proBNP within only 7 h, without prior extraction of the plasma. The detection limit was 0.4 pmol/L for a 50-µl plasma sample. Within-run CVs ranged from 5.2%–8.0% in proBNP assay and from 7.0%–8.4% in total BNP assay, and between-run CVs ranged from 5.3–7.4% in proBNP assay and from 2.9%–9.5% in total BNP assay, respectively. The dilution curves for plasma samples showed good linearity (correlation coefficients = 0.998–1.00), and analytical recovery was 90–101%. The mean total BNP and proBNP in plasma from 116 healthy subjects were 1.4±1.2 pM and 1.0±0.7 pM, respectively, and were 80±129 pM and 42±70 pM in 32 heart failure patients. Plasma proBNP levels significantly correlate with age in normal subjects.

Conclusions/Significance

Our immunochemiluminescent assay is sufficiently rapid and precise for routine determination of total BNP and proBNP in human plasma.     

Critical role of ABCA1 transporter in sphingosine 1-phosphate release from astrocytes

Sphingosine 1-phosphate (S1P) is accumulated in lipoproteins, especially high-density lipoprotein (HDL), in plasma. However, it remains uncharacterized how extracellular S1P is produced in the CNS. The treatment of rat astrocytes with retinoic acid and dibutyryl cAMP, which induce apolipoprotein E (apoE) synthesis and HDL-like lipoprotein formation, stimulated extracellular S1P accumulation in the presence of its precursor sphingosine. The released S1P was present together with apoE particles in the HDL fraction. S1P release from astrocytes was inhibited by the treatment of the cells with glybenclamide or small interfering RNAs specific to ATP-binding cassette transporter A1 (ABCA1). Astrocytes from Abca1−/− mice also showed impairment of retinoic acid/dibutyryl cAMP-induced S1P release in association with the blockage of HDL-like lipoprotein formation. However, the formation of either apoE or lipoprotein itself was not sufficient, and additional up-regulation of ABCA1 was requisite to stimulate S1P release. We conclude that the S1P release from astrocytes is coupled with lipoprotein formation through ABCA1.     

Artificial model of photosynthetic oxygen evolving complex: Catalytic O2 production from water by di-μ-oxo manganese dimers supported by clay compounds

Adsorption of [(OH₂)(terpy)Mn(μ-O)₂Mn(terpy)(OH₂)]³⁺ (terpy = 2,2′:6′,2?-terpyridine) (1) onto montmorillonite K10 (MK10) yielded catalytic dioxygen (O₂) evolution from water using a Ce^IV oxidant. The Mn K-edge X-ray absorption near edge structure (XANES) of the 1/MK10 hybrid suggested that the oxidation state of the di-μ-oxo Mn₂ core could be Mn^III-Mn^IV. However the pre-edge peak in the XANES spectrum of 1 adsorbed on MK10 is different from the neat 1 powder. The kinetic analysis of O₂ evolution showed that the catalysis requires cooperation of two equivalents of 1 adsorbed on MK10. The reaction of the [(bpy)₂Mn(μ-O)₂Mn(bpy)₂]³⁺ (bpy = 2,2′-bipyridine) (2)/MK10 hybrid with a Ce^IV oxidant evolved O₂. However, the turnover number value was less than unity for 2/MK10, showing that 2 adsorbed on MK10 does not work as a catalyst. The terminal water ligands could be an important for the catalysis by adsorbed 1. The mechanism of O₂ production by photosynthetic oxygen evolving complex is discussed based on catalytic O₂ evolution by 1 adsorbed on MK10.     

Role of scavenger receptor class B type I and sphingosine 1-phosphate receptors in high density lipoprotein-induced inhibition of adhesion molecule expression in endothelial cells

We characterized the molecular mechanisms by which high density lipoprotein (HDL) inhibits the expression of adhesion molecules, including vascular cell adhesion molecule-1 and intercellular adhesion molecule-1, induced by sphingosine 1-phosphate (S1P) and tumor necrosis factor (TNF) alpha in endothelial cells. HDL inhibited S1P-induced nuclear factor kappaB activation and adhesion molecule expression in human umbilical vein endothelial cells. The inhibitory HDL actions were associated with nitric-oxide synthase (NOS) activation and were reversed by inhibitors for phosphatidylinositol 3-kinase and NOS. The HDL-induced inhibitory actions were also attenuated by the down-regulation of scavenger receptor class B type I (SR-BI) and its associated protein PDZK1. When TNFalpha was used as a stimulant, the HDL-induced NOS activation and the inhibitory action on adhesion molecule expression were, in part, attenuated by the down-regulation of the expression of S1P receptors, especially S1P(1), in addition to SR-BI. Reconstituted HDL composed mainly of apolipoprotein A-I and phosphatidylcholine mimicked the SR-BI-sensitive part of HDL-induced actions. Down-regulation of S1P(3) receptors severely suppressed the stimulatory actions of S1P. Although G(i/o) proteins may play roles in either stimulatory or inhibitory S1P actions, as judged from pertussis toxin sensitivity, the coupling of S1P(3) receptors to G(12/13) proteins may be critical to distinguish the stimulatory pathways from the inhibitory ones. In conclusion, even though S1P alone stimulates adhesion molecule expression, HDL overcomes S1P(3) receptor-mediated stimulatory actions through SR-BI/PDZK1-mediated signaling pathways involving phosphatidylinositol 3-kinase and NOS. In addition, the S1P component of HDL plays a role in the inhibition of TNFalpha-induced actions through S1P receptors, especially S1P(1).     

Luteolin, a flavonoid, inhibits AP-1 activation by basophils

Flavonoids including luteolin, apigenin, and fisetin are inhibitors of IL-4 synthesis and CD40 ligand expression by basophils. This study was done to search for compounds with greater inhibitory activity of IL-4 expression and to clarify the molecular mechanisms through which flavonoids inhibit their expression. Of the 37 flavonoids and related compounds examined, ayanin, luteolin, and apigenin were the strongest inhibitors of IL-4 production by purified basophils in response to anti-IgE antibody plus IL-3. Luteolin did not suppress Syk or Lyn phosphorylation in basophils, nor did suppress p54/46 SAPK/JNK, p38 MAPK, and p44/42 MAPK activation by a basophilic cell line, KU812 cells, stimulated with A23187 and PMA. However, luteolin did inhibit phosphorylation of c-Jun and DNA binding activity of AP-1 in nuclear lysates from stimulated KU812 cells. These results provide a fundamental structure of flavonoids for IL-4 inhibition and demonstrate a novel action of flavonoids that suppresses the activation of AP-1.     

Functional Characteristics of Enhanced Fc Receptor Expression of β2 Integrin-Deficient Bovine Mononuclear Phagocytes

Fc receptor expression, cytoplasmic Ca²⁺ signaling, chemiluminescent (CL) response, and electron spin resonance (ESR) combined with spin trapping of blood mononuclear phagocytes from control heifers and a heifer with leukocyte adhesion deficiency (LAD) were evaluated to elucidate the relationships between complement receptor type 3 (CR3) and Fc receptor expression and their functional responses. The mean fluorescence intensity of fluorescein isothiocyanate (FITC)-conjugated anti-bovine IgG bound to mononuclear phagocytes from the heifer with LAD was 1.8-fold higher than that of control heifers. The mean increments of cytoplasmic Ca²⁺ concentrations of mononuclear phagocytes from the heifer with LAD stimulated with OPZ, Agg-IgG, and PMA were 39.4 (P<0.05), 118, and 71.6% compared with those of control heifers. A 1.27-fold increase in the CL response relative to control heifers was detected when mononuclear phagocytes from the heifer with LAD were stimulated with Agg-IgG. The OPZ-induced CL response of mononuclear phagocytes from the heifer with LAD was significantly (P<0.05) decreased, whereas the PMA-induced CL response was similar to that of control heifers. The ESR spectrum of mononuclear phagocytes from the heifer with LAD was increased when stimulated with Agg-IgG, and was impaired when stimulated by OPZ compared with that of control heifers. The ESR spectrum of mononuclear phagocytes stimulated with PMA was similar in control heifers and the heifer with LAD. Fc receptors on mononuclear phagocytes from the heifer with LAD were enhanced, and their cytoplasmic Ca²⁺ signaling, CL response, and ESR-spin trapping when stimulated with Agg-IgG and OPZ appeared to be associated with enhanced Fc receptors.