Differences between group X and group V secretory phospholipase A2 in lipolytic modification of lipoproteins

Secretory phospholipases A(2) (sPLA(2)s) are a diverse family of low molecular mass enzymes (13-18 kDa) that hydrolyze the sn-2 fatty acid ester bond of glycerophospholipids to produce free fatty acids and lysophospholipids. We have previously shown that group X sPLA(2) (sPLA(2)-X) had a strong hydrolyzing activity toward phosphatidylcholine in low-density lipoprotein (LDL) linked to the formation of lipid droplets in the cytoplasm of macrophages. Here, we show that group V sPLA(2) (sPLA(2)-V) can also cause the lipolysis of LDL, but its action differs remarkably from that of sPLA(2)-X in several respects. Although sPLA(2)-V released almost the same amount of fatty acids from LDL, it released more linoleic acid and less arachidonic acid than sPLA(2)-X. In addition, the requirement of Ca(2+) for the lipolysis of LDL was about 10-fold higher for sPLA(2)-V than sPLA(2)-X. In fact, the release of fatty acids from human serum was hardly detectable upon incubation with sPLA(2)-V in the presence of sodium citrate, which contrasted with the potent response to sPLA(2)-X. Moreover, sPLA(2)-X, but not sPLA(2)-V, was found to specifically interact with LDL among the serum proteins, as assessed by gel-filtration chromatography as well as sandwich enzyme-immunosorbent assay using anti-sPLA(2)-X and anti-apoB antibodies. Surface plasmon resonance studies have revealed that sPLA2-X can bind to LDL with high-affinity (K(d) = 3.1 nM) in the presence of Ca(2+). Selective interaction of sPLA(2)-X with LDL might be involved in the efficient hydrolysis of cell surface or intracellular phospholipids during foam cell formation.     

p62/SQSTM1-Dependent Autophagy of Lewy Body-Like α-Synuclein Inclusions

α-Synuclein is the main component of Lewy bodies, the intraneuronal inclusion bodies characteristic of Parkinson’s disease. Although α-synuclein accumulation is caused by inhibition of proteasome and autophagy-lysosome, the degradation of α-synuclein inclusions is still unknown. Formation of Lewy body-like inclusions can be replicated in cultured cells by introducing α-synuclein fibrils generated in vitro. We used this cell culture model to investigate the autophagy of α-synuclein inclusions and impaired mitochondria. The intracellular α-synuclein inclusions immediately underwent phosphorylation and ubiquitination. Simultaneously they were encircled by an adaptor protein p62/SQSTM1 and directed to the autophagy-lysosome pathway in HEK293 cell line. Most phospho-α-synuclein-positive inclusions were degraded in 24 h, however, lysosomal dysfunction with bafilomycin A1 significantly affected their clearance. Moreover, inhibition of autophagy by Atg-5 siRNA treatment reduced the incorporation of α-synuclein inclusions into LC3-positive autophagosomes. Knockdown experiments demonstrated the requirement of p62 for α-synuclein autophagy. These results demonstrate that α-synuclein inclusions are preferred targets for p62-dependent autophagy. Next, we investigated the autophagic clearance of impaired mitochondria in α-synuclein inclusion-containing cells. Impaired mitochondria were almost completely eliminated after mitochondrial uncoupling even in the presence of α-synuclein inclusions, suggesting that mitochondrial clearance is not prevented by α-synuclein inclusions in HEK293 cells.     

Amyloid β Levels in Human Red Blood Cells

Amyloid β-peptide (Aβ) is hypothesized to play a key role by oxidatively impairing the capacity of red blood cells (RBCs) to deliver oxygen to the brain. These processes are implicated in the pathogenesis of Alzheimer''s disease (AD). Although plasma Aβ has been investigated thoroughly, the presence and distribution of Aβ in human RBCs are still unclear. In this study, we quantitated Aβ40 and Aβ42 in human RBCs with ELISA assays, and provided evidence that significant amounts of Aβ could be detected in RBCs and that the RBC Aβ levels increased with aging. The RBC Aβ levels increased with aging. On the other hand, providing an antioxidant supplement (astaxanthin, a polar carotenoid) to humans was found to decrease RBC Aβ as well as oxidative stress marker levels. These results suggest that plasma Aβ40 and Aβ42 bind to RBCs (possibly with aging), implying a pathogenic role of RBC Aβ. Moreover, the data indicate that RBC Aβ40 and Aβ42 may constitute biomarkers of AD. As a preventive strategy, therapeutic application of astaxanthin as an Aβ-lowering agent in RBCs could be considered as a possible anti-dementia agent.

Trial Registration

Controlled-Trials.com ISRCTN42483402     

A novel class of sticky peel and light green mutations causes cuticle deficiency in leaves and fruits of tomato (Solanum lycopersicum)

The plant cuticle consists of aliphatic wax and cutin, and covers all the aerial tissues, conferring resistance to both biotic and abiotic stresses. In this study, we performed phenotypic characterizations of tomato mutants having both sticky peel (pe) and light green (lg) mutations. Our genetic analysis showed that these two mutations are tightly linked and behave like a monogenic recessive mutation. The double mutant (pe lg) produced glossy soft fruits with light green leaves, most likely due to defects in cuticle formation. Cytological analysis revealed that the thickness of the fruit cuticle layer was dramatically reduced in the pe lg mutant. The epidermal cells of the leaves were also deformed in the pe lg mutant, suggesting that leaf cuticle formation was also disrupted in the mutant. Consistent with this, transmission electron microscopic analysis showed that the electron density of the cuticle layer of the adaxial surface of the leaf was reduced in the pe lg mutant compared to WT, suggesting that there are changes in cuticle structure and/or composition in the pe lg mutant. Both physiological analysis to measure the rate of transpiration, and staining of the fruits and leaves with toluidine blue, revealed that water permeability was enhanced in the pe lg mutant, consistent with the reduced thickness of its cuticle layer. Taken together the preliminary analyses of the cuticle components, the PE LG is most likely involved in proper cuticle formation.     

Identification of vitamin A-free cells in a stellate cell-enriched fraction of normal rat liver as myofibroblasts

Myofibroblasts (MFs) as well as hepatic stellate cells (HSCs) are known to be involved in liver fibrogenesis. Quiescent HSCs (qHSCs) in culture have been thought to differentiate to replicative activated HSCs (aHSCs). In this study a qHSC-enriched fraction isolated by Nycodenz-isodensity centrifugation was separated with a fluorescence-activated cell sorter, which revealed the presence of a small fraction (occupancy rate = 0.4%) of cells that did not show vitamin A-autofluorescence under ultraviolet (UV)-irradiation (UV⁻ cells). The remaining vitamin A-containing cells were autofluorescent (UV⁺) and originally expressed markers of qHSCs, and, in culture, did not grow, lost vitamin A, and expressed markers of aHSCs. UV⁻ cells showed morphology of MFs, and, in culture, grew to form colonies and expressed markers of MFs. These results indicated that UV⁺ and UV⁻ cells represent qHSCs and MFs, respectively, and that aHSCs have no growth potential and are a cell-type distinct from proliferative MFs. Gene expression profiles of UV⁻ cells (MFs) newly identified gremlin as one of MF-preferential genes and its proteins were localized around fibrotic septa in rat and human livers. In addition, we suggested that the qHSC-enriched fraction included approximately 6% of liver MFs.