Characterization of microsomal and mitochondrial phospholipase D activities and cloning of a phospholipase D alpha cDNA from strawberry fruits.

Phospholipase D alpha (PLD, EC 3.1.4.4)) is a key enzyme involved in membrane deterioration that occurs during fruit ripening and senescence. The biochemical and molecular characteristics of PLD was studied in strawberry (Fragaria ananassa Duch) fruits, which are non-climacteric fruits. PLD activity was primarily associated with the mitochondrial and microsomal fractions and showed increased activity during development. Optimal pH levels of activity were observed at 5.5 and 6.5 for mitochondrial PLD and at 5 and 7 for microsomal PLD. Calcium enhanced microsomal PLD activity at 1-40 microM levels. PLD activity followed Michaelis-Menten kinetics. Lineweaver-Burk analysis gave Km values in the range of 114 and 277 microM using dipalmitoylphosphatidylcholine (DPPC) as substrate for mitochondrial and microsomal PLD, respectively. The Vmax value for the microsomal PLD was nearly 12-fold higher than that of mitochondrial PLD. A 2874 bp full-length cDNA for PLD alpha was amplified from strawberry fruit mRNA using RT-PCR and 5'- and 3'-RACE encoding an 810 amino acid-polypeptide. The predicted strawberry PLD sequence showed the characteristic C2 domain and the phospholipase domains conferring calcium sensitivity and the enzyme activity, respectively. The strawberry PLD alpha showed a high degree of similarity to other PLD alphas from plants. The implications of PLD regulation during ripening of fruits are discussed.     

Chemerin‐induced mitochondrial dysfunction in skeletal muscle

Chemerin is a novel adipocyte‐derived factor that induces insulin resistance in skeletal muscle. However, the effect of chemerin on skeletal muscle mitochondrial function has received little attention. In the present study, we investigated whether mitochondrial dysfunction is involved in the pathogenesis of chemerin‐mediated insulin resistance. In this study, we used recombinant adenovirus to express murine chemerin in C57BL/6 mice. The mitochondrial function and structure were evaluated in isolated soleus muscles from mice. The oxidative mechanism of mitochondrial dysfunction in cultured C2C12 myotubes exposed to recombinant chemerin was analysed by western blotting, immunofluorescence and quantitative real‐time polymerase chain reaction. The overexpression of chemerin in mice reduced the muscle mitochondrial content and increased mitochondrial autophagy, as determined by the increased conversion of LC3‐I to LC3‐II and higher expression levels of Beclin1 and autophagy‐related protein‐5 and 7. The chemerin treatment of C2C12 myotubes increased the generation of mitochondrial reactive oxygen species, concomitant with a reduced mitochondrial membrane potential and increased the occurrence of mitochondrial protein carbonyls and mitochondrial DNA deletions. Knockdown of the expression of chemokine‐like receptor 1 or the use of mitochondria‐targeting antioxidant Mito‐TEMPO restored the mitochondrial dysfunction induced by chemerin. Furthermore, chemerin exposure in C2C12 myotubes not only reduced the insulin‐stimulated phosphorylation of protein kinase B (AKT) but also dephosphorylated forkhead box O3α (FoxO3α). Chemerin‐induced mitochondrial autophagy likely through an AKT‐FoxO3α‐dependent signalling pathway. These findings provide direct evidence that chemerin may play an important role in regulating mitochondrial remodelling and function in skeletal muscle.     

Prohibitin involvement in the generation of mitochondrial superoxide at complex I in human sperm

Prohibitin (PHB), a major mitochondrial membrane protein, has been shown earlier in our laboratoryto regulate sperm motility via an alteration in mitochondrial membrane potential (MMP) in infertile men with poor sperm quality. To test if PHB expression is associated with sperm mitochondrial superoxide (mROS) levels, here we examined sperm mROS levels, high MMP and lipid peroxidation in infertile men with poor sperm motility (asthenospermia, A) and/or low sperm concentrations (oligoasthenospermia, OA). The diaphorase‐type activity of sperm mitochondrial complex I (MCI) and PHB expression were also determined. We demonstrate that mROS and lipid peroxidation levels are significantly higher in sperm from A and OA subjects than in normospermic subjects, whereas high MMP and PHB expression are significantly lower. A positive correlation between mROS and lipid peroxidation and a negative correlation of mROS with PHB expression, high MMP, and sperm motility were found in these subjects. The finding of similar diaphorase‐type activity levels of sperm MCI in the three groups studied suggests that the catalytic subunits of MCI in the matrix arm may produce mROS on its own. There may be a dysfunction of electron transport at MCI associated with decreased expression of PHB in sperm with poor quality. We conclude that mROS level is increased and associated with decreased PHB expression, and it may regulate sperm motility via increases in low MMP and lipid peroxidation. This is the first report on the involvement of PHB in human sperm motility loss associated with increased generation of mROS at MCI.     

Enhanced seed viability and lipid compositional changes during natural ageing by suppressing phospholipase Dα in soybean seed

Changes in phospholipid composition and consequent loss of membrane integrity are correlated with loss of seed viability. Furthermore, phospholipid compositional changes affect the composition of the triacylglycerols (TAG), i.e. the storage lipids. Phospholipase D (PLD) catalyses the hydrolysis of phospholipids to phosphatidic acid, and PLDα is an abundant PLD isoform. Although wild‐type (WT) seeds stored for 33 months were non‐viable, 30%–50% of PLDα‐knockdown (PLD‐KD) soybean seeds stored for 33 months germinated. WT and PLD‐KD seeds increased in lysophospholipid levels and in TAG fatty acid unsaturation during ageing, but the levels of lysophospholipids increased more in WT than in PLD‐KD seeds. The loss of viability of WT seeds was correlated with alterations in ultrastructure, including detachment of the plasma membrane from the cell wall complex and disorganization of oil bodies. The data demonstrate that, during natural ageing, PLDα affects the soybean phospholipid profile and the TAG profile. Suppression of PLD activity in soybean seed has potential for improving seed quality during long‐term storage.     

Changes in mitochondrial membrane composition and oxidative status during rapid growth,maturation and aging in zebrafish, Danio rerio

Considering membranes and membrane components as possible pacemakers of the main processes taking place inside mitochondria, changes in phospholipids or fatty acids could play a central role linking different mechanisms involved in cumulative damage to cell molecules and dysfunction during periods of high stress, such as rapid growth and aging. Changes affecting either lipid class or fatty acid compositions could affect phospholipid and membrane properties and alter mitochondrial function and cell viability. In the present study, mitochondrial oxidative status and mitochondrial membrane phospholipid compositions were analyzed throughout the life-cycle of zebrafish. TBARS content significantly increased in 18-month-old fish while aconitase activity decreased in 24-month-old fish, which have been related with oxidative damage to molecules. Mitochondria-specific superoxide dismutase decreased in 24-month-old animals although this change was not statistically significant. Age affected both mitochondrial phospholipid content and the peroxidation index of most phospholipid classes suggesting that oxidative damage to mitochondrial lipids was occurring.     

Parkinson's disease-associated DJ-1 mutations impair mitochondrial dynamics and cause mitochondrial dysfunction

Mitochondrial dysfunction represents a critical event during the pathogenesis of Parkinson's disease (PD) and expanding evidences demonstrate that an altered balance in mitochondrial fission/fusion is likely an important mechanism leading to mitochondrial and neuronal dysfunction/degeneration. In this study, we investigated whether DJ-1 is involved in the regulation of mitochondrial dynamics and function in neuronal cells. Confocal and electron microscopic analysis demonstrated that M17 human neuroblastoma cells over-expressing wild-type DJ-1 (WT DJ-1 cells) displayed elongated mitochondria while M17 cells over-expressing PD-associated DJ-1 mutants (R98Q, D149A and L166P) (mutant DJ-1 cells) showed significant increase of fragmented mitochondria. Similar mitochondrial fragmentation was also noted in primary hippocampal neurons over-expressing PD-associated mutant forms of DJ-1. Functional analysis revealed that over-expression of PD-associated DJ-1 mutants resulted in mitochondria dysfunction and increased neuronal vulnerability to oxidative stress (H(2) O(2)) or neurotoxin. Further immunoblot studies demonstrated that levels of dynamin-like protein (DLP1), also known as Drp1, a regulator of mitochondrial fission, was significantly decreased in WT DJ-1 cells but increased in mutant DJ-1 cells. Importantly, DLP1 knockdown in these mutant DJ-1 cells rescued the abnormal mitochondria morphology and all associated mitochondria/neuronal dysfunction. Taken together, these studies suggest that DJ-1 is involved in the regulation of mitochondrial dynamics through modulation of DLP1 expression and PD-associated DJ-1 mutations may cause PD by impairing mitochondrial dynamics and function.     

植物磷脂酶D基因表达与衰老的关系

磷脂酶D (PLD)是一种重要的磷脂水解酶,在植物细胞中普遍存在。磷脂酶D能激活许多重要的细胞生理功能,包括调控细胞膜的重建、跨膜信号传导及细胞内调控、细胞骨架组装、防御反应以及种子萌发和植物的衰老等。对磷脂酶D的基本特性、磷脂酶D基因特异性表达模式及其活性抑制与植物衰老的关系进行了综述,并探讨和展望了今后植物磷脂酶D基因的研究方向。     

Vildagliptin improves high glucose‐induced endothelial mitochondrial dysfunction via inhibiting mitochondrial fission

The dipeptidyl peptidase 4 inhibitor vildagliptin (VLD), a widely used anti‐diabetic drug, exerts favourable effects on vascular endothelium in diabetes. We determined for the first time the improving effects of VLD on mitochondrial dysfunction in diabetic mice and human umbilical vein endothelial cells (HUVECs) cultured under hyperglycaemic conditions, and further explored the mechanism behind the anti‐diabetic activity. Mitochondrial ROS (mtROS) production was detected by fluorescent microscope and flow cytometry. Mitochondrial DNA damage and ATP synthesis were analysed by real time PCR and ATPlite assay, respectively. Mitochondrial network stained with MitoTracker Red to identify mitochondrial fragmentation was visualized under confocal microscopy. The expression levels of dynamin‐related proteins (Drp1 and Fis1) were determined by immunoblotting. We found that VLD significantly reduced mtROS production and mitochondrial DNA damage, but enhanced ATP synthesis in endothelium under diabetic conditions. Moreover, VLD reduced the expression of Drp1 and Fis1, blocked Drp1 translocation into mitochondria, and blunted mitochondrial fragmentation induced by hyperglycaemia. As a result, mitochondrial dysfunction was alleviated and mitochondrial morphology was restored by VLD. Additionally, VLD promoted the phosphorylation of AMPK and its target acetyl‐CoA carboxylase in the setting of high glucose, and AMPK activation led to a decreased expression and activation of Drp1. In conclusion, VLD improves endothelial mitochondrial dysfunction in diabetes, possibly through inhibiting Drp1‐mediated mitochondrial fission in an AMPK‐dependent manner.     

Warburg effect—damping of electromagnetic oscillations

Mitochondrial dysfunction is a central defect in cells creating the Warburg and reverse Warburg effect cancers. However, the link between mitochondrial dysfunction and cancer has not yet been clearly explained. Decrease of mitochondrial oxidative energy production to about 50 % in comparison with healthy cells may be caused by inhibition of pyruvate transfer into mitochondrial matrix and/or disturbed H⁺ ion transfer across inner mitochondrial membrane into cytosol. Lowering of the inner membrane potential and shifting of the working point of mitochondria to high values of pH above an intermediate point causes reorganization of the ordered water layer at the mitochondrial membrane. The reorganized ordered water layers at high pH values release electrons which are transferred to the cytosol rim of the layer. The electrons damp electromagnetic activity of Warburg effect cancer cells or fibroblasts associated with reverse Warburg effect cancer cells leading to lowered electromagnetic activity, disturbed coherence, increased frequency of oscillations and decreased level of biological functions. In reverse Warburg effect cancers, associated fibroblasts supply energy-rich metabolites to the cancer cell resulting in increased power of electromagnetic field, fluctuations due to shift of oscillations to an unstable nonlinear region, decreased frequency and loss of coherence.     

Phospholipid and triacylglycerol profiles modified by PLD suppression in soybean seed

Phospholipase D (PLD) is capable of hydrolyzing membrane phospholipids, producing phosphatidic acid. To alter phospholipid profiles in soybean seed, we attenuated PLD enzyme activity by an RNA interference construct using the partial sequence from a soybean PLDα gene. Two transgenic soybean lines were established by particle inflow gun (PIG) bombardment by co‐bombarding with pSPLDi and pHG1 vectors. The lines were evaluated for the presence and expression of transgenes thoroughly through the T₄ generation. PLD‐suppressed soybean lines were characterized by decreased PLDα enzyme activity and decreased PLDα protein both during seed development and in mature seeds. There was no change in total phospholipid amount; however, the PLD‐attenuated transgenic soybean seed had higher levels of di18 : 2 (dilinoleoyl)‐phosphatidylcholine (PC) and ‐phosphatidylethanolamine (PE) in seeds than the non‐transgenic lines. The increased polyunsaturation was at the expense of PC and PE species containing monounsaturated or saturated fatty acids. In addition to increased unsaturation in the phospholipids, there was a decrease in unsaturation of the triacylglycerol (TAG) fraction of the soybean seeds. Considering recent evidence for the notion that desaturation of fatty acids occurs in the PC fraction and that the PC → DAG (diacylglycerol) → TAG pathway is the major route of TAG biosynthesis in developing soybean seed, the current data suggest that PLDα suppression slows the conversion of PC to TAG. This would be consistent with PLD playing a positive role in that conversion. The data indicate that soybean PLD attenuation is a potentially useful approach to altering properties of edible and industrial soybean lecithin.     

Ageing-Induced Alterations in Lipid/Phospholipid Profiles of Rat Brain and Liver Mitochondria: Implications for Mitochondrial Energy-Linked Functions

Effects of ageing on the lipid/phospholipid profile of brain and liver mitochondria from rats were examined. In the brain mitochondria the contents of total phospholipid (TPL) and cholesterol (CHL) increased with simultaneous increase in the TPL/CHL (mole:mole) ratio. The proportion and contents of lysophospholipid (Lyso), sphingomyelin (SPM), phosphatidylinositol (PI), phosphatidylserine (PS) and diphosphatidylglycerol (DPG) components increased, with maximal increases seen for PS and PI; phosphatidylcholine (PC) and phosphatidylethanolamine (PE) components registered decrease. In the liver mitochondria contents of TPL and CHL increased. However, the TPL/CHL (mole:mole) ratio was not altered. Lyso, PI and PS increased. However, the magnitude of increase was competitively lower; PE and DPG decreased. SPM and PC did not change as a consequence of ageing. These changes altered the contents of individual phospholipids in the two membrane systems. Respiration with glutamate, pyruvate + malate, succinate and ascorbate + N,N,N’,N’-tetramethyl-p-phenylenediamine was significantly impaired in brain mitochondria from old animals. For liver mitochondria the respiratory activity declined with glutamate and succinate. Correlation studies by regression analysis revealed that the lipid/phospholipid classes regulate respiratory function differently in the mitochondria from the two tissues. The respiration-related parameters in the brain mitochondria were dependent on multiple lipid/phospholipid components, and the process of regulation was complex compared to the liver mitochondrial functions.     

Alterations of sarcolemmal phospholipase D and phosphatidate phosphohydrolase in congestive heart failure

Phospholipase D 2 (PLD2) is the major PLD isozyme associated with the cardiac sarcolemmal (SL) membrane. Hydrolysis of SL phosphatidylcholine (PC) by PLD2 produces phosphatidic acid (PA), which is then converted to 1,2 diacylglycerol (DAG) by the action of phosphatidate phosphohydrolase type 2 (PAP2). In view of the role of both PA and DAG in the regulation of Ca²⁺ movements and the association of abnormal Ca²⁺ homeostasis with congestive heart failure (CHF), we examined the status of both PLD2 and PAP2 in SL membranes in the infarcted heart upon occluding the left coronary artery in rats for 1, 2, 4, 8 and 16 weeks. A time-dependent increase in both SL PLD2 and PAP2 activities was observed in the non-infarcted left ventricular tissue following myocardial infarction (MI); however, the increase in PAP2 activity was greater than that in PLD2 activity. Furthermore, the contents of both PA and PC were reduced, whereas that of DAG was increased in the failing heart SL membrane. Treatment of the CHF animals with imidapril, an angiotensin-converting enzyme (ACE) inhibitor, attenuated the observed changes in heart function, SL PLD2 and PAP2 activities, as well as SL PA, PC and DAG contents. The results suggest that heart failure is associated with increased activities of both PLD2 and PAP2 in the SL membrane and the beneficial effect of imidapril on heart function may be due to its ability to prevent these changes in the phospholipid signaling molecules in the cardiac SL membrane.     

Full recovery of the NADH:ubiquinone activity of complex I (NADH:ubiquinone oxidoreductase) from Yarrowia lipolytica by the addition of phospholipids

Phospholipase D 2 (PLD2) is the major PLD isozyme associated with the cardiac sarcolemmal (SL) membrane. Hydrolysis of SL phosphatidylcholine (PC) by PLD2 produces phosphatidic acid (PA), which is then converted to 1,2 diacylglycerol (DAG) by the action of phosphatidate phosphohydrolase type 2 (PAP2). In view of the role of both PA and DAG in the regulation of Ca(2+) movements and the association of abnormal Ca(2+) homeostasis with congestive heart failure (CHF), we examined the status of both PLD2 and PAP2 in SL membranes in the infarcted heart upon occluding the left coronary artery in rats for 1, 2, 4, 8 and 16 weeks. A time-dependent increase in both SL PLD2 and PAP2 activities was observed in the non-infarcted left ventricular tissue following myocardial infarction (MI); however, the increase in PAP2 activity was greater than that in PLD2 activity. Furthermore, the contents of both PA and PC were reduced, whereas that of DAG was increased in the failing heart SL membrane. Treatment of the CHF animals with imidapril, an angiotensin-converting enzyme (ACE) inhibitor, attenuated the observed changes in heart function, SL PLD2 and PAP2 activities, as well as SL PA, PC and DAG contents. The results suggest that heart failure is associated with increased activities of both PLD2 and PAP2 in the SL membrane and the beneficial effect of imidapril on heart function may be due to its ability to prevent these changes in the phospholipid signaling molecules in the cardiac SL membrane.     

Evidence of mitochondrial involvement in scrapie infection.

Two cDNA libraries were constructed from brain membrane and cytoskeletal preparations purified from scrapie-infected hamster brains. Four recombinants strongly preferential to the scrapie cytoskeletal preparation were identified by the differential hybridization of 7,000 recombinants. These clones were not, however, preferential to total nucleic acids extracted from scrapie-infected hamster brains. DNA sequence analysis revealed all four clones to have significant sequence similarities to the mouse mitochondrial genome. This correlation led us to consider a mitochondrial association with scrapie infectivity. Brain mitochondria were purified by sucrose gradient density centrifugation and found to contain high infectivity. Removal of mitochondrial outer membranes by osmotic shock or digitonin treatment resulted in no detectable loss of titer.     

Effect of β-amyloid (25-35) on mitochondrial function and expression of mitochondrial permeability transition pore proteins in rat hippocampal neurons

The aim of this study was to assess the effect of the β‐amyloid fragment Aβ_25–35 on mitochondrial structure and function and on the expression of proteins associated with the mitochondrial permeability transition pore (MPTP) in rat hippocampal neurons. Ninety clean‐grade Sprague–Dawley rats were randomly assigned to six groups (n = 15 per group). Aβ_25–35 (1, 5, or 10 µg/rat) was injected into hippocampal area CA1. Normal saline was injected as a control. The effect of Aβ_25–35 injection on hippocampal structure was assessed by transmission electron microscopy. Ca²⁺‐ATPase activity, [Ca²⁺]_i, and mitochondrial membrane potential were measured. The expression of genes associated with the MPTP, including the voltage‐dependent anion channel (VDAC), adenine nucleotide translocator (ANT), and cyclophilin D (Cyp‐D), were evaluated. Results showed that Aβ_25–35 injection damaged the mitochondrial structure of hippocampal neurons, decreased Ca²⁺‐ATPase activity and mitochondrial membrane potential, and increased [Ca²⁺]_i. The expression levels for VDAC, ANT, and Cyp‐D in all groups were significantly (P < 0.05) higher than those in the normal control group after Aβ_25–35 injection. These results indicate that Aβ_25–35 damages mitochondria in rat hippocampal neurons and effects mitochondrial dysfunction, as well as increasing the expression of genes associated with the MPTP. Mitochondrial dysfunction may result in increased MPTP gene expression, leading to neurodegenerative effects. J. Cell. Biochem. 112: 1450–1457, 2011. © 2011 Wiley‐Liss, Inc.     

Effect of Dietary Lipids on Phospholipase D Activity in Rat Brain

Phospholipase D (PLD) is emerging as a major player in many novel signaling pathways. Based on recent studies correlating membrane composition with enzyme function, we speculated that feeding of dietary lipids to the newborns has a major impact on brain PLD activity. To test this hypothesis, the rat dams were fed fat-free powder containing either safflower oil or fish oil, and a control powdered chow. The pups were weaned onto the diet and sacrificed at 30 days of age. PLD activity was measured by transphosphatidylation assays using rat brain membranes. This study shows that microsome GTPS-dependent PLD activity in rats fed safflower oil or fish oil was significantly reduced by 38% and 30% respectively compared to controls. Oleate-dependent PLD activity in the safflower oil group, however, was significantly increased by 38%. In contrast, synaptosome membrane (P2) GTPS-dependent PLD activity in rats consuming safflower oil was significantly increased by 29%, but there was no difference in oleate-dependent PLD activity. Likewise, no difference was observed in microsome oleate-dependent PLD and P2 GTPS-dependent PLD activity between the fish oil and the control groups. These results indicate that dietary lipid intake appears to modulate phospholipid metabolism and differential expression of PLD isozymes in the brain.