Hydrolysis Of Phosphatidylcholine Research Articles

Lipidomics of sheep and goat Milk-based infant formulae during in vitro dynamic digestion

Lipid hydrolysis process during IF digestion, particularly the characterization of the lipidome and the resulting lipid breakdown products, has not been thoroughly investigated. This study aimed to compare the lipid hydrolysis profiles during the in vitro dynamic digestion of IFs made from whole sheep and goat milk. Using a lipidomics platform and multivariate statistical analysis, we observed changes in complex lipid levels during digestion. In the gastric compartment, we noted a progressive hydrolysis of triacylglycerols, phosphatidylcholines, and sphingomyelins. Conversely, lipolysis breakdown products like monoacylglycerols (e.g., MG(16:0), MG(18:0)), diacylglycerols, lysophosphatidylcholines (LPC 16:0, LPC 18:1, LPC 18:2), and free fatty acids increased in the intestinal compartment. The lipolysis trends were similar for both types of infant formulas, with long-chain fatty acid triglycerides (C > 46) exhibiting lower digestibility compared to medium-chain fatty acid triglycerides. Overall, these results indicate that sheep milk can be used as an ingredient in the manufacturing of IF.

Baicalein loaded liposome with hyaluronic acid and Polyhexamethylene guanidine modification for anti methicillin-resistant Staphylococcus aureus infection

Targeting delivery to the infection site and good affinity of vehicle to the bacterial are two main concerns in therapy of bacterial infection, and on-demand release of drug is another important issue. In this work, a liposome drug delivery system (HA/P/BAI-lip) incorporated with baicalein and modified by PHMG and HA was prepared. Several characterizations were conducted to examine the physical properties of liposome. Then it was applied to treatments of MRSA induced dorsal subcutaneous abscess model and the thigh muscle infected model. The presence of guanidine group in HA/P/BAI-lip rendered the liposome satisfactory bacterial target ability and good pH sensitive properties. The lipase secreted by bacterial could promote the hydrolysis of soybean phosphatidylcholine (SPC) in liposome. The modification of HA in HA/P/BAI-lip could lead the drug system to the exact infected site where CD44 was abundant because of inflammation. The low pH microenvironment characteristic of bacterial infection could induce the swelling of liposome following by degradation. Taken together, baicalein could be released selectively at the infected site to exert antibacterial capacity. HA/P/BAI-lip showed impressive antibacterial ability and dramatically decrease the bacterial burden of infection site and alleviate the infiltration of inflammatory cells, facilitating the recovery of infection.

The role of lysosomal phospholipase A2 in the catabolism of bis(monoacylglycerol)phosphate and association with phospholipidosis

Bis(monoacylglycerol)phosphate (BMP) is an acidic glycerophospholipid localized to late endosomes and lysosomes. However, the metabolism of BMP is poorly understood. Because many drugs that cause phospholipidosis inhibit lysosomal phospholipase A2 (LPLA2, PLA2G15, LYPLA3) activity, we investigated whether this enzyme has a role in BMPcatabolism. The incubation of recombinant human LPLA2 (hLPLA2) and liposomes containing the naturally occurring BMP (sn-(2-oleoyl-3-hydroxy)-glycerol-1-phospho-sn-1’-(2′-oleoyl-3′-hydroxy)-glycerol (S,S-(2,2′,C18:1)-BMP) resulted in the deacylation of this BMP isomer. The deacylation rate was 70 times lower than that of dioleoyl phosphatidylglycerol (DOPG), an isomer and precursor of BMP. The release rates of oleic acid from DOPG and four BMP stereoisomers by LPLA2 differed. The rank order of the rates of hydrolysis were DOPG>S,S-(3,3′,C18:1)-BMP>R,S-(3,1′,C18:1)-BMP>R,R-(1,1′,C18:1)>S,S-(2,2′)-BMP. The cationic amphiphilic drug amiodarone (AMD) inhibited the deacylation of DOPG and BMP isomers by hLPLA2 in a concentration-dependent manner. Under these experimental conditions, the IC50s of amiodarone-induced inhibition of the four BMP isomers and DOPG were less than 20 μM and approximately 30 μM, respectively. BMP accumulation was observed in AMD-treated RAW 264.7 cells. The accumulated BMP was significantly reduced by exogenous treatment of cells with active recombinant hLPLA2 but not with diisopropylfluorophosphate-inactivated recombinant hLPLA2. Finally, a series of cationic amphiphilic drugs known to cause phospholipidosis were screened for inhibition of LPLA2 activity as measured by either the transacylation or fatty acid hydrolysis of BMP or phosphatidylcholine as substrates. Fifteen compounds demonstrated significant inhibition with IC50s ranging from 6.8 to 63.3 μM. These results indicate that LPLA2 degrades BMP isomers with different substrate specificities under acidic conditions and may be the key enzyme associated with BMP accumulation in drug-induced phospholipidosis.

Application of the thin layer chromatography method for the determination of the degree of hydrolysis of soy lecithin

The aim of this study was the determination of the degree of enzymatic hydrolysis of soy lecithin using thin layer chromatography in combination with a modern imaging data processing program. Hydrolysis of phospholipids was carried by the action of the enzyme phospholipase A2 (PLA2) in the presence of calcium ions for 2 h at a temperature of 50°C and pH 5.7. Chromatographic separation of the components of the reaction medium was carried out 10, 20, 30, 60, 90, and 120 min after adding the PLA2 in the chloroform: methanol: ammonium hydroxide system in a ratio of 6.5 : 2.5 : 0.4 (v/v/v). The following phospholipids and their lysoforms were identified: Rf = 0.41±0.03 for phosphatidylethanolamine (PE), Rf = 0.28±0.02 for phosphatidylcholine (PC), Rf = 0.09±0.01 for phosphatidylinositol (PI), Rf = 0.06±0.01 for phosphatidic acid (PA), Rf = 0.15±0.02 for lysophosphatidylethanolamine (LPE), Rf = 0.08±0.01 for lysophosphatidylcholine (LPC), Rf = 0.03±0.01 for lysophosphatidylinositol (LPI) and Rf = 0.02±0.01 for lysophosphatidic acid (LPA). It was shown that soy lecithin contained PC, PE, PI, and PA before the enzymatic reaction, the content of these compounds after the addition of the PLA2 decreases due to the formation of LPC, LPE, LPI, and LPA, respectively. The dependence of the degree of hydrolysis (α, %) of PC on the duration of the enzymatic reaction was obtained. It was established that the intensive conversion of PC to LPC under the action of the PLA2 occurred during 60 min (α = 82%). Further conversion of PC occurred insignificantly: 84% after 90 min and 87% after 120 min, which was due to the inhibition of enzyme activity by the final reaction product.

Hydrolysis of Phospholipids in Presence of Phospholipase D:Thrmodynamic and Kinetic Studies of Hydrolysis in Water and Alcoholic Environments

Phospholipase D(PLD)catalyzes the hydrolysis of phospholipids to produce phosphatidic acid and hydroxyl compounds. Phosphatidylcholine (PC) is the most abundant phospholipid in animals and plants, often constituting nearly 50% of total complex lipids in animals and plants. However, Phosphatidylserine (PS) makes up around 5–10% of all phospholipids. This work includes the monitoring thermodynamic and kinetic of the enzymatic hydrolysis of PC and PS in both water and ethanol. All experiments were performedusing the Schlink line technique inN2present as an inert gas to prevent the oxidative stress. UV/Vis. spectrophotometer was used to observe the kinetic of all enzymatic reactions. In addition, the Origin 2019 software was used to analyze and find the kinetic parameters of the enzymatic hydrolysis reactions. The results show that the enzymatic hydrolys is reactions of PC in water and in ethanol occurredat the same conditions, enzymatic activity 1.752 U/mg, temperature 37 ᵒC, and pH =7. However, the enzymatic hydrolysis reactions of PS in water occurred at different conditions than in ethanol. The enzymatic substitution reaction of PSinethanol was thermodynamic favorable reaction due to the value of the ∆G =_164.868 J, but in water was thermodynamically unfavorable ΔG = 65.048 J. However, the enzymatic hydrolysis reaction of PC in water was thermodynamically unfavorable ∆G = 345.319 Jaswellas in ethanol ∆G = 74.433 J. The study shows that there is clear impact of present nitrogen bases of PC and the environment of the hydrolysis on the activity of the enzymatic catalyzing.

The wide world of non-mammalian phospholipase D enzymes

Phospholipase D (PLD) hydrolyses phosphatidylcholine (PtdCho) to produce free choline and the critically important lipid signaling molecule phosphatidic acid (PtdOH). Since the initial discovery of PLD activities in plants and bacteria, PLDs have been identified in a diverse range of organisms spanning the taxa. While widespread interest in these proteins grew following the discovery of mammalian isoforms, research into the PLDs of non-mammalian organisms has revealed a fascinating array of functions ranging from roles in microbial pathogenesis, to the stress responses of plants and the developmental patterning of flies. Furthermore, studies in non-mammalian model systems have aided our understanding of the entire PLD superfamily, with translational relevance to human biology and health. Increasingly, the promise for utilization of non-mammalian PLDs in biotechnology is also being recognized, with widespread potential applications ranging from roles in lipid synthesis, to their exploitation for agricultural and pharmaceutical applications.

Multi‐omics approach reveals disturbances in brain phosphatidylcholine metabolism in Alzheimer’s Disease

AbstractBackgroundLipid dysregulation is implicated in Alzheimer’s disease (AD) pathology, given its involvement in amyloid β processing, myelination, and cell membrane structure. Studies have demonstrated increased phosphatidylcholine (PC) hydrolysis in the brains of AD individuals, but the connections between AD and PC metabolism are not fully understood.MethodTo investigate the alterations in PC metabolism in AD, we analyzed publicly available transcriptomic, metabolomic, and lipidomic data from the ROSMAP study, which included dorsolateral prefrontal cortex (DLPFC) tissue and serum samples. DLPFC and serum samples were classified based on clinical consensus diagnosis of cognitive status at the time of death and the last valid clinical diagnosis, respectively. Concentration differences were determined with a linear model using the Limma R package.ResultAccording to the lipidomic results, the concentration of total PC‐containing glycerophospholipid was significantly decreased in AD DLPFC samples (P < 0.05). Specifically, diacyl‐PC and acyl‐alkyl‐PC, the two most abundant subclasses, were significantly decreased in the brains of AD patients (P < 0.05). The concentration of the PC breakdown product, glycerophosphocholine (GPC), was increased in AD patient brains (P‐value < 0.05). However, GPC phosphodiesterase 1 (GPCPD1), the enzyme that further hydrolyzes GPC to free choline, was downregulated and choline abundance was reduced in AD DLPFC samples (P < 0.05). The expression of phosphatidylethanolamine N‐methyltransferase (PEMT), which converts PE to PC, was reduced as was the abundance of PEMT products (PC 18:2/20:4 and PC 18:0/20:4, P < 0.05). These alterations in the brain were not observed in the serum.ConclusionThese results indicate that PC metabolism is disturbed in the DLPFC of AD patients, with evidence of increased PC breakdown and reduced GPC hydrolysis as well as reduced conversion of PE to PC in AD brains. However, these changes in the brain are not reflected in serum. Further studies are needed to better understand changes in phospholipid metabolism in the brains of AD patients.

Preparation of vacuum-assisted conjugated linoleic acid phospholipids under nitrogen: Mechanism of acyl migration of lysophospholipids

Sn-Glycerol-3-phosphatidylcholine (GPC) was prepared by hydrolysis of phosphatidylcholine (PC) catalyzed by phospholipase A1 (PLA1). Nitrogen flow assisted the esterification of conjugated linoleic acid (CLA) and GPC to produce conjugated linoleic acid lysophosphatidylcholine (LPC − CLA). The effects of different reaction conditions on the PC conversion and acyl migration rates were investigated, and the acyl migration mechanism under acidic and alkaline conditions was studied. In addition, the optimum conditions for the esterification of CLA and GPC were selected. The optimal condition for the hydrolysis of PC was an enzyme loading of 5 %, pH of 5, reaction temperature of 50 ℃, and reaction time of 3 h. The results also showed that the maximum esterification rate reached 82.37 % at an enzyme loading of 15 %, CLA/GPC molar ratio of 50:1, and vacuum pressure of 13.3 kPa. This study not only improved the bioavailability of PC but also effectively increased the content of LPC − CLA.

Human sphingomyelin synthase 1 generates diacylglycerol in the presence and absence of ceramide via multiple enzymatic activities.

Sphingomyelin (SM) synthase 1 (SMS1), which is involved in lipodystrophy, deafness, and thrombasthenia, generates diacylglycerol (DG) and SM using phosphatidylcholine (PC) and ceramide as substrates. Here, we found that SMS1 possesses DG-generating activities via hydrolysis of PC and phosphatidylethanolamine (PE) in the absence of ceramide and ceramide phosphoethanolamine synthase (CPES) activity. In the presence of the same concentration (4.7 mol%) of PC and ceramide, the amounts of DG produced by SMS and PC-phospholipase C (PLC) activities of SMS1 were approximately 65% and 35% of total DG production, respectively. PC-PLC activity showed substrate selectivity for saturated and/or monounsaturated fatty acid-containing PC species. A PC-PLC/SMS inhibitor, D609, inhibited only SMS activity. Mn2+ inhibited only PC-PLC activity. Intriguingly, DG attenuated SMS/CPES activities. Our study indicates that SMS1 is a unique enzyme with PC-PLC/PE-PLC/SMS/CPES activities.

A newly developed PLD1 inhibitor ameliorates rheumatoid arthritis by regulating pathogenic T and B cells and inhibiting osteoclast differentiation

Phospholipase D1 (PLD1), which catalyzes the hydrolysis of phosphatidylcholine to phosphatidic acid and choline, plays multiple roles in inflammation. We investigated the therapeutic effects of the newly developed PLD1 inhibitors A2998, A3000, and A3773 in vitro and in vivo rheumatoid arthritis (RA) model. A3373 reduced the levels of LPS-induced TNF-α, IL-6, and IgG in murine splenocytes in vitro. A3373 also decreased the levels of IFN-γ and IL-17 and the frequencies of Th1, Th17 cells and germinal-center B cells, in splenocytes in vitro. A3373 ameliorated the severity of collagen-induced arthritis (CIA) and suppressed infiltration of inflammatory cells into the joint tissues of mice with CIA compared with vehicle-treated mice. Moreover, A3373 prevented systemic bone demineralization in mice with CIA and suppressed osteoclast differentiation and the mRNA levels of osteoclastogenesis markers in vitro. These results suggest that A3373 has therapeutic potential for RA.

Potential risk resulting from the influence of static magnetic field upon living organisms. Numerically simulated effects of the static magnetic field upon model complex lipids

Background: Recognising effects of static magnetic field (SMF) of varying flux density on flora and fauna is attempted. For this purpose, the influence of static magnetic field is studied for molecules of five complex lipids i.e. such as β-carotene, sphingosine, ceramide, cholesterol and phosphatidylcholine. Methods: Computations of the effect of real SMF 0.0, 0.1, 1, 10 and 100 AMFU (Arbitrary Magnetic Field Unit; here 1AMFU > 1000 T) flux density were performed in silico (computer vacuum), involving advanced computational methods. Results: SMF polarises molecules depending on applied flux density. Only β-carotene survives exposure to SMF of 10 and 100 AMFU without radical splitting of some valence bonds. Molecules of remaining lipids suffered radical cleavage of some bonds on exposure to SMF of 10 and 100 AMFU. Manipulation with applied flux density provides either inhibition or stimulation of biological functions of the lipids under study. Conclusions: SMF destabilises complex lipids to the extent depending applied flux density. Biological functions of β-carotene are fairly sensitive to SMF, whereas only slight response to the effect of SMF is observed in case of sphingosine, ceramide and cholesterol. Enzymatic hydrolysis of phosphatidylcholine is stimulated by SMF regardless of the catalysed enzyme employed.

Transesterification of phosphatidylcholine with DHA-rich algal oil using immobilized Candida antarctica lipase B to produce DHA-phosphatidylcholine

Docosahexaenoic acid (DHA) enriched with phospholipids (PLs) (DHA-PLs) is a type of structured PL with good physicochemical and nutritional properties. Compared to PLs and DHA, DHA-PLs has higher bioavailability and structural stability and many nutritional benefits. To improve the enzymatic synthesis of DHA-PLs, this study investigated the preparation of phosphatidylcholine (PC) enriched with DHA (DHA-PC) via enzymatic transesterification of algal oil, which is rich in DHA-triglycerides, using immobilized Candida antarctica lipase B (CALB). The optimized reaction system incorporated 31.2% DHA into the acyl chain of PC and converted 43.6% PC to DHA-PC within 72 h at 50 °C, 1:8 PC: algal oil mass ratio, 25% enzyme load (based on total substrate mass), and 0.02 g/mL molecular sieve concentration. Consequently, the side reactions of PC hydrolysis were effectively suppressed and products with high PC content (74.8%) were produced. Molecular structure analysis showed that exogenous DHA was specifically incorporated into the sn-1 site of the PC by immobilized CALB. Furthermore, the evaluation of reusability with eight cycles showed that the immobilized CALB had good operational stability in the present reaction system. Collectively, this study demonstrated the applicability of immobilized CALB as a biocatalyst for synthesizing DHA-PC and provided an improved enzyme-catalyzed method for future DHA-PL synthesis.

Activity-based directed evolution of a membrane editor in mammalian cells.

Cellular membranes contain numerous lipid species, and efforts to understand the biological functions of individual lipids have been stymied by a lack of approaches for controlled modulation of membrane composition in situ. Here we present a strategy for editing phospholipids, the most abundant lipids in biological membranes. Our membrane editor is based on a bacterial phospholipase D (PLD), which exchanges phospholipid head groups through hydrolysis or transphosphatidylation of phosphatidylcholine with water or exogenous alcohols. Exploiting activity-dependent directed enzyme evolution in mammalian cells, we have developed and structurally characterized a family of 'superPLDs' with up to a 100-fold enhancement in intracellular activity. We demonstrate the utility of superPLDs for both optogenetics-enabled editing of phospholipids within specific organelle membranes in live cells and biocatalytic synthesis of natural and unnatural designer phospholipids in vitro. Beyond the superPLDs, activity-based directed enzyme evolution in mammalian cells is a generalizable approach to engineer additional chemoenzymatic biomolecule editors.

Fatty acid desaturation by stearoyl-CoA desaturase-1 controls regulatory T cell differentiation and autoimmunity

The imbalance between pathogenic and protective T cell subsets is a cardinal feature of autoimmune disorders such as multiple sclerosis (MS). Emerging evidence indicates that endogenous and dietary-induced changes in fatty acid metabolism have a major impact on both T cell fate and autoimmunity. To date, however, the molecular mechanisms that underlie the impact of fatty acid metabolism on T cell physiology and autoimmunity remain poorly understood. Here, we report that stearoyl-CoA desaturase-1 (SCD1), an enzyme essential for the desaturation of fatty acids and highly regulated by dietary factors, acts as an endogenous brake on regulatory T-cell (Treg) differentiation and augments autoimmunity in an animal model of MS in a T cell-dependent manner. Guided by RNA sequencing and lipidomics analysis, we found that the absence of Scd1 in T cells promotes the hydrolysis of triglycerides and phosphatidylcholine through adipose triglyceride lipase (ATGL). ATGL-dependent release of docosahexaenoic acid enhanced Treg differentiation by activating the nuclear receptor peroxisome proliferator-activated receptor gamma. Our findings identify fatty acid desaturation by SCD1 as an essential determinant of Treg differentiation and autoimmunity, with potentially broad implications for the development of novel therapeutic strategies and dietary interventions for autoimmune disorders such as MS.

Enzyme mediated production of asymmetrically charged liposomes.

In asymmetric liposomes, the inner and outer leaflets of the lipid bilayer have different compositions. As such, these liposomes are appealing platforms for modeling and studying membrane asymmetry in biological membranes. In addition, asymmetric liposomes can serve as innovative drug delivery vehicles in which, the inner leaflet can be designed for maximal cargo encapsulation while the outer leaflet can be optimized for minimal toxicity. This study aims to explore the use of a membrane-active enzyme, phospholipase D (PLD), to produce liposomes with asymmetrically-charged membranes. PLD is an interfacial enzyme that catalyzes the hydrolysis of zwitterionic phosphatidylcholine (PC) lipids to produce choline and negatively-charged phosphatidic acid (PA) lipids. For this work, we introduced PLD to pre-formed PC liposomes and monitored the zeta potential of these liposomes at different time points. Zeta potential of these liposomes showed a time-dependent drop, presumably due to PLD activity and production of negatively-charged PA lipids in the membrane. Using a calibration curve, the zeta potential values were converted to the amount of PA formed in the outer leaflet of liposomes. Notably, the use of heat-deactivated PLD enzyme did not have an impact on liposomal zeta values. In addition, the results of a choline assay that was used to quantify the produced choline and thus PA in the system were comparable to those from zeta measurements. Furthermore, performing these studies with different PLD concentrations showed that the rate of PA production varied with the concentration of enzyme. Lastly, experiments of dye leakage from liposomes suggested that this enzymatic activity did not have a significant impact on liposomal membrane integrity. Together, these findings indicate that PLD enzymatic activity can be utilized to generate liposomes with asymmetrically charged leaflets, providing exciting opportunities for membrane biophysical studies as well as drug delivery applications.

Alleviation of pulp breakdown in harvested longan fruit by acidic electrolyzed water in relation to membrane lipid metabolism

This study investigated the effect of acidic electrolyzed water (AEW) on the inhibition of pulp breakdown of postharvest longan fruit. The harvested longan fruit were immersed in AEW (pH 2.5 and available chlorine concentration 80 mg L−1) for 10 min, then air-dried and stored at 25 °C. It was revealed that the AEW treatment reduced the cell membrane permeability and breakdown incidence of pulp by modulating membrane lipid metabolism. Compared to the untreated fruit, AEW treatment significantly suppressed the activities of lipoxygenase (LOX), lipase, and phospholipase D (PLD), down-regulated the expression of DlPLD1, DlPLD2, Dllipase1, Dllipase2, DlLOX1 and DlLOX2, and reduced the hydrolysis of phosphatidylinositol (PI) and phosphatidylcholine (PC) and the accumulation of phosphatidic acid (PA). In addition, a lower saturated fatty acids (SFAs) proportion, but a higher unsaturated fatty acids (USFAs) proportion, the index of unsaturated fatty (IUFA), and unsaturated to saturated fatty acids ratio (U/S) were found in AEW-treated longan fruit. These findings indicated that AEW treatment inhibited the activities of membrane lipid metabolism-related enzymes (LOX, lipase, and PLD) and their expressions of associated genes, and altered the cell membrane lipid composition, which was conductive to maintaining the integrity of cell membrane, ultimately alleviating the progress of pulp breakdown in harvested longan fruit. Consequently, AEW treatment may be a reliable method to reduce the occurrence of pulp breakdown and improve the storability of harvested longan fruit.

An Investigation of the Altered Textural Property in Woody Breast Myopathy Using an Integrative Omics Approach.

Woody breast (WB) is a myopathy observed in broiler Pectoralis major (PM) characterized by its tough and rubbery texture with greater level of calcium content. The objective of this study was to investigate the functionality/integrity of WB sarcoplasmic reticulum (SR), which may contribute to the elevated calcium content observed in WB and other factors that may influence WB texture. Fourteen Ross line broiler PM [7 severe WB and 7 normal (N)] were selected, packaged, and frozen at −20°C at 8 h postmortem from a commercial processing plant. Samples were used to measure pH, sarcomere length, proteolysis, calpain activity, collagenase activity, collagen content, collagen crosslinks density, and connective tissue peak transitional temperature. Exudate was also collected from each sample to evaluate free calcium concentration. The SR fraction of the samples was separated and utilized for proteomic and lipidomic analysis. The WB PM had a higher pH, shorter sarcomeres, lower % of intact troponin-T, more autolyzed μ/m calpain, more activated collagenase, greater collagen content, greater mature collagen crosslinks density, and higher connective tissue peak transitional temperature than the N PM (p ≤ 0.05). Exudate from WB PM had higher levels of free calcium than those from N PM (p < 0.05). Proteomics data revealed an upregulation of calcium transport proteins and a downregulation of proteins responsible for calcium release (p < 0.05) in WB SR. Interestingly, there was an upregulation of phospholipase A2 (PLA2), and cholinesterase exhibited a 7.6-fold increase in WB SR (p < 0.01). Lipidomics data revealed WB SR had less relative % of phosphatidylcholine (PC) and more lysophosphatidylcholine (LPC; p < 0.05). The results indicated that upregulation of calcium transport proteins and downregulation of calcium-release proteins in WB SR may be the muscle’s attempt to regulate this proposed excessive signaling of calcium release due to multiple factors, such as upregulation of PLA2 resulting in PC hydrolysis and presence of cholinesterase inhibitors in the system prolonging action potential. In addition, the textural abnormality of WB may be the combined effects of shorter sarcomere length and more collagen with greater crosslink density being deposited in the broiler PM.

24-Epibrassinolide improves chilling tolerance by regulating PpCBF5-mediated membrane lipid metabolism in peach fruit

24-Epibrassinolide plays an important role in regulating abiotic stress, which may enhance cold resistance of fruits and vegetables. C-repeat binding factor (CBF) has a rapid response to cold stress and is a crucial transcription factor for cold signal pathway. This study investigated the potential effect of EBR treatment on membrane lipid metabolism and the possible regulation mechanism of PpCBF5 to membrane lipid metabolism in peach fruit. The results showed that EBR treatment effectively suppressed the increase in activities and gene expression of phospholipase D, lipoxygenase and lipase, inhibited the hydrolysis of phosphatidylcholine and phosphatidylinositol to pohosphatidic acid, reduced the malondialdehyde content, and enhanced unsaturated fatty acids contents, thus alleviated the membrane lipid peroxidation and maintained the stability of cell membrane. In addition, the results of molecular assays in vivo, including dual luciferase reporter assay, in vivo bioluminescence imaging, yeast one hybrid and transient overexpression assay, showed that PpCBF5 negatively regulated the transcription of PpPLD and PpLipase. In conclusion, EBR treatment enhanced chilling tolerance of peach fruit through regulating membrane lipid metabolism and PpCBF5 might act as a crucial regulatory factor on membrane lipid metabolism.

A secretory phospholipase D hydrolyzes phosphatidylcholine to suppress rice heading time.

Phospholipase D (PLD) hydrolyzes membrane phospholipids and is crucial in various physiological processes and transduction of different signals. Secretory phospholipases play important roles in mammals, however, whose functions in plants remain largely unknown. We previously identified a rice secretory PLD (spPLD) that harbors a signal peptide and here we reported the secretion and function of spPLD in rice heading time regulation. Subcellular localization analysis confirmed the signal peptide is indispensable for spPLD secretion into the extracellular spaces, where spPLD hydrolyzes substrates. spPLD overexpression results in delayed heading time which is dependent on its secretory character, while suppression or deficiency of spPLD led to the early heading of rice under both short-day and long-day conditions, which is consistent with that spPLD overexpression/suppression indeed led to the reduced/increased Hd3a/RFT1 (Arabidopsis Flowing Locus T homolog) activities. Interestingly, rice Hd3a and RFT1 bind to phosphatidylcholines (PCs) and a further analysis by lipidomic approach using mass spectrometry revealed the altered phospholipids profiles in shoot apical meristem, particularly the PC species, under altered spPLD expressions. These results indicate the significance of secretory spPLD and help to elucidate the regulatory network of rice heading time.

Sphingomyelin synthases 1 and 2 exhibit phosphatidylcholine phospholipase C activity

Many studies have confirmed the enzymatic activity of a mammalian phosphatidylcholine (PC) phospholipase C (PLC) (PC-PLC), which produces diacylglycerol (DAG) and phosphocholine through the hydrolysis of PC in the absence of ceramide. However, the protein(s) responsible for this activity have never yet been identified. Based on the fact that tricyclodecan-9-yl-potassium xanthate can inhibit both PC-PLC and sphingomyelin synthase (SMS) activities, and SMS1 and SMS2 have a conserved catalytic domain that could mediate a nucleophilic attack on the phosphodiester bond of PC, we hypothesized that both SMS1 and SMS2 might have PC-PLC activity. In the present study, we found that purified recombinant SMS1 and SMS2 but not SMS-related protein have PC-PLC activity. Moreover, we prepared liver-specific Sms1/global Sms2 double-KO mice. We found that liver PC-PLC activity was significantly reduced and steady-state levels of PC and DAG in the liver were regulated by the deficiency, in comparison with control mice. Using adenovirus, we expressed Sms1 and Sms2 genes in the liver of the double-KO mice, respectively, and found that expressed SMS1 and SMS2 can hydrolyze PC to produce DAG and phosphocholine. Thus, SMS1 and SMS2 exhibit PC-PLC activity in vitro and in vivo.