Effects of curcumin-mediated photodynamic treatment on lipid degradation of oysters during refrigerated storage.

BACKGROUND: Oyster's lipid degradation leads to a decrease in edible and nutritional value. Curcumin-mediated photodynamic treatment (PDT) is an innovative non-thermal technology, although evaluation of the oyster's lipid degradation has been scarce. In the present study, we investigated peroxide value, thiobarbituric acid reactive substance, triacylglycerol and free fatty acids to evaluate the effect of curcumin-mediated PDT on lipid degradation of oysters during refrigerated storage. RESULTS: The results showed that curcumin-mediated PDT could delay oyster's lipid degradation. Next, the activities of enzymes were detected to determine the mechanisms behind the effects of curcumin-mediated PDT. It was revealed that the activities of lipase, phospholipase A2 (PLA2 ), phospholipase C (PLC), phospholipase D (PLD) and lipoxygenase (LOX) were significantly inhibited after curcumin-mediated PDT (P < 0.05). Furthermore, 16 s rRNA analysis established that the relative abundances of Pseudoalteromonas and Psychrilyobacter were reduced by 51.58% and 43.82%, respectively, after curcumin-mediated PDT. CONCLUSION: Curcumin-mediated PDT could delay oyster's lipid degradation by inhibiting the activities of lipase, PLA2 , PLC, PLD and LOX, as well as by changing the oyster's microbial composition, reducing the relative abundance of Pseudoalteromonas and Psychrilyobacter. © 2021 Society of Chemical Industry.

Phospholipase A2 products predict the hematopoietic support capacity of horse serum.

Horse serum is commonly used as an additive to support the maintenance of hematopoietic progenitor cells in culture. However, the wide variability in the performance of different lots calls for parallel testing of multiple batches over extended periods of culture. Identification of the serum components that determine hematopoietic support would therefore save considerable time and effort and would help to standardize culture procedures. We report here that the ability of horse serum to support the self-renewal of multipotent murine hematopoietic progenitor FDCP-Mix cells is correlated to the concentration of specific fatty acid products of phospholipase A2 and more closely to the spectrum of eicosanoids generated by their further processing through cyclooxygenase and lipoxygenase pathways. Supportive sera have low levels of lysophosphatidylcholine and inflammatory eicosanoids. This links known markers of inflammation, infection and platelet activation to the ability of serum to maintain progenitor cells in an undifferentiated state, providing a means for prospective identification of suitable sera as well as quality control of the production process.

19F MRI Nanoprobes for the Turn-On Detection of Phospholipase A2 with a Low Background.

The highly sensitive detection and imaging of enzymatic activities in vivo could provide effective information about biological functions for monitoring the process of disease and evaluating the effect of therapy. 19F magnetic resonance imaging (MRI) has attracted wide interest because of its deep tissue imaging capability and negligible endogenous background interference, which are suitable for the visualization of enzymatic activities in vivo, but the fabrication of this probe faces challenges. Here, we report nanoprobes with turn-on 19F MRI for sensing the activity of phospholipase A2 (PLA2). These nanoprobes are composed of Gd3+-exchanged NaYF4:Yb3+/Er3+ upconversion luminescent nanoparticles grafted with perfluoro-15-crown-5-ether (PFCE) as the hydrophobic core with a phospholipid shell in which the 19F MRI signal of PFCE is obviously quenched by adjacent Gd3+. The shielded 19F MRI signal of these nanoprobes is then turned on by the nanoprobe dissolution stemming from phospholipid hydrolysis by PLA2 and increases linearly along with the increment of PLA2 in the range of 5.0-200 U/L. Apart from the in vitro detection of PLA2 by 19F NMR, these nanoprobes show great potential for both the in vivo 19F MRI sensing of PLA2 and activity screening of PLA2 inhibitors with a high signal-to-noise ratio and a high penetration depth.

Cooperative Substrate-Cofactor Interactions and Membrane Localization of the Bacterial Phospholipase A2 (PLA2) Enzyme, ExoU.

The ExoU type III secretion enzyme is a potent phospholipase A2 secreted by the Gram-negative opportunistic pathogen, Pseudomonas aeruginosa Activation of phospholipase activity is induced by protein-protein interactions with ubiquitin in the cytosol of a targeted eukaryotic cell, leading to destruction of host cell membranes. Previous work in our laboratory suggested that conformational changes within a C-terminal domain of the toxin might be involved in the activation mechanism. In this study, we use site-directed spin-labeling electron paramagnetic resonance spectroscopy to investigate conformational changes in a C-terminal four-helical bundle region of ExoU as it interacts with lipid substrates and ubiquitin, and to examine the localization of this domain with respect to the lipid bilayer. In the absence of ubiquitin or substrate liposomes, the overall structure of the C-terminal domain is in good agreement with crystallographic models derived from ExoU in complex with its chaperone, SpcU. Significant conformational changes are observed throughout the domain in the presence of ubiquitin and liposomes combined that are not observed with either liposomes or ubiquitin alone. In the presence of ubiquitin, two interhelical loops of the C-terminal four-helix bundle appear to penetrate the membrane bilayer, stabilizing ExoU-membrane association. Thus, ubiquitin and the substrate lipid bilayer act synergistically to induce a conformational rearrangement in the C-terminal domain of ExoU.

A fluorometric assay for lysosomal phospholipase A2 activity using fluorescence-labeled truncated oxidized phospholipid.

Lysosomal phospholipase A2 (LPLA2) is a key enzyme involved in the homeostasis of cellular phospholipids. Recently, LPLA2 was reported to preferentially degrade some truncated oxidized phospholipids at the sn-1 position. A commercially available, truncated oxidized phospholipid conjugated with a fluorescent dye, 1-palmitoyl-2-glutaroyl-sn-glycero-3-phosphoethanolamine-N-[4-(dipyrrometheneboron difluoride) butanoyl] (PGPE-BODIPY), was used to develop a specific assay for this enzyme. When recombinant mouse LPLA2 was incubated with liposomes consisting of 1,2-O-octadecyl-sn-glycero-3-phosphocholine/PGPE-BODIPY under acidic conditions, PGPE-BODIPY was converted to palmitic acid and a polar BODIPY-product. After phase partitioning by chloroform/methanol, the polar BODIPY-product was recovered in the aqueous phase and identified as 1-lyso-PGPE-BODIPY. The formation of 1-lyso-PGPE-BODIPY was quantitatively determined by fluorescent measurements. The Km and Vmax values of the recombinant LPLA2 for PGPE-BODIPY were 5.64 µM and 20.7 µmol/min/mg protein, respectively. Detectable activity against PGPE-BODIPY was present in LPLA2 deficient mouse sera, but the deacylase activity was completely suppressed by treatment with 4-(2-aminoethyl)benzenesulfonyl fluoride (AEBSF). AEBSF had no effect on LPLA2 activity. The LPLA2 activity of mouse serum pre-treated with AEBSF was specifically and quantitatively determined by this assay method. The PGPE-BODIPY and AEBSF based LPLA2 assay is convenient and can be used to measure LPLA2 activity in a variety of biological specimens.

Computational investigations of physicochemical, pharmacokinetic, toxicological properties and molecular docking of betulinic acid, a constituent of Corypha taliera (Roxb.) with Phospholipase A2 (PLA2).

BACKGROUND: Betulinic acid (BA) is a natural triterpenoid compound and exhibits a wide range of biological and medicinal properties including anti-inflammatory activity. Therefore, this theoretical investigation is performed to evaluate (a) physicochemical properties such as acid dissociation constant (pKa), distribution coefficient (logD), partition coefficient (logP), aqueous solubility (logS), solvation free energy, dipole moment, polarizability, hyperpolarizability and different reactivity descriptors, (b) pharmacokinetic properties like human intestinal absorption (HIA), cellular permeability, skin permeability (PSkin), plasma protein binding (PPB), penetration of the blood brain barrier (BBB), (c) toxicological properties including mutagenicity, carcinogenicity, risk of inhibition of hERG gene and (d) molecular mechanism of anti-inflammatory action which will aid the development of analytical method and the synthesis of BA derivatives. METHODS: The physicochemical properties were calculated using MarvinSketch 15.6.29 and Gaussian 09 software package. The pharmacokinetic and toxicological properties were calculated on online server PreADMET. Further, the molecular docking study was conducted on AutoDock vina in PyRx 0.8. RESULTS: The aqueous solubility increased with increasing pH due to the ionization of BA leading to decrease in distribution coefficient. The solvation energies in water, dimethyl sulfoxide (DMSO), acetonitrile, n-octanol, chloroform and carbon tetrachloride were - 41.74 kJ/mol, - 53.80 kJ/mol, - 66.27 kJ/mol, - 69.64 kJ/mol, - 65.96 kJ/mol and - 60.13 kJ/mol, respectively. From the results of polarizability and softness, it was clear that BA is less stable and hence, kinetically more reactive in water. BA demonstrated good human intestinal absorption (HIA) and moderate cellular permeability. Further, BA also exhibited positive CNS activity due to high permeability through BBB. The toxicological study revealed that BA was a mutagenic compound but noncarcinogenic in mice model. Moreover, molecular docking study of BA with PLA2 revealed that BA interacts with GLY22 & GLY29 through hydrogen bond formation and LEU2, PHE5, HIS6, ALA17, ALA18, HIS47 and TYR51 through different types of hydrophobic interactions. The binding affinity of BA was - 41.00 kJ/mol which is comparable to the binding affinity of potent inhibitor 6-Phenyl-4(R)-(7-Phenyl-heptanoylamino)-hexanoic acid (BR4) (- 33.89 kJ/mol). CONCLUSIONS: Our computed properties may assist the development of analytical method to assay BA or to develop BA derivatives with better pharmacokinetic and toxicological profile.

A constant area monolayer method to assess optimal lipid packing for lipolysis tested with several secreted phospholipase A2.

We present an analysis of lipid monolayer hydrolysis at a constant area to assess the optimal lateral surface pressure value (Πopt) and thus, the surface packing density of the lipid, at which the activity of a given lipolytic enzyme is maximal. This isochoric method consists of a measurement of the decrease down to zero of the Πopt of phospholipid substrate monolayer due to continuous hydrolysis using only one reaction compartment. We performed the comparison of both approaches using several commercially available and literature-evaluated sPLA2s. Also, we characterized for the first time the profile of hydrolysis of DLPC monolayers catalyzed by a sPLA2 from Streptomyces violaceoruber and isoenzymes purified from Bothrops diporus venom. One of these viper venom enzymes is a new isoenzyme, partially sequenced by a mass spectrometry approach. We also included the basic myotoxin sPLA2-III from Bothrops asper. Results obtained with the isochoric method and the standard isobaric one produced quite similar values of Πopt, validating the proposal. In addition, we propose a new classification parameter, a lipolytic ratio of hydrolysis at two lateral pressures, 20 mN·m(-1) and 10 mN·m(-1), termed here as LR20/10 index. This index differentiates quite well "high surface pressure" from "low surface pressure" sPLA2s and, by extension; it can be used as a functional criterion for the quality of a certain enzyme. Also, this index could be added to the grouping systematic criteria for the superfamily proposed for phospholipase A2.

Cytosolic phospholipase A2 (cPLA2) IVA as a potential signature molecule in cigarette smoke condensate induced pathologies in alveolar epithelial lineages.

BACKGROUND: Smoking is one of the leading causes of millions of deaths worldwide. During cigarette smoking, most affected and highly exposed cells are the alveolar epithelium and generated oxidative stress in these cells leads to death and damage. Several studies suggested that oxidative stress causes membrane remodeling via Phospholipase A2s but in the case of cigarette smokers, mechanistically study is not yet fully defined. In view of present perspective, we evaluated the involvement of cytosolic phospholipase A2 (cPLA2) IVA as therapeutic target in cigarette smoke induced pathologies in transformed type I and type II alveolar epithelial cells. METHODS: Transformed type I (WI26) and type II (A549) alveolar epithelial cells were used for the present study. Cigarette smoke condensate (CSC) was prepared from most commonly used cigarette (Gold Flake with filter) by the Indian population. CSC-induced molecular changes were evaluated through cell viability using MTT assay, reactive oxygen species (ROS) measurement using 2,7 dichlorodihydrofluorescin diacetate (DCFH-DA), cell membrane integrity using fluorescein diacetate (FDA) and ethidium bromide (EtBr) staining, super oxide dismutase (SOD) levels, cPLA2 activity and molecular involvement of specific cPLA2s at selected 24 h time period. RESULTS: CSC-induced response on both type of epithelial cells shown significantly reduction in cell viability, declined membrane integrity, with differential escalation of ROS levels in the range of 1.5-15 folds and pointedly increased cPLA2 activity (p < 0.05). Likewise, we observed distinction antioxidant potential in these two types of lineages as type I cells had considerably higher SOD levels when compared to type II cells (p < 0.05). Further molecular expression of all cPLA2s increased significantly in a dose dependent manner, specifically cytosolic phospholipase A2 IVA with maximum manifestation of 3.8 folds. Interestingly, CSC-induced ROS levels and cPLA2s expression were relatively higher in A549 cells as compared to WI26 cells. CONCLUSIONS: The present study indicates that among all cPLA2s, specific cPLA2 IVA are the main enzymes involved in cigarette smoke induced anomalies in type I and type II lung epithelial cells and targeting them holds tremendous possibilities in cigarette smoke induced lung pathologies.

Synthesis and Evaluation of Cytosolic Phospholipase A(2) Activatable Fluorophores for Cancer Imaging.

Activatable fluorophores selective to cytosolic phospholipase A2 (cPLA2) were synthesized and evaluated for their ability to image triple negative breast cancer cells. The activatable constructs were synthesized by esterification of a small molecule fluorophore with a fatty acid resulting in ablated fluorescence. Selectivity for cPLA2 was generated through the choice of fluorophore and fatty acid. Esterification with arachidonic acid was sufficient to impart specificity to cPLA2 when compared to esterification with palmitic acid. In vitro analysis of probes incorporated into phosphatidylcholine liposomes demonstrated that a nonselective phospholipase (sPLA2 group IB) was able to hydrolyze both arachidonate and palmitate coupled fluorophores resulting in the generation of fluorescence. Of the four fluorophores tested, DDAO (7-hydroxy-9H-(1,3-dichloro-9,9-dimethylacridin-2-one)) was observed to perform optimally in vitro and was analyzed further in 4175-Luc+ cells, a metastatic triple negative human breast cancer cell line expressing high levels of cPLA2. In contrast to the in vitro analysis, DDAO arachidonate was shown to activate selectively in 4175-Luc+ cells compared to the control DDAO palmitate as measured by fluorescence microscopy and quantitated with fluorescence spectroscopy. The addition of two agents known to activate cPLA2 enhanced DDAO arachidonate fluorescence without inducing any change to DDAO palmitate. Inhibition of cPLA2 resulted in reduced fluorescence of DDAO arachidonate but not DDAO palmitate. Together, we report the synthesis of a cPLA2 selective activatable fluorophore capable of detecting cPLA2 in triple negative breast cancer cells.

Cholesterol gallstones and bile host diverse bacterial communities with potential to promote the formation of gallstones.

The prevalence of cholesterol gallstones has increased in recent years. Bacterial infection correlates with the formation of gallstones. We studied the composition and function of bacterial communities in cholesterol gallstones and bile from 22 cholesterol gallstone patients using culture-dependent and culture-independent methods. Altogether fourteen and eight bacterial genera were detected in cholesterol gallstones and bile, respectively. Pseudomonas spp. were the dominant bacteria in both cholesterol gallstones and bile. As judged by diversity indices, hierarchical clustering and principal component analysis, the bacterial communities in gallstones were different from those in bile. The gallstone microbiome was considered more stable than that of bile. The different microbial communities may be partially explained by differences in their habitats. We found that 30% of the culturable strains from cholesterol gallstones secreted ß-glucuronidase and phospholipase A2. Pseudomonas aeruginosa strains showed the highest ß-glucuronidase activity and produced the highest concentration of phospholipase A2, indicating that Ps. aeruginosa may be a major agent in the formation of cholesterol gallstones.

Reporter-encapsulated liposomes on graphene field effect transistors for signal enhanced detection of physiological enzymes.

A novel approach for enzymatic assay using reporter-encapsulated liposomes on graphene field effect transistors (FET) is proposed. This approach involves real time monitoring of drain current (Id) of reduced graphene oxide (rGO) upon rupture of reporter-encapsulated 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC) liposomes triggered by enzymes. For validation of the proposed approach, 2,4,6-trinitrophenol (TNP) is used as the reporter for specific detection of phospholipase A2 (PLA2), a key enzyme in various membrane related physiological processes. Experimental results revealed that Id increased with PLA2 concentration, which is attributed to the interaction between released TNP and rGO. The limit of detection (LOD) achieved by the proposed approach was 80 pM, which is superior to most assays reported previously and much lower than the cut-off level of circulating secretory PLA2 (2.07 nM). Besides the high accuracy of the electronic detection methodology, the signal enhancement effect realized by the excess concentration of TNP (approximately 1 mM) in liposomes is believed to be the main reason for the significantly enhanced sensitivity of the proposed assay, indicating great potential for further improvement in the sensitivity by increasing the concentration of TNP. In addition, the proposed approach is rapid (incubation time ≤ 10 min) and label-free, thus showing great potential for practical applications in the future.

A fluorogenic phospholipid for the detection of lysosomal phospholipase A2 activity.

Lysosomal phospholipase A2 (group XV PLA2, LPLA2) is a lysosomal enzyme linked to drug-induced phospholipidosis. We developed phospholipid "smart probes" based on the conversion of a quenched fluorogenic substrate to a fluorescent product. Due to the preference of LPLA2 for phosphatidylglycerol, three fluorogenic phosphatidylglycerols were synthesized. Two fluorogenic phosphatidylglycerols were conjugated with one FAM (fluorescein amidite) group and one DABCYL [4-(4-dimethylaminophenylazo)-benzoyl] group; the third substrate consisted of two FAM groups conjugated at the sn-1 and sn-2 positions. The sn-1 ester linkage was replaced with an amide linkage. 1-FAM-2-DABCYL-PG was degraded by recombinant LPLA2 and mouse serum but not by the serum obtained from LPLA2-deficient mice when 1,2-dioleoyl-PG/1-FAM-2-DABCYL-PG liposomes were used. The formation of 1-FAM-lyso-PG generated from 1-FAM-2-DABCYL-PG in the presence of LPLA2 was quantitatively determined by fluorescent measurements. The 1-FAM-2-DABCYL-PG incorporated into 1,2-dioleoyl-phosphatidylcholine/sulfatide liposomes was used to evaluate the effect of the cationic amphiphilic drugs amiodarone and fluoxetine on LPLA2 activity. The IC(50) values of amiodarone and fluoxetine estimated by fluorescent measurement were 10 and 19µM, respectively. These results indicate that 1-FAM-2-DABCYL-PG is a specific substrate for LPLA2 and a useful reagent for the detection of LPLA2 activity from multiple sources.

Evidence supporting a key role of Lp-PLA2-generated lysophosphatidylcholine in human atherosclerotic plaque inflammation.

OBJECTIVE: To determine whether the level of lysophosphatidylcholine (lysoPC) generated by lipoprotein-associated phospholipase A2 (Lp-PLA2) is associated with severity of inflammation in human atherosclerotic plaques. Elevated plasma Lp-PLA2 is associated with increased cardiovascular risk. Lp-PLA2 inhibition reduces atherosclerosis. Lp-PLA2 hydrolyzes low-density lipoprotein-oxidized phospholipids generating lysoPCs. According to in vitro studies, lysoPCs are proinflammatory but the association between their generation and plaque inflammation remains unknown. METHODS AND RESULTS: Inflammatory activity in carotid plaques (162 patients) was determined immunohistochemically and by analyzing cytokines in homogenates (multiplex immunoassay). LysoPCs were quantified using mass spectrometry and Lp-PLA2 and the lysoPC metabolite lysophosphatidic acid (LPA) by ELISA. There was a strong correlation among lysoPC 16:0, 18:0, 18:1, LPA, and Lp-PLA2 in plaques. LysoPC 16:0, 18:0, 18:1, LPA, and Lp-PLA2 correlated with interleukin-1ß, interleukin-6, monocyte chemoattractant protein-1, macrophage inflammatory protein-1ß, regulated on activation normal T-cell expressed and secreted, and tumor necrosis factor-α in plaques. High lysoPC and Lp-PLA2 correlated with increased plaque macrophages and lipids and with low content of smooth muscle cells, whereas LPA only correlated with plaque macrophages. Lp-PLA2, lysoPC 16:0, 18:0, and 18:1, but not LPA were higher in symptomatic than in asymptomatic plaques. CONCLUSIONS: The associations among Lp-PLA2, lysoPCs, LPA, and proinflammatory cytokines in human plaques suggest that lysoPCs play a key role in plaque inflammation and vulnerability. Our findings support Lp-PLA2 inhibition as a possible strategy for the prevention of cardiovascular disease.

Quantum dot-NBD-liposome luminescent probes for monitoring phospholipase A2 activity.

In this paper we describe the fabrication and characterization of new liposome encapsulated quantum dot-fluorescence resonance energy transfer (FRET)-based probes for monitoring the enzymatic activity of phospholipase A2. To fabricate the probes, luminescent CdSe/ZnS quantum dots capped with trioctylphosphine oxide (TOPO) ligands were incorporated into the lipid bilayer of unilamellar liposomes with an average diameter of approximately 100 nm. Incorporating TOPO capped quantum dots in liposomes enabled their use in aqueous solution while maintaining their hydrophobicity and excellent photophysical properties. The phospholipid bilayer was labeled with the fluorophore NBD C6-HPC (2-(6-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)hexanoyl-1-hexa decanoyl-sn-glycero-3-phosphocholine). The luminescent quantum dots acted as FRET donors and the NBD dye molecules acted as FRET acceptors. The probe response was based on FRET interactions between the quantum dots and the NBD dye molecules. The NBD dye molecules were cleaved and released to the solution in the presence of the enzyme phospholipase A2. This led to an increase of the luminescence of the quantum dots and to a corresponding decrease in the fluorescence of the NBD molecules, because of a decrease in FRET efficiency between the quantum dots and the NBD dye molecules. Because the quantum dots were not attached covalently to the phospholipids, they did not hinder the enzyme activity as a result of steric effects. The probes were able to detect amounts of phospholipase A2 as low as 0.0075 U mL(-1) and to monitor enzyme activity in real time. The probes were also used to screen phospholipase A2 inhibitors. For example, we found that the inhibition efficiency of MJ33 (1-hexadecyl-3-(trifluoroethyl)-sn-glycero-2-phosphomethanol) was higher than that of OBAA (3-(4-octadecyl)benzoylacrylic acid).

Phylogeny-Related Variations in Venomics: A Test in a Subset of Habu Snakes (Protobothrops).

We conducted a comparative analysis to unveil the divergence among venoms from a subset of Old World habu snakes (Protobothrops) in terms of venomic profiles and toxicological and enzymatic activities. A total of 14 protein families were identified in the venoms from these habu snakes, and 11 of them were shared among these venoms. The venoms of five adult habu snakes were overwhelmingly dominated by SVMP (32.56 ± 13.94%), PLA2 (22.93 ± 9.26%), and SVSP (16.27 ± 4.79%), with a total abundance of over 65%, while the subadult P. mangshanensis had an extremely low abundance of PLA2 (1.23%) but a high abundance of CTL (51.47%), followed by SVMP (22.06%) and SVSP (10.90%). Apparent interspecific variations in lethality and enzymatic activities were also explored in habu snake venoms, but no variations in myotoxicity were found. Except for SVSP, the resemblance of the relatives within Protobothrops in other venom traits was estimated to deviate from Brownian motion evolution based on phylogenetic signals. A comparative analysis further validated that the degree of covariation between phylogeny and venom variation is evolutionarily labile and varies among clades of closely related snakes. Our findings indicate a high level of interspecific variation in the venom proteomes of habu snakes, both in the presence or absence and the relative abundance of venom protein families, and that these venoms might have evolved under a combination of adaptive and neutral mechanisms.

Interpopulational variation and ontogenetic shift in the venom composition of Lataste's viper (Vipera latastei, Boscá 1878) from northern Portugal.

Lataste's viper (Vipera latastei) is a venomous European viper endemic to the Iberian Peninsula, recognised as medically important by the World Health Organization. To date, no comprehensive characterisation of this species' venom has been reported. Here, we analysed the venoms of juvenile and adult specimens of V. latastei from two environmentally different populations from northern Portugal. Using bottom-up venomics, we produced six venom proteomes (three per population) from vipers belonging to both age classes (i.e., two juveniles and four adults), and RP-HPLC profiles of 54 venoms collected from wild specimens. Venoms from juveniles and adults differed in their chromatographic profiles and relative abundances of their toxins, suggesting the occurrence of ontogenetic changes in venom composition. Specifically, snake venom metalloproteinase (SVMP) was the most abundant toxin family in juvenile venoms, while snake venom serine proteinases (SVSPs), phospholipases A2 (PLA2s), and C-type lectin-like (CTLs) proteins were the main toxins comprising adult venoms. The RP-HPLC venom profiles were found to vary significantly between the two sampled localities, indicating geographic variability. Furthermore, the presence/absence of certain peaks in the venom chromatographic profiles appeared to be significantly correlated also to factors like body size and sex of the vipers. Our findings show that V. latastei venom is a variable phenotype. The intraspecific differences we detected in its composition likely mirror changes in the feeding ecology of this species, taking place during different life stages and under different environmental pressures. SIGNIFICANCE: Lataste's viper (Vipera latastei) is a medically important viper endemic to the Iberian Peninsula, inhabiting different habitats and undergoing a marked ontogenetic dietary shift. In the current study, we report the first proteomic analysis of V. latastei venom from two environmentally different localities in northern Portugal. Our bottom-up venomic analyses show that snake venom serine proteinases (SVSPs), phospholipases A2 (PLA2s), and C-type lectin-like (CTLs) proteins are the major components of adult V. latastei venom. The comparative analysis of young and adult venoms suggests the occurrence of ontogenetic shift in toxin abundances, with snake venom metalloproteinases (SVMPs) being the predominant toxins in juvenile venoms. Moreover, geographic venom variation between the two studied populations is also detected, with our statistical analyses suggesting that factors like body size and sex of the vipers are possibly at play in its determination. Our work represents the first assessment of the composition of V. latastei venom, and the first step towards a better understanding of the drivers behind its variability.

Expression of Physaria longchain acyl-CoA synthetases and hydroxy fatty acid accumulation in transgenic Arabidopsis.

Hydroxy fatty acids (HFA) are industrially useful chemical feedstocks that accumulate in seed-storage triacylglycerols (TAG) of several plant species, including castor (Ricinus communis) and Physaria (Physaria fendleri). For researchers, HFA also offer a unique opportunity to trace fatty acid metabolism and modification. Past work producing HFA in Arabidopsis (Arabidopsis thaliana) has demonstrated the importance of isozymes of TAG synthesis from plants that evolved to store HFA and as a result have a high degree of specificity towards HFA substrates. Castor phospholipase A2α (RcPLA2) has specificity for HFA-containing phosphatidylcholine. However, expression of RcPLA2 in HFA-accumulating Arabidopsis line CL37-PLA2 reduced HFA content of TAG. This loss was interpreted as being due to poor ability of Arabidopsis longchain acyl-CoA synthetases (LACSs) to utilize HFAs substrates. LACS enzymes are essential to activate HFA to HFA-CoA for TAG synthesis. Physaria is a close relative of Arabidopsis in the Brassicaceae family. To test the hypothesis that this close relatedness would allow Physaria LACSs to interface successfully with Arabidopsis enzymes of seed lipid metabolism and thereby restore HFA accumulation, we transformed PfLACS4 and PfLACS8 constructs into the CL37-PLA2 line. However, HFA content was not recovered, and biochemical characterization of recombinant PfLACS4 and PfLACS8 indicated that these isozymes have substrate specificities and selectivities that are similar to their Arabidopsis orthologues. These and other results pose an important question about how HFA synthesized on phosphatidylcholine can be transferred into the acyl-CoA pool for TAG synthesis.

New CZE-DAD method for honeybee venom analysis and standardization of the product.

The aim of this study was to develop a new precise and accurate CZE-DAD method for honeybee venom analysis using cytochrome c as an internal standard. The 64.5 cm total length, 56 cm effective length, 75 µm ID, and 360 µm OD uncoated fused-silica capillary was used. The samples were injected into the capillary under a 50-mbar pressure for 7 s. There were 15 kV of electric field across the capillary applied. The current intensity was 26 µA. The separation was carried out at 25 °C. The analysis was run with the normal electrode polarity. The following steps and parameters were taken into account for the validation of the developed method: selectivity, precision, accuracy, linearity, limit of detection and limit of quantitation. All steps of the validation procedure proved that the developed analytical procedure was suitable for its intended purpose. Possibly this was the first study in which several honeybee venom components were separated and five of them were identified by capillary zone electrophoresis. In addition, the developed method was applied for quantitative analysis of 38 honeybee venom samples. The content (relative to the dry venom mass) of analyzed peptides in honeybee venom samples collected in 2002-2007 was as follows: apamine from 0.93% to 4.34% (mean, 2.85 ± 0.79%); mast cell degranulating peptide (MCDP) from 1.46% to 4.37% (mean, 2.82 ± 0.64%); phospholipase A(2) from 7.41% to 20.25% (mean, 12.95 ± 3.09%); melittin from 25.40% to 60.27%, (mean, 45.91 ± 9.78%). The results were compared with the experimental data obtained for the same venom samples analyzed earlier by the HPLC method. It was stated that HPCE and HPLC data did not differ significantly and that the HPCE method was the alternative for the HPLC method. Moreover, using the results obtained principal component analysis (PCA) was applied to clarify the general distribution patterns or similarities of four major honeybee venom constituents collected from two different bee strains in various months and years. PCA has shown that the strain of bee appears to be the only criteria for bee venom sample classification. Strong correlations between apamine, MCDP, phospholipase A(2), and melittin were confirmed. These correlations have to be taken into account in the honeybee venom standardization. The developed method due to its simplicity can be easily automated and incorporated into routine operations both in the bee venom identification, quality control, and standardization of the product.

Antiasthmatic action of dibenzylbutyrolactone lignans from fruits of Forsythia viridissima on asthmatic responses to ovalbumin challenge in conscious guinea-pigs.

It was reported previously that dibenzylbutyrolactone lignans from Forsythia viridissima fruits, which are traditional medicines for the treatment of inflammatory diseases, have antiinflammatory effects. In this study, the effects on the immediate-phase response (IAR) and late-phase response (LAR) following aerosolized-ovalbumin challenge in ovalbumin-sensitized guinea-pigs were evaluated by measuring the specific airway resistance (sRaw), recruitment of leukocytes and chemical mediators in the bronchoalveolar lavage fluids (BALF) as well as a histopathological survey. Arctiin and matairesinol at 12.5 mg/kg significantly (p < 0.05) decreased sRaw by 51.83% and 43.15% in IAR and by 47.41% and 35.43% in LAR, respectively, whereas arctigenin at 25 mg/kg was significantly active, compared with the controls. Furthermore, arctiin and arctigenin dose-dependently inhibited histamine, and the activities of phospholipase A2 (PLA2) and eosinophil peroxidase (EPO) in BALF, respectively, whereas matairesinol inhibited EPO and PLA2 at 12.5 mg/kg and histamine at 50 mg/kg, in addition, they moderately improved the infiltration of eosinophils, compared with controls. Dexamethasone, cromolyn and salbutamol significantly inhibited sRaw in both IAR and LAR, and the recruitment of leukocytes and chemical mediators, whereas salbutamol did not alter chemical mediators, in BALF. These results indicate the three lignans have antiasthmatic effects which were less active than those of the reference drugs.

[Effects of polydatin on bleomycin-induced pulmonary fibrosis in rats].

OBJECTIVE: To observe the effects of three different doses of polydatin (PD) on pulmonary interstitial fibrosis in rats induced by bleomycin. METHOD: One hundred and twenty-nine healthy Sprague-Dawley rats three months old, were randomly divided into six groups. Group A: normal control group; group B: model group treated with bleomycin (pretreatment with saline 1 mL x kg(-1) intraperitoneally before bleomycin); group C: PD 10 mg x kg(-1) (pretreatment with PD 10 mg x kg(-1) intraperitoneally before bleomycin); group D: PD 20 mg x kg(-1) (pretreatment with PD 20 mg x kg(-1) intraperitoneally before bleomycin); group E: PD 40 mg x kg(-1) (pretreatment with PD 40 mg x kg(-1) intraperitoneally before bleomycin), group F: dexamethason (DXM) treated group (pretreatment with saline 1 mL x kg(-1) intraperitoneally before bleomycin and then with DXM 1 mg x kg(-1) x d(-1)). At day 3, 7, 14, 28 after injection of bleomycin, eight rats in each group were randomly chosen to be killed. The right lungs of dead rats were removed and appropriately processed for hematoxylin and eosin (H&E) stain, histologically observed under light microscope. The hydroxyproline content and the PLA2 activity in pulmonary homogenate were measured with alkaline hydrolysis assay and acid modified microtitrimetic method. The levels of leukotriene C4 (LTC4), prostaglandin E2 (PGE2), transforming growth factor-beta1 (TGF-beta1) in bronchoalveolar lavage fluid (BALF) were measured with enzyme-linked immunosorbent assay (ELISA). RESULT: At day 3, 7, 14, 28 after intratracheal instillation of bleomycin in rats of group B, the PLA2 activity in lung homogenate and the levels of its metabolic products PGE2, LTC4 as well as TGF-beta1 in BALF increased significantly compared with those in group A (P < 0.01). And lung hydroxyproline concentration began to grow up markedly at day 7 compared with those in group A (P < 0.05), reaching its maximum at day 28. Compared with group B, three different doses of PD and DXM significantly reduced the activity of the PLA2 and hydroxyproline concentration in lung homogenate as well as the levels of PGE2, LTC4, TGF-beta1 in BALF at various periods (P < 0.05). There was statistically significant difference between three different doses of PD groups (P < 0.05). And the group E (PD 40 mg x kg(-1)) was lower than group D (PD 20 mg x kg(-1)), group D was lower than group C (PD 10 mg x kg(-1)) (respectively, P < 0.01). Group E and DXM group were no significant difference. However, all these observation parameters were higher than the normal level (compared with group A, P < 0.01). Histological studies revealed that it was showed less inflammation and a lower degree of fibrosis in the lungs treated with PD than bleomycin model group. CONCLUSION: PD has the protective effect on pulmonary interstitial fibrosis. However, it can't completely block the process of pulmonary fibrosis.