Case study of chemical and enzymatic degumming processes in soybean oil production at an industrial plant.

The vegetable oil degumming process plays a critical role in refining edible oil. Phospholipids (PL) removal from crude extracted soybean oil (SBO) by the enzymatic degumming process has been investigated in this work. Enzymatic degumming of extracted SBO with microbial phospholipase A1 PLA-1 Quara LowP and Lecitase Ultra enzymes have also been studied comparatively. The main novelty of our work is the use of the enzymatic degumming process on an industrial scale (600 tons a day). Many parameters have been discussed to understand in detail the factors affecting oil losses during the degumming process. The factors such as chemical conditioning (CC) by phosphoric acid 85%, the enzyme dosage mg/kg (feedstock dependent), the enzymatic degumming reaction time, and the characteristics of the plant-processed SBO have been discussed in detail. As a main point, the degummed oil with a phosphorus content of < 10 mg/kg increases yield. Quara LowP and Lecitase Ultra enzymes are not specific for certain phospholipids PL; however, the conversion rate depends on the SBO phospholipid composition. After 4 h, over 99% of Phospholipids were degraded to their lysophospholipid LPL (lysolecithin). The results showed a significant effect of operating parameters and characteristics of different origins of SBO, fatty acids FFA content, Phosphorus content and total divalent metals (Calcium Ca, Magnesium Mg and Iron Fe mg/kg) content on the oil loss. The benefit of using enzymatic degumming of vegetable oils rather than traditional chemical refining is that the enzymatic degumming process reduces total oil loss. This decrease is known as enzymatic yield. The enzymatic degumming also decreases wastewater and used chemicals and running costs; moreover, it enables physical refining by lowering the residue phosphorus to < 10 mg/kg.

Phospholipase A1 Member A Activates Fibroblast-like Synoviocytes through the Autotaxin-Lysophosphatidic Acid Receptor Axis.

Lysophosphatidylserine (lysoPS) is known to regulate immune cell functions. Phospholipase A1 member A (PLA1A) can generate this bioactive lipid through hydrolysis of sn-1 fatty acids on phosphatidylserine (PS). PLA1A has been associated with cancer metastasis, asthma, as well as acute coronary syndrome. However, the functions of PLA1A in the development of systemic autoimmune rheumatic diseases remain elusive. To investigate the possible implication of PLA1A during rheumatic diseases, we monitored PLA1A in synovial fluids from patients with rheumatoid arthritis and plasma of early-diagnosed arthritis (EA) patients and clinically stable systemic lupus erythematosus (SLE) patients. We used human primary fibroblast-like synoviocytes (FLSs) to evaluate the PLA1A-induced biological responses. Our results highlighted that the plasma concentrations of PLA1A in EA and SLE patients were elevated compared to healthy donors. High concentrations of PLA1A were also detected in synovial fluids from rheumatoid arthritis patients compared to those from osteoarthritis (OA) and gout patients. The origin of PLA1A in FLSs and the arthritic joints remained unknown, as healthy human primary FLSs does not express the PLA1A transcript. Besides, the addition of recombinant PLA1A stimulated cultured human primary FLSs to secrete IL-8. Preincubation with heparin, autotaxin (ATX) inhibitor HA130 or lysophosphatidic acid (LPA) receptor antagonist Ki16425 reduced PLA1A-induced-secretion of IL-8. Our data suggested that FLS-associated PLA1A cleaves membrane-exposed PS into lysoPS, which is subsequently converted to LPA by ATX. Since primary FLSs do not express any lysoPS receptors, the data suggested PLA1A-mediated pro-inflammatory responses through the ATX-LPA receptor signaling axis.

Elevated phosphatidylserine-specific phospholipase A1 level in hyperthyroidism.

OBJECTIVES: Although a single nucleotide polymorphism in a specific receptor for lysophosphatidylserine, a lysophospholipid mediator involved in the immune system, is reportedly associated with Graves' disease, the association between lysophosphatidylserine and thyroid disorders remains to be elucidated. Therefore, we aimed to investigate the association between the level of phosphatidylserine-specific phospholipase A1 (PS-PLA1), which produces lysophosphatidylserine, and thyroid disorders. METHODS: We measured serum PS-PLA1 levels in the patients with various thyroid disorders (n = 120) and normal subjects (n = 58). RESULTS: We observed that the serum PS-PLA1 levels were higher in the subjects with Graves' disease, subacute thyroiditis, or silent thyroiditis, while they were not modulated in the patients with hypothyroidism. The serum PS-PLA1 levels were strongly correlated with the levels of thyroid hormones, especially in the subjects with Graves' disease. Moreover, we found that the serum PS-PLA1 levels were lowered by treatment with anti-thyroid reagents in subjects with Graves' disease and that the changes in PS-PLA1 were strongly correlated with those in thyroid hormones. CONCLUSION: These results suggest that PS-PLA1 might be a novel target in the treatment of hyperthyroidism, especially Graves' disease, and that its measurement might be useful as a supplementary diagnostic test for thyroid function.

Lipid composition and emulsifying properties of Camelina sativa seed lecithin.

There is no information on the chemical composition of camelina seed lecithin; therefore, the objective of this study was to investigate the chemical composition and emulsifying properties of lecithin recovered from camelina seed oil by water (WDCL) and enzymatic degumming (EDCL) using phospholipase A1 (PLA1). The lecithin obtained by both WDLC and EDLC was rich in phosphatidylinositol (PI), and contents were 37.8 and 25.2wt%, respectively. Lecithin recovered by enzymatic degumming contained more lysophospholipids compared to water degumming. The saturated fatty acid content of the EDCL was significantly higher than that of the WDCL. Emulsions stabilized using EDCL resulted in the highest stability when deionized water was used as the aqueous phase (original pH); however, at pH=7.5, emulsions stabilized using EDCL and WDCL were less stable compared to the emulsion stabilized with soy lecithin. Results showed that camelina seed lecithin is a promising alternative PI-rich emulsifier for various food applications.

A Plastid Phosphatidylglycerol Lipase Contributes to the Export of Acyl Groups from Plastids for Seed Oil Biosynthesis.

The lipid composition of thylakoid membranes inside chloroplasts is conserved from leaves to developing embryos. A finely tuned lipid assembly machinery is required to build these membranes during Arabidopsis thaliana development. Contrary to thylakoid lipid biosynthetic enzymes, the functions of most predicted chloroplast lipid-degrading enzymes remain to be elucidated. Here, we explore the biochemistry and physiological function of an Arabidopsis thylakoid membrane-associated lipase, PLASTID LIPASE1 (PLIP1). PLIP1 is a phospholipase A1 In vivo, PLIP1 hydrolyzes polyunsaturated acyl groups from a unique chloroplast-specific phosphatidylglycerol that contains 16:1 Δ3trans as its second acyl group. Thus far, a specific function of this 16:1 Δ3trans -containing phosphatidylglycerol in chloroplasts has remained elusive. The PLIP1 gene is highly expressed in seeds, and plip1 mutant seeds contain less oil and exhibit delayed germination compared with the wild type. Acyl groups released by PLIP1 are exported from the chloroplast, reincorporated into phosphatidylcholine, and ultimately enter seed triacylglycerol. Thus, 16:1 Δ3trans uniquely labels a small but biochemically active plastid phosphatidylglycerol pool in developing Arabidopsis embryos, which is subject to PLIP1 activity, thereby contributing a small fraction of the polyunsaturated fatty acids present in seed oil. We propose that acyl exchange involving thylakoid lipids functions in acyl export from plastids and seed oil biosynthesis.

Lipid composition and emulsifying properties of canola lecithin from enzymatic degumming.

This study investigated the polar lipid composition and emulsifying properties of canola lecithin from enzymatic degumming (CLED). Phospholipase A1 was used for enzymatic degumming of crude canola oil to collect lecithin sample. Canola lecithin from water degumming (CLWD) was also collected and served as the control. The results showed that the contents of phosphatidylethanolamine (PE) (2.99%) and phosphatidylcholine (PC) (6.59%) in CLED were significantly lower than that in CLWD (PE 15.55% and PC 21.93%); while the content of lysophosphatidylcholine (LPC) (19.45%) in CLED was significantly higher than that in CLWD (3.27%). Unsaturated fatty acids accounted for a higher percentage of the total fatty acids in CLED than in CLWD. CLED promoted more stable o/w emulsions than CLWD. This study provides a better understanding of the chemical nature of CLED, and important information for utilization of CLED as o/w emulsifier.

Selective Enrichment of Omega-3 Fatty Acids in Oils by Phospholipase A1.

Omega fatty acids are recognized as key nutrients for healthier ageing. Lipases are used to release ω-3 fatty acids from oils for preparing enriched ω-3 fatty acid supplements. However, use of lipases in enrichment of ω-3 fatty acids is limited due to their insufficient specificity for ω-3 fatty acids. In this study use of phospholipase A1 (PLA1), which possesses both sn-1 specific activity on phospholipids and lipase activity, was explored for hydrolysis of ω-3 fatty acids from anchovy oil. Substrate specificity of PLA1 from Thermomyces lenuginosus was initially tested with synthetic p-nitrophenyl esters along with a lipase from Bacillus subtilis (BSL), as a lipase control. Gas chromatographic characterization of the hydrolysate obtained upon treatment of anchovy oil with these enzymes indicated a selective retention of ω-3 fatty acids in the triglyceride fraction by PLA1 and not by BSL. 13C NMR spectroscopy based position analysis of fatty acids in enzyme treated and untreated samples indicated that PLA1 preferably retained ω-3 fatty acids in oil, while saturated fatty acids were hydrolysed irrespective of their position. Hydrolysis of structured triglyceride,1,3-dioleoyl-2-palmitoylglycerol, suggested that both the enzymes hydrolyse the fatty acids at both the positions. The observed discrimination against ω-3 fatty acids by PLA1 appears to be due to its fatty acid selectivity rather than positional specificity. These studies suggest that PLA1 could be used as a potential enzyme for selective concentrationof ω-3 fatty acids.

Práticas de cuidado ao idoso indígena - atuação dos profissionais de saúde / The elderly care practices of indigenous-performance of health / Las prácticas de cuidado de ancianos indígenas de rendimiento de la salud

Este estudo objetivou compreender as práticas de cuidado dos profissionais de saúde que assistem os idosos Kaingang. Estudo qualitativo, apoiado na etnografia, realizado com dez profissionais à que atuam na atenção primária saúde da Terra Indígena Faxinal, Paraná, Brasil. Os dados foram coletados no período de novembro de 2010 a fevereiro de 2012 por meio da observação participante e entrevistas, e, analisados à luz da Teoria Transcultural do Cuidado. Identificaram-se como práticas de cuidado a medicação e imunização, bem como, cuidados da medicina tradicional. Para realização destes cuidados, os profissionais dispunham de estratégias que proporcionavam manutenção dos idosos na assistência. Conclui-se que valores culturais e científicos necessitam integrar a assistência para melhoria da saúde dos idosos indígenas.

This research aims to understand the care practices of health professionals who assist the elderly Kaingang. It is a qualitative study, supported in ethnography, conducted by ten professionals working in primary health care in the indigenous land of Faxinal, Paraná, Brazil. The data was collected from November 2010 to February 2012 by participant observation and interviews, and analyzed based on the Transcultural Care Theory. Was identified the preoccupation of the carers practices with the medication and immunization, as well as traditional medical care. To achieve these, care professionals had strategies that implemented maintenance of older people in care. We conclude that cultural values and integrate scientific need assistance to improve the health of elderly indigenous.

Este estudio tuvo como objetivo entender las prácticas de cuidado de los profesionales de la salud que asisten a los ancianos Kaingang. Estudio cualitativo, apoyado en la etnografía, llevado a cabo con diez profesionales que trabajan en la atención primaria de la salud de la tierra indígena de Faxinal, Paraná, Brasil. Los datos fueron recogidos a partir de noviembre 2010 a febrero 2012 a través de la observación participante y las entrevistas, y analizado con base en la Teoría del Cuidado Transcultural. Se identificaron las prácticas de atención médica y imunizacion,el cuidado de la medicina, así tradicional. Para lograrlo, los profesionales tenían estrategias que proporcionaban el mantenimiento de las personas mayores en su atención. Se concluye que los valores culturales y científicos necesitan ayuda para mejorar la salud de los ancianos indígenas.

The effect of ultrasound on enzymatic degumming process of rapeseed oil by the use of phospholipase A(1).

Comparative studies of enzymatic degumming process of rapeseed oil were carried out in mechanical-stirring and ultrasonic-assisted mechanical-stirring systems. The influences of enzyme dosage (10-50 mg/kg), pH (4.5-6), temperature (45-65 °C), water amount (1-3%), ultrasonic power (0.06-0.09 W/cm(3)) and reaction time were investigated subsequently. A suitable ultrasonic power of 0.07 W/cm(3) was determined to guarantee satisfactory degumming efficiency and enzyme activity. Compared to the mechanical-stirring system, optimum temperature of phospholipase A (PLA) in the ultrasonic-assisted mechanical-stirring system was about 5 °C higher, while the effects of pH on both of the two systems were quite similar. Less time and water were used in the ultrasonic-assisted mechanical-stirring system for enzymatic degumming. The study on the quality changes of degummed oils showed that ultrasound could accelerate the oxidation of edible oils due to the effect of cavitation, thus more attention should be paid on the oxidative stability in the further application.

Purification of L-alpha glycerylphosphorylcholine by column chromatography.

Colorless L-alpha glycerylphosphorylcholine (L-α-GPC) was obtained at 99.8% purity, 69.8% recovery, and with a specific rotation of -2.5° via a five-step procedure. L-α-GPC was first produced by phospholipase A(1) hydrolysis of soy lecithin powder. Ca(2+) and Cl(-) were then effectively removed using two successive 001×7 cation and D311 anion exchange resin column chromatography procedures. Silica gel column chromatography and decoloration with active carbon were then applied to remove remaining impurities and colorant. Characterization of the L-α-GPC product was well in agreement with the standard. The resin and silica gel showed remarkable ability for L-α-GPC isolation after 10 uses. Thus, this study presents a simple and cost-effective method for preparing L-α-GPC with high yield and purity, low cost, and environmental friendliness, and encourages future investigation into its adaptation for industrial applications.

AtDSEL, an Arabidopsis cytosolic DAD1-like acylhydrolase, is involved in negative regulation of storage oil mobilization during seedling establishment.

Mobilization of seed storage reserves is essential for seed germination and seedling establishment. Here, we report that AtDSEL, an Arabidopsis thalianaDAD1-like Seedling Establishment-related Lipase, is involved in the mobilization of storage oils for early seedling establishment. AtDSEL is a cytosolic member of the DAD1-like acylhydrolase family encoded by At4g18550. Bacterially expressed AtDSEL preferentially hydrolyzed 1,3-diacylglycerol and 1-monoacylglycerol, suggesting that AtDSEL is an sn-1-specific lipase. AtDSEL-overexpressing transgenic Arabidopsis plants (35S:AtDSEL) were defective in post-germinative seedling growth in medium without an exogenous carbon source. This phenotype was rescued by the addition of sucrose to the growth medium. In contrast, loss-of-function mutant plants (atdsel-1 and atdsel-2) had a mildly fast-growing phenotype regardless of the presence of an exogenous carbon source. Electron microscopy revealed that 5-day-old 35S:AtDSEL cotyledons retained numerous peroxisomes and oil bodies, which were exhausted in wild-type and mutant cotyledons. The impaired seedling establishment of 35S:AtDSEL was not rescued by the addition of an exogenous fatty acid source, and 35S:AtDSEL seedling growth was insensitive to 2,4-dichlorophenoxybutyric acid, indicating that ß-oxidation was blocked in AtDSEL-overexpressers. These results suggest that AtDSEL is involved in the negative regulation of seedling establishment by inhibiting the breakdown of storage oils.

Activation tagging of an Arabidopsis SHI-RELATED SEQUENCE gene produces abnormal anther dehiscence and floral development.

The tapetum is a layer of cells covering the inner surface of pollen sac wall. It contributes to anther development by providing enzymes and materials for pollen coat biosynthesis and nutrients for pollen development. At the end of anther development, the tapetum is degenerated, and the anther is dehisced, releasing mature pollen grains. In Arabidopsis, several genes are known to regulate tapetum formation and pollen development. However, little is known about how tapetum degeneration and anther dehiscence are regulated. Here, we show that an activation-tagged mutant of the S HI-R ELATED S EQUENCE 7 (SRS7) gene exhibits disrupted anther dehiscence and abnormal floral organ development in addition to its dwarfed growth with small, curled leaves. In the mutant hypocotyls, cell elongation was reduced, and gibberellic acid sensitivity was diminished. Whereas anther development was normal, its dehiscence was suppressed in the dominant srs7-1D mutant. In wild-type anthers, the tapetum disappeared at anther development stages 11 and 12. In contrast, tapetum degeneration was not completed at these stages, and anther dehiscence was inhibited, causing male sterility in the mutant. The SRS7 gene was expressed mainly in the filaments of flowers, where the DEFECTIVE-IN-ANTHER-DEHISCENCE 1 (DAD1) enzyme catalyzing jasmonic acid (JA) biosynthesis is accumulated immediately before flower opening. The DAD1 gene was induced in the srs7-1D floral buds. In fully open flowers, the SRS7 gene was also expressed in pollen grains. It is therefore possible that the abnormal anther dehiscence and floral development of the srs7-1D mutant would be related with JA.

Expression of phosphatidylserine-specific phospholipase A(1) mRNA in human THP-1-derived macrophages.

The expression of phosphatidylserine-specific phospholipase A(1) (PS-PLA(1)) is most upregulated in the genes of peripheral blood cells from chronic rejection model rats bearing long-term surviving cardiac allografts. The expression profile of PS-PLA(1) in peripheral blood cells responsible for the immune response may indicate a possible biological marker for rejection episodes. In this study, PS-PLA(1) mRNA expression was examined in human THP-1-derived macrophages. The effects of several immunosuppressive agents on this expression were also examined in in vitro experiments. A real-time RT-PCR analysis revealed that PS-PLA(1) mRNA expression was found in human THP-1-derived macrophages. This expression was enhanced in the cells stimulated with lipopolysaccharide (LPS), a toll-like receptor (TLR) 4 ligand. Other TLR ligands (TLR2, 3, 5, 7, and 9) did not show a significant induction of PS-PLA(1) mRNA. The time course of the mRNA expression profiles was different between PS-PLA(1) and tumor necrosis factor-α (TNF-α), which showed a maximal expression at 12 and 1 h after LPS stimulation, respectively. Among the observed immunosuppressive agents, corticosteroids, prednisolone, 6α-methylprednisolone, dexamethasone, and beclomethasone inhibited PS-PLA(1) expression with half-maximal inhibitory concentrations less than 3.0 nM, while methotrexate, cyclosporine A, tacrolimus, 6-mercaptopurine, and mycophenoic acid showed either a weak or moderate inhibition. These results suggest that the expression of PS-PLA(1) mRNA in THP-1-derived macrophages is activated via TLR4 and it is inhibited by corticosteroids, which are used at high dosages to suppress chronic allograft rejection.

The tumor suppressor gene H-Rev107 functions as a novel Ca2+-independent cytosolic phospholipase A1/2 of the thiol hydrolase type.

H-Rev107 is a protein that was previously cloned as a negative regulator of proto-oncogene Ras and classified as a class II tumor suppressor. Its structural similarity to lecithin retinol acyltransferase and Ca2+-independent phosphatidylethanolamine (PE) N-acyltransferase led us to analyze H-Rev107 as an enzyme involved in phospholipid metabolism. Here, we show that recombinant H-Rev107s from rat, human, and mouse possess phospholipase (PL) A1 or A2 activity toward phosphatidylcholine (PC). Further examination with purified recombinant protein revealed that H-Rev107 functions as a cytosolic Ca2+-independent PLA(1/2) for PC and PE with higher PLA1 activity than PLA2 activity. Dithiothreitol and iodoacetic acid exhibited stimulatory and inhibitory effects, respectively. Histidine-21 and cysteine-111 of rat H-Rev107 were presumed to form a catalytic dyad based on database analysis, and their single mutants were totally inactive. These results suggested that H-Rev107 is a hydrolase of the thiol type. The N-terminal proline-rich and C-terminal hydrophobic domains of H-Rev107 were earlier reported to be responsible for the regulation of cell proliferation. Analysis of deletion mutants indicated that these domains are also catalytically essential, suggesting relevance of the catalytic activity to the anti-proliferative activity.

Expression in yeast of a novel phospholipase A1 cDNA from Arabidopsis thaliana.

During a search for cDNAs encoding plant sterol acyltransferases, we isolated four full-length cDNAs from Arabidopsis thaliana that encode proteins with substantial identity with animal lecithin : cholesterol acyltransferases (LCATs). The expression of one of these cDNAs, AtLCAT3 (At3g03310), in various yeast strains resulted in the doubling of the triacylglycerol content. Furthermore, a complete lipid analysis of the transformed wild-type yeast showed that its phospholipid content was lower than that of the control (void plasmid-transformed) yeast whereas lysophospholipids and free fatty acids increased. When microsomes from the AtLCAT3-transformed yeast were incubated with di-[1-14C]oleyl phosphatidylcholine, both the lysophospholipid and free fatty acid fractions were highly and similarly labelled, whereas the same incubation with microsomes from the control yeast produced a negligible labelling of these fractions. Moreover when microsomes from AtLCAT3-transformed yeast were incubated with either sn-1- or sn-2-[1-14C]acyl phosphatidylcholine, the distribution of the labelling between the free fatty acid and the lysophosphatidylcholine fractions strongly suggested a phospholipase A1 activity for AtLCAT3. The sn-1 specificity of this phospholipase was confirmed by gas chromatography analysis of the hydrolysis of 1-myristoyl, 2-oleyl phosphatidylcholine. Phosphatidylethanolamine and phosphatidic acid were shown to be also hydrolysed by AtLCAT3, although less efficiently than phosphatidylcholine. Lysophospatidylcholine was a weak substrate whereas tripalmitoylglycerol and cholesteryl oleate were not hydrolysed at all. This novel A. thaliana phospholipase A1 shows optimal activity at pH 6-6.5 and 60-65 degrees C and appears to be unaffected by Ca2+. Its sequence is unrelated to all other known phospholipases. Further studies are in progress to elucidate its physiological role.

Application of high-density array-based signature-tagged mutagenesis to discover novel Yersinia virulence-associated genes.

Yersinia pestis, the causative agent of plague, and the enteropathogen Yersinia pseudotuberculosis have nearly identical nucleotide similarity yet cause markedly different diseases. To investigate this conundrum and to study Yersinia pathogenicity, we developed a high-density oligonucleotide array-based modification of signature-tagged mutagenesis (STM). Y. pseudotuberculosis YPIII mutants constructed with the tagged transposons were evaluated in the murine yersiniosis infection model. The DNA tags were amplified using biotinylated primers and hybridized to high-density oligonucleotide arrays containing DNA complementary to the tags. Comparison of the hybridization signals from input pools and output pools identified a mutant whose relative abundance was significantly reduced in the output pool. Sequence data from 31 transposon insertion regions was compared to the complete Y. pestis CO92 genome sequence. The 26 genes present in both species were found to be almost identical, but five Y. pseudotuberculosis genes identified through STM did not have counterparts in the Y. pestis genome and may contribute to the different tropisms in these closely related pathogens. Potential virulence genes identified include those involved in lipopolysaccharide biosynthesis, adhesion, phospholipase activity, iron assimilation, and gene regulation. The phospholipase A (PldA) mutant exhibited reduced phospholipase activity compared to the wild-type strain and in vivo attenuation of the mutant was confirmed. The combination of optimized double tag sequences and high-density array hybridization technology offers improved performance, efficiency, and reliability over classical STM and permits quantitative analysis of data.

The DEFECTIVE IN ANTHER DEHISCIENCE gene encodes a novel phospholipase A1 catalyzing the initial step of jasmonic acid biosynthesis, which synchronizes pollen maturation, anther dehiscence, and flower opening in Arabidopsis.

The Arabidopsis mutant defective in anther dehiscence1 (dad1) shows defects in anther dehiscence, pollen maturation, and flower opening. The defects were rescued by the exogenous application of jasmonic acid (JA) or linolenic acid, which is consistent with the reduced accumulation of JA in the dad1 flower buds. We identified the DAD1 gene by T-DNA tagging, which is characteristic to a putative N-terminal transit peptide and a conserved motif found in lipase active sites. DAD1 protein expressed in Escherichia coli hydrolyzed phospholipids in an sn-1-specific manner, and DAD1-green fluorescent protein fusion protein expressed in leaf epidermal cells localized predominantly in chloroplasts. These results indicate that the DAD1 protein is a chloroplastic phospholipase A1 that catalyzes the initial step of JA biosynthesis. DAD1 promoter::beta-glucuronidase analysis revealed that the expression of DAD1 is restricted in the stamen filaments. A model is presented in which JA synthesized in the filaments regulates the water transport in stamens and petals.

Molecular characterization of cytosolic phospholipase A2-beta.

We have isolated a cDNA encoding a 1012-amino acid polypeptide cPLA2-beta, that has significant homology with cPLA2-alpha in both the calcium-dependent lipid binding domain as well as in the catalytic domain. Transient expression of cPLA2-beta cDNA in COS cells results in an increase in calcium-dependent phospholipase A1 (PLA1) and PLA2 activities compared with vector-transfected cells. cPLA2-beta is markedly less selective for cleavage at sn-2 as compared with cPLA2-alpha and cPLA2-gamma. Northern analysis reveals a cPLA2-beta transcript of 8 kilobase pairs that is expressed in all the human tissues examined. With the identification of cPLA2-beta, the newly defined cPLA2 family now comprises three members that may have dramatically different mechanisms for regulation of expression and enzymatic activation.

Molecular cloning and expression of the gene encoding a phospholipase A1 from Aspergillus oryzae.

Phospholipase A1 (PLA1) is a hydrolytic enzyme that catalyzes removal of the acyl group from position 1 of lecithin to form lysolecithin. The genomic DNA and cDNA encoding PLA1 from Aspergillus oryzae were cloned with the mixed deoxyribonucleotide-primed polymerase chain reaction. The PLA1 gene is composed of 1,056 bp and has four exons and three short introns (63, 54, and 51 bp). The deduced amino acid sequence of PLA1 contained the N-terminal sequence of the mature PLA1 analyzed by Edman degradation. PLA1 cDNA has an open reading frame of 885 bp encoding the PLA1 precursor of 295 amino acid residues. The mature PLA1 is composed of 269 amino acid residues, and a prepro-sequence of 26 amino acid residues is at the N-terminal region of the PLA1 precursor. PLA1 has two possible N-glycosylation sites (Asn27 and Asn55). PLA1 has a consensus pentapeptide (-Gly-His-Ser-Xaa-Gly-), which is conserved in lipases. The amino acid sequence of PLA1 showed 47% identity with that of mono- and diacylglycerol lipase from Penicillium camembertii. The PLA1 cDNA was expressed in Saccharomyces cerevisiae KS58-2D, indicating the cloned gene to be functional.

An alternative splicing form of phosphatidylserine-specific phospholipase A1 that exhibits lysophosphatidylserine-specific lysophospholipase activity in humans.

Phosphatidylserine-specific phospholipase A1 (PS-PLA1), which acts specifically on phosphatidylserine (PS) and 1-acyl-2-lysophosphatidylserine (lyso-PS) to hydrolyze fatty acids at the sn-1 position of these phospholipids, was first identified in rat platelets (Sato, T., Aoki, J., Nagai, Y., Dohmae, N., Takio, K., Doi, T., Arai, H., and Inoue, K. (1997) J. Biol. Chem. 272, 2192-2198). In this study we isolated and sequenced cDNA clones encoding human PS-PLA1, which showed 80% homology with rat PS-PLA1 at the amino acid level. In addition to an mRNA encoding a 456-amino acid product (PS-PLA1), an mRNA with four extra bases inserted at the boundary of the exon-intron junction was detected in human tissues and various human cell lines. This mRNA is most probably produced via an alternative use of the 5'-splicing site (two consensus sequences for RNA splicing occur at the boundary of the exon-intron junction) and encodes a 376-amino acid product (PS-PLA1DeltaC) that lacks two-thirds of the C-terminal domain of PS-PLA1. Unlike PS-PLA1, PS-PLA1DeltaC hydrolyzed exclusively lyso-PS but not PS appreciably. Any other phospholipids such as phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidic acid (PA), and their lyso derivatives were not hydrolyzed at all. These data demonstrated that PS-PLA1DeltaC exhibits lyso-PS-specific lysophospholipase activity and that the C-terminal domain of PS-PLA1 is responsible for recognizing diacylphospholipids. In addition, human PS-PLA1 gene was mapped to chromosome 3q13.13-13.2 and was unexpectedly identical to the nmd gene, which is highly expressed in nonmetastatic melanoma cell lines but poorly expressed in metastatic cell lines (van Groningen, J. J., Bloemers, H. P., and Swart, G. W. (1995) Cancer Res. 55, 6237-6243).