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The food enzyme with phospholipase A1 (phosphatidycholine 1‐acylhydrolase, EC 3.1.1.32) and lysophospholipase (2‐lysophosphatidylcholine acylhydrolase, EC 3.1.1.5) activities is produced with the genetically modified Aspergillus niger strain PLN by DSM. The genetic modifications do not give rise to safety concerns. The food enzyme is free from viable cells of the production organism and its DNA. It is intended to be used for the production of refined edible fats and oils by degumming. Since residual amounts of total organic solids are removed during this process, dietary exposure was not calculated and toxicological studies were considered unnecessary for the assessment of this food enzyme. A search for the similarity of the amino acid sequence of the food enzyme to known allergens was made and no matches were found. The Panel considered that the risk of allergic reactions upon dietary exposure cannot be excluded, but the likelihood is low. Based on the data provided, the Panel concluded that this food enzyme does not give rise to safety concerns, under the intended conditions of use. [ABSTRACT FROM AUTHOR]

To understand the lifespan of higher organisms, including humans, it is important to understand lifespan at the cellular level as a prerequisite. So, fission yeast is a good model organism for the study of lifespan. To identify the novel factors involved in longevity, we are conducting a large-scale screening of long-lived mutant strains that extend chronological lifespan (cell survival in the stationary phase) using fission yeast. One of the newly acquired long-lived mutant strains (No.98 mutant) was selected for analysis and found that the long-lived phenotype was due to a missense mutation (92Phe → Ile) in the plb1+ gene. plb1+ gene in fission yeast is a nonessential gene encoding a homolog of phospholipase B, but its functions under normal growth conditions, as well as phospholipase B activity, remain unresolved. Our analysis of the No.98 mutant revealed that the plb1 mutation reduces the integrity of the cellular membrane and cell wall and activates Sty1 via phosphorylation. [ABSTRACT FROM AUTHOR]

The phospholipase B homolog Plb1 and the cAMP-dependent protein kinase (PKA) pathway are required by fission yeast, also known as to Schizosaccharomyces pombe, to grow under KCl-stress conditions. Here, we report the relative contributions of Plb1 and the cAMP/PKA pathway during the hypertonic stress response. We show that the plb1∆, cyr1∆, and pka1∆ single mutants are sensitive to high concentrations of KCl but insensitive to sorbitol-induced osmotic stress. In contrast, the plb1∆ cyr1∆ and plb1∆ pka1∆ double mutants are hypersensitive to KCl and sorbitol. The cyr1∆ pka1∆ double mutants showed the same phenotype of each single mutant. Growth inhibition due to hypertonic stress in the plb1∆, plb1∆ cyr1∆, and plb1∆ pka1∆ strains was partially rescued by cgs1 deletion—cgs1∆ has constitutively active Pka1—or by the deletion of transcription factor Rst2, which is negatively regulated by Pka1. Pka1-GFP localized in the nucleus and cytoplasm in plb1∆, whereas it is localized only in the cytoplasm in cyr1∆, indicating that Plb1 does not regulate Pka1 localization. Glucose limitation downregulates the PKA pathway, and it was accordingly observed that glucose limitation in plb1∆ further increased the strain's sensitivity to KCl. Growth inhibition by KCl in plb1∆ under glucose-limited conditions was significantly rescued by cgs1∆ and slightly rescued by rst2∆. These findings indicate that, in fission yeast, Plb1 and the glucose-sensing cAMP/PKA pathway play a synergistic role in responding to hypertonic stress. [ABSTRACT FROM AUTHOR]

Phospholipase B (PLB) harbors three distinct activities with broad substrate specificities and application fields. Its hydrolyzing of sn-1 and sn-2 acyl ester bonds enables it to catalyze the production of L-alpha-glycerylphosphorylcholine (L-α-GPC) from phosphatidylcholine (PC) without speed-limiting acyl migration. This work was intended to obtain high-level active PLB and apply it to establish an efficient system for L-α-GPC synthesis. PLB from Pseudomonas fluorescens was co-expressed with five different molecular chaperones, including trigger factor (Tf), GroEL-GroES (GroELS), DnaK-DnaJ-GrpE (DnaKJE), GroELS and DnaKJE, or GroELS and Tf or fused with maltose binding protein (MBP) in Escherichia coli BL21(DE3) to improve PLB expression. PLB with DnaKJE-assisted expression exhibited the highest catalytic activity. Further optimization of the expression conditions identified an optimal induction OD600 of 0.8, IPTG concentration of 0.3 mmol/L, induction time of 9 h, and temperature of 25 °C. The PLB activity reached a maximum of 524.64 ± 3.28 U/mg under optimal conditions. Subsequently, to establish an efficient PLB-catalyzed system for L-α-GPC synthesis, a series of organic-aqueous mixed systems and surfactant-supplemented aqueous systems were designed and constructed. Furthermore, the factors of temperature, reaction pH, metal ions, and substrate concentration were further systematically identified. Finally, a high yield of 90.50 ± 2.21% was obtained in a Span 60-supplemented aqueous system at 40 °C and pH 6.0 with 0.1 mmol/L of Mg2+. The proposed cost-effective PLB production and an environmentally friendly PLB-catalyzed system offer a candidate strategy for the industrial production of L-α-GPC. [ABSTRACT FROM AUTHOR]

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Abstract The food enzyme is a lysophospholipase (2‐lysophosphatidylcholine acylhydrolase; EC 3.1.1.5) produced with a genetically modified Aspergillus niger strain NZYM‐LP by Novozymes A/S. The genetic modifications do not give rise to safety concerns. The food enzyme is free from viable cells of the production organism and its DNA. The lysophospholipase food enzyme is intended to be used in starch processing for glucose syrups production, and for degumming of fats and oils. Residual amounts of total organic solids (TOS) are removed by the purification steps applied during the production of glucose syrups, and washing and purification steps applied during degumming, consequently, dietary exposure estimation was considered not necessary. Genotoxicity tests did not raise safety concerns. The repeated dose 90‐day oral toxicity study was carried out with a phospholipase A1 from A. niger (strain NZYM‐FP). The Panel considered this enzyme as a suitable substitute to be used in this toxicity study in rats, because they derive from the same recipient strain, the location of the inserts are comparable, no partial inserts were present and the production methods are essentially the same. The Panel identified a no observed adverse effect level (NOAEL) at the highest dose tested of 1,356 mg TOS/kg body weight (bw) per day. Similarity of the amino acid sequence to those of known allergens was searched and no match was found. The Panel considered that, under the intended conditions of use, the risk of allergic sensitisation and elicitation reactions by dietary exposure cannot be excluded, but the likelihood for this occurring is considered to be low. Based on the data provided, the removal of TOS during the starch processing for the production of glucose syrups and during the degumming of fats and oils, the Panel concluded that this food enzyme does not give rise to safety concerns under the intended conditions of use.

l-alpha-glycerylphosphorylcholine (GPC) has been shown to enhance cognitive performance. Meanwhile, vegetable oils must be refined to remove the impurities for them to be edible. Phospholipase B (PLB), having the ability of hydrolyzing both the sn-1 and sn-2 acyl ester bonds of phospholipids, can produce GPC using PC as substrate and transform the non-hydratable phospholipids into their hydratable forms. The Saccharomyces cerevisiae plb gene, which encodes PLB, was cloned and expressed in Pichia pastoris GS115 to produce recombinant PLB (rPLB). Fermentation optimisation yielded rPLB activity levels as high as 1723 U/mL. rPLB demonstrated maximum enzymatic activity at 40 °C and pH 5.5 and was stable at temperatures between 30 and 40 °C and pH values between 5.0 and 6.0. rPLB synthesised GPC with a conversion rate of 17% (w/w) and exhibited high degumming activity towards peanut oil, decreasing the phosphorus content from 91.8 to 3.7 mg/kg within 3 h. This study describes a candidate phospholipase for potential applications involving the modification of phospholipids and vegetable oil degumming.

The food enzyme is a lysophospholipase (2‐lysophosphatidylcholine acylhydrolase; EC 3.1.1.5) produced with a genetically modified Aspergillus niger strain NZYM‐LP by Novozymes A/S. The genetic modifications do not give rise to safety concerns. The food enzyme is free from viable cells of the production organism and its DNA. The lysophospholipase food enzyme is intended to be used in starch processing for glucose syrups production, and for degumming of fats and oils. Residual amounts of total organic solids (TOS) are removed by the purification steps applied during the production of glucose syrups, and washing and purification steps applied during degumming, consequently, dietary exposure estimation was considered not necessary. Genotoxicity tests did not raise safety concerns. The repeated dose 90‐day oral toxicity study was carried out with a phospholipase A1 from A. niger (strain NZYM‐FP). The Panel considered this enzyme as a suitable substitute to be used in this toxicity study in rats, because they derive from the same recipient strain, the location of the inserts are comparable, no partial inserts were present and the production methods are essentially the same. The Panel identified a no observed adverse effect level (NOAEL) at the highest dose tested of 1,356 mg TOS/kg body weight (bw) per day. Similarity of the amino acid sequence to those of known allergens was searched and no match was found. The Panel considered that, under the intended conditions of use, the risk of allergic sensitisation and elicitation reactions by dietary exposure cannot be excluded, but the likelihood for this occurring is considered to be low. Based on the data provided, the removal of TOS during the starch processing for the production of glucose syrups and during the degumming of fats and oils, the Panel concluded that this food enzyme does not give rise to safety concerns under the intended conditions of use. [ABSTRACT FROM AUTHOR]

Measuring the production of Candida dubliniensis (C. dubliniensis) phospholipase B (PLase B) by the Price's method has long been considered to be unattainable because the levels of PLase produced are undetectable. In this study, C. dubliniensis, C. glabrata, C. guilliermondii, C. krusei, C. parapsilosis and C. tropicalis were shown to produce PLase B and form clear white zones around their colonies when peptone, a component of the original Price's egg yolk (OP) agar, is replaced with a yeast nitrogen base (YNB). This new medium is named modified Price's (MP) agar. Based on this finding, we propose a new modified Price's (NMP) agar containing 0.75% peptone and 0.25% YNB, which enabled measurement of PLase B production by C. dubliniensis and C. albicans with results consistent with those obtained for C. albicans grown on OP agar. We strongly believe that the MP and NMP agars are very useful for screening PLase B production by C. dubliniensis and non-albicans Candida spp. Moreover, the addition of several bioactive agents (the proteinase inhibitors pepstatin A and saquinavir, the calcineurin inhibitors cyclosporine A and tacrolimus, the cell-permeable cAMP analog dBcAMP, and the quorum-sensing molecule farnesol) to the OP agar enhanced PLase B production by C. dubliniensis. During the course of our study to clarify the reason why PLase B was not produced, we found that C. dubliniensis cells grown on OP agar undergo a white-to-opaque transition, which may explain why they showed minimal production of PLase B on this medium. [ABSTRACT FROM AUTHOR]

Phospholipase B is a virulence factor for several clinically important pathogenic fungi, including Candida albicans, Cryptococcus neoformans and Aspergillus fumigatus, but its role in the thermally dimorphic fungus Talaromyces marneffei remains unclear. Here, we provide the first report of the expression of a novel phospholipase gene, designated TmPlb1, from T. marneffei in the eukaryotic expression system of Pichia pastoris GS115. Sensitive real-time quantitative reverse-transcription PCR (qRT-PCR) demonstrated that the expression of TmPlb1 increased 1.85-fold in the yeast phase compared with the mycelial phase. TmPlb1 contains an open reading frame (ORF) of 732 bp that encodes a protein of 243 amino acids. The conserved serine, aspartate and histidine catalytic triad and the G-X-S-X-G domain of TmPLB1 provide the structural basis for its molecular activity. The ORF of TmPlb1 was successfully cloned into a pPIC9K vector containing an α-mating factor secretion signal that allowed the secretory expression of TmPLB1 in P. pastoris. The heterologous protein expression began 12 h after methanol induction and peaked at 96 h. Through analysis with SDS–polyacrylamide gel electrophoresis (SDS-PAGE), western blotting and mass spectrometry, we confirmed that TmPLB1 was successfully expressed. Through Ni-affinity chromatography, TmPLB1 was highly purified, and its concentration reached 240.4 mg/L of culture medium. With specific substrates, the phospholipase A1 and phospholipase A2 activities of TmPLB1 were calculated to be 5.96 and 1.59 U/mg, respectively. The high purity and activity of the TmPLB1 obtained here lay a solid foundation for further investigation.Emerging Microbes & Infections (2016) 5, e120; doi:10.1038/emi.2016.119; published online 23 November 2016

L-alpha-glycerylphosphorylcholine (GPC) has been shown to enhance cognitive performance. Meanwhile, vegetable oils must be refined to remove the impurities for them to be edible. Phospholipase B (PLB), having the ability of hydrolyzing both the sn-1 and sn-2 acyl ester bonds of phospholipids, can produce GPC using PC as substrate and transform the non-hydratable phospholipids into their hydratable forms. The Saccharomyces cerevisiae plb gene, which encodes PLB, was cloned and expressed in Pichia pastoris GS115 to produce recombinant PLB (rPLB). Fermentation optimisation yielded rPLB activity levels as high as 1723 U/mL. rPLB demonstrated maximum enzymatic activity at 40 °C and pH 5.5 and was stable at temperatures between 30 and 40 °C and pH values between 5.0 and 6.0. rPLB synthesised GPC with a conversion rate of 17% (w/w) and exhibited high degumming activity towards peanut oil, decreasing the phosphorus content from 91.8 to 3.7 mg/kg within 3 h. This study describes a candidate phospholipase for potential applications involving the modification of phospholipids and vegetable oil degumming. [ABSTRACT FROM AUTHOR]

Phospholipase B (EC 3.1.1.5) are a distinctive group of enzymes that catalyzes the hydrolysis of fatty acids esterified at the sn-1 and sn-2 positions forming free fatty acids and lysophospholipids. The structural information and catalytic mechanism of phospholipase B are still not clear. Herein, we reported a putative phospholipase B (TmPLB1) from Talaromyces marneffei GD-0079 synthesized by genome mining library. The gene (TmPlb1) was expressed and the TmPLB1 was purified using E. coli shuffle T7 expression system. The putative TmPLB1 was purified by affinity chromatography with a yield of 13.5%. The TmPLB1 showed optimum activity at 35 °C and pH 7.0. The TmPLB1 showed enzymatic activity using Lecithin (soybean > 98% pure), and the hydrolysis of TmPLB1 by 31P NMR showed phosphatidylcholine (PC) as a major phospholipid along with lyso-phospholipids (1-LPC and 2-LPC) and some minor phospholipids. The molecular modeling studies indicate that its active site pocket contains Ser125, Asp183 and His215 as the catalytic triad. The structure dynamics and simulations results explained the conformational changes associated with different environmental conditions. This is the first report on biochemical characterization and structure dynamics of TmPLB1 enzyme. The present study could be helpful to utilize TmPLB1 in food industry for the determination of food components containing phosphorus. Additionally, such enzyme could also be useful in Industry for the modifications of phospholipids.

In this study, a gene encoding a putative lipase from Fusarium oxysporum was optimized via codon optimization and expressed in Pichia pastoris KM71. The gene product was identified as a phospholipase B (PLB). The engineered P. pastoris was further cultured in a 3.6-L bioreactor. After optimization of the induction conditions, this system produced 6.6 mg mL −1 protein and 6503.8 U mL −1 PLB activity in the culture medium. Efficient expression of this PLB in P. pastoris should reduce the costs of production and application. The purified enzyme, with a specific activity of 1170 U mg −1 , was optimally active at pH 5.0 and 55 °C. The results of a degumming experiment performed using the recombinant PLB showed that the phosphorus content of a test oil was decreased from 75.88 ppm to 3.3 ppm in 2 h under optimal reaction conditions. This study provides a basis for the industrial use of F. oxysporum PLB in oil degumming applications. [ABSTRACT FROM AUTHOR]

Phospholipases play a key role in the metabolism of phospholipids and in cell signaling. They are also a very useful tool to explore phospholipid structure and metabolism as well as membrane organization. They are at the center of this review, covering a period starting in 1971 and focused on a number of subjects in which my colleagues and I have been involved. Those include determination of phospholipid asymmetry in the blood platelet membrane, biosynthesis of lysophosphatidic acid, biochemistry of platelet-activating factor, first attempts to define the role of phosphoinositides in cell signaling, and identification of novel digestive (phospho)lipases such as pancreatic lipase-related protein 2 (PLRP2) or phospholipase B. Besides recalling some of our contributions to those various fields, this review makes an appraisal of the impressive and often unexpected evolution of those various aspects of membrane phospholipids and lipid mediators. It is also the occasion to propose some new working hypotheses. [ABSTRACT FROM AUTHOR]

Neuropathy target esterase (NTE) has been proven to act as a lysophospholipase (LysoPLA) and phospholipase B (PLB) in mammalian cells. In this study, we took human neuroblastoma SK-N-SH cells as the research object and explored the effect of NTE on phospholipid homeostasis. The results showed that phosphatidylcholine (PC) and lysophosphatidylcholine (LPC) levels significantly increased (> 40%), while glycerophosphocholine (GPC) decreased (below 60%) after NTE gene was knockdown in the cells (NTE < 30% of control), which were prepared by gene silencing with dsRNA-NTE. However, in the NTE-overexpressed cells (NTE > 50% of control), which were prepared by expressing recombinant catalytic domain of NTE, LPC remarkably decreased (below 80%) and GPC enhanced (> 40%). Mipafox, a neuropathic organophosphorus compound (OP), significantly inhibited NTE-LysoPLA and NTE-PLB activities (> 95–99% inhibition at 50 μM), which was accompanied with a decreased GPC level (below 40%) although no change of the PC and LPC levels was observed; while paraoxon, a non-neuropathic OP, suppresses neither the activities of NTE-phospholipases nor the levels of PC, LPC, and GPC. Thus, we concluded that both the stable up- or down-regulated expression of NTE gene and the loss of NTE-LysoPLA/PLB activities disrupts phospholipid homeostasis in the cells although the inhibition of NTE activity only decreased GPC content without altering PC and LPC levels. • Over- or down-expression of NTE disrupts phospholipids homeostasis in SK-N-SH cells • PC and LPC levels enhanced and GPC decreased in the NTE-knockeddown cells • LPC was decreased and GPC was enhanced in the NTE-overexpressed cells • Inhibition of NTE activity decreased GPC content without altering PC and LPC levels • Loss of NTE activity does not affect the cell proliferation [ABSTRACT FROM AUTHOR]

Hyaluronidases contribute to local and systemic damages after envenoming, since they act as spreading factors cleaving the hyaluronan presents in the connective tissues of the victim, facilitating the diffusion of venom components. Although hyaluronidases are ubiquitous in snake venoms, they still have not been detected in transcriptomic analysis of the Lachesis venom gland and neither in the proteome of its venom performed previously. This work purified a hyaluronidase from Lachesis muta rhombeata venom whose molecular mass was estimated by SDS-PAGE to be 60 kDa. The hyaluronidase was more active at pH 6 and 37 °C when salt concentration was kept constant and more active in the presence of 0.15 M monovalent ions when the pH was kept at 6. Venom was fractionated by reversed-phase liquid chromatography (RPLC). Edman sequencing after RPLC failed to detect hyaluronidase, but identified a new serine proteinase isoform. The hyaluronidase was identified by mass spectrometry analysis of the protein bands in SDS-PAGE. Additionally, phospholipase B was identified for the first time in Lachesis genus venom. The discovery of new bioactive molecules might contribute to the design of novel drugs and biotechnology products as well as to development of more effective treatments against the envenoming. [ABSTRACT FROM AUTHOR]

A novel phospholipase B (TLPLB) from Thermotoga lettingae TMO has been cloned, functionally overexpressed in Escherichia coli and purified to homogeneity. Gas chromatography indicated that the enzyme could efficiently hydrolyze both the sn-1 and sn-2 ester bonds of 1-palmitoyl-2-oleoyl phosphatidylcholine as phospholipase B. TLPLB was optimally active at 70 °C and pH 5.5, respectively. Its thermostability is relatively high with a half-life of 240 min at 90 °C. TLPLB also displayed remarkable organic solvent tolerance and maintained approximately 91-161 % of its initial activity in 20 and 50 % (v/v) hydrophobic organic solvents after incubation for 168 h. Furthermore, TLPLB exhibited high degumming activity towards rapeseed, soybean, peanut and sunflower seed oils, where the phosphorus contents were decreased from 225.2, 189.3, 85.6 and 70.4 mg/kg to 4.9, 4.7, 3.2 and 2.2 mg/kg within 5 h, respectively. TLPLB could therefore be used for the degumming of vegetable oils. [ABSTRACT FROM AUTHOR]

Highlights: [•] The recombinant phospholipase B displayed 2-fold higher activity compared to native strain. [•] The recombinant enzyme could degum the phosphorous content of vegetable oils <5mg/kg. [•] Phospholipase B from Pseudomonas fluorescens BIT-18 was overexpressed in Pichia pastoris. [Copyright &y& Elsevier]

A novel metal ion-independent phospholipase B ( PLB684) from Streptomyces sp. strain NA684 was purified 264-fold from the culture supernatant with 2.85% recovery (6330 U·mg protein−1). The enzyme functions as a monomer with a molecular mass of 38.9 kDa. Maximum activity was found at pH 8.4 and 50 °C. The substrate specificity was in the order: phosphatidylcholine ≥ phosphatidic acid ≥ lysophosphatidylcholine > phosphatidylserine > phosphatidylinositol > phosphatidylglycerol. The enzyme did not hydrolyze phosphatidylethanolamine, tristearin and dipalmitin. PLB684 hydrolyzed lysophosphatidylcholine and diacylphosphatidylcholine, and lysophosphatidylcholine was primarily produced during the early stages of phosphatidylcholine hydrolysis. The apparent Km, Vmax and kcat for hydrolysis of dimyristoyl phosphatidic acid were 14.5 m m, 15.8 mmol·min−1·mg protein−1 and 1.02 × 104 s−1, respectively. The positional specificity of 1-palmitoyl-2-oleoyl- sn-glycero-3-phosphocholine hydrolysis was investigated using GC. In the reaction equilibrium, the molar ratio of released fatty acids ( sn-1 : sn-2) was 45 : 55. The ORF of the gene is 1239 bp in length and codes for a 30-amino acid signal peptide and a 382-amino acid mature enzyme. The deduced amino acid sequence of PLB684 shows 60% identity to a uncharacterized protein of Streptomyces auratus AGR0001 (UniProt accession number: J1RQY0). The extracellular production of PLB684 was achieved using a pUC702 expression vector and Streptomyces lividans as the host. Mutagenesis analysis showed that Ser12 is essential for the catalytic function of PLB684 and that the active site may include residues Ser330 and His332. [ABSTRACT FROM AUTHOR]

Abstract: A gene from Pseudomonas fluorescens BIT-18 encoding a protein with phospholipase B activity (Pf-PLB) was cloned in E. coli BL21 (DE3). The open reading frame consists of 1272bp and potentially encodes a protein of 423 amino acid residues with a calculated molecular mass of 45.8kDa. The nucleotide sequence of Pf-PLB is 45%, 42%, 41%, 40%, 33%, and 31% identical to that of Bifidobacterium animals, Mycobacterium parascrofulaceum, Acidobacterium capsulatum, Lactobacillus johnsonii, Moraxella bovis, and Moraxella catarrhalis, respectively. The His-tagged protein was purified by affinity chromatography and the eluted protein hydrolyzed both the 1- and 2-ester bond of phosphatidylcholine. The recombinant Pf-PLB had optimal activity at pH 6.0 and 30°C, and it showed 20.1% higher efficiency in the conversion rate of the phosphorus content than the wild-type. [Copyright &y& Elsevier]

Abstract: Pseudomonas fluorescens BIT-18 was isolated from soil near a vegetable oil factory and shown to produce a B-type phospholipase. The enzyme was partially purified by ammonium sulfate precipitation. Gas chromatography demonstrated that the enzyme preparation hydrolyzed both the 1- and 2-ester bonds of phosphatidylcholine. When degumming of soybean, rapeseed, and peanut oil was performed with this enzyme preparation, oils with phosphorous contents lower than 5mg/kg were obtained after 5h of enzyme treatment at 40°C. The enzyme preparation did not show lipase activity, thus free fatty acids were only generated from the phospholipids. Therefore, this novel phospholipase B is potentially useful for the refining of high-quality oils with attractive yields. [Copyright &y& Elsevier]

Tobacco products and cigarette smoke cause many respiratory diseases including cancer. 4-(Methyl nitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is a potent carcinogen in cigarette smoke, but its effect on lipid metabolism remains enigmatic. Hence, Saccharomyces cerevisiae was exposed to different concentrations of NNK (0-400 µmol L-1) to elucidate its role in lipid metabolism. Exposure to NNK substantially decreases (about 60%) of the phospholipid content with a concomitant increase in lysophospholipids. Significant reduction was observed in the phosphatidylcholine followed by phosphatidylethanolamine with NNK-treated cells. On the contrary, cells accumulated significant amount of neutral lipids and free fatty acids. Exposure of yeast cells (wild-type cells and three plbΔ mutant strains) to NNK greatly enhances the hydrolysis of phospholipid in the presence of calcium. We are the first to report that exposure to NNK enhances phospholipase B (PLB), particularly plb1p activity. Furthermore, NNK also promotes the alteration of phospholipid fatty acid (FA) content. These results suggest that NNK aids in the degradation of phospholipids by enhanced PLB activity and is accompanied with FA alteration. Understanding the altered phospholipid metabolism in the presence of NNK remains a worthy pursuit. [ABSTRACT FROM AUTHOR]

tobacco-specific nitrosamine (TSNA), N-nitrosonornicotine (NNN), is a potent carcinogen present in cigarette smoke, and chronic exposure to it can lead to pulmonary cancer. NNN causes changes in phospholipid metabolism and the mechanism is yet to be elucidated. Exposure of Saccharomyces cerevisiae to 50 μM NNN leads to a substantial decrease in phosphatidylserine (PS) by 63%, phosphatidylcholine (PC) by 42% and phosphatidylethanolamine (PE) by 36% with a concomitant increase in lysophospholipids (LPL) by 25%. The alteration in phospholipid content was dependent on increasing NNN concentration. Reduced phospholipids were accompanied with increased neutral lipid content. Here we report for the first time that NNN exposure, significantly increases phospholipase B (PLB) activity and the preferred substrate is PC, a major phospholipid responsible for a series of metabolic functions. Furthermore, NNN also promotes the alteration of fatty acid (FA) composition; it increases the long chain fatty acid (C18 series) in phospholipids specifically phosphatidylethanolamine (PE) and PS; while on the contrary it increases short chain fatty acids in cardiolipin (CL). NNN mediated degradation of phospholipids is associated with enhanced PLB activity and alteration of phospholipid composition is accompanied with acyl chain remodelling. Understanding the altered phospholipid metabolism produced by NNN exposure is a worthwhile pursuit because it will help to understand the toxicity of tobacco smoke. [ABSTRACT FROM AUTHOR]

Abstract: Human phospholipase B-precursor (PLB-P) is a newly identified and purified protein from human neutrophils. The precise function of PLB-P in vivo is not yet known. Its existence in neutrophils and the enzymatic activity against phospholipids imply a role in the defence against invading microorganisms and in the generation of lipid mediators of inflammation. We describe here the generation of specific antibodies against PLB-P, the tissue localizations of PLB-P and the establishment of an accurate, specific, and reproducible radioimmunoassay (RIA). A survey of normal and malignant tissues showed strong immunostaining of PLB-P in neuronal and myeloid cells and in adrenal glands. Elevated levels were found in sera of patients with influenza A infection i.e. >1μg/L and in gut fluids of patients with inflammatory bowel disease i.e. >20μg/L. The levels correlated to markers of neutrophil activation, suggesting a neutrophil origin of PLB-P in these conditions. The antibodies and the assay will be useful in the future basic and clinical investigations of PLB-P. [Copyright &y& Elsevier]

The article presents a study which determines the physiological role of LipC, a lipase of the spore coat in Bacillus subtilis. It says that LipC gene was cloned and overexpressed in Escherichia coli. The study used thin-layer chromatography and gas chromatography-mass spectrometry analysis which indicated that LipC cleaves the fatty acids of phospholipids as phospholipase B. It suggests that LipC is relevant in the degradation of the outer spore membrane during sporulation.

Purpose: To determine the virulence of extracellular phospholipase B (PLB) of Candida albicans in keratomycosis. Methods: A model of keratomycosis was established in 48 New Zealand albino rabbits covered with contact lenses. Effects of PLB-deficient mutant strain of C. albicans and its isogenic parental strain on the keratomycosis were compared. In vitro, these two strains were incubated with corneal stromal cells respectively (37°C, 5% CO2). The influence of the strains on monolayer keratocytes was detected by scanning electron microscopy, enzyme-linked immunosorbent assay, and flow cytometry with Annexin-V/propidium iodide. Results: Fungal hyphae grew perpendicularly to the corneal stromal lamellae. The difference of the two strains in hyphal invasion was significant at two days after inoculation (p = 0.002) but not significant at the other timepoints. Severity of inflammation in rabbits with the parental strain was the same as that with the PLB null strain (p > 0.05). The morphogenesis and number of adherent germ tubes of the two strains were similar (p > 0.05), but the number of germ tubes penetrating cell monolayer was significantly different (p = 0.009). More prostaglandin E2 was detected in the culture supernatants of the parental strain group than the null strain group. The percentages of cells with damaged cellular membrane were 3.02%, 2.04%, and 0.12% in the parental group, the PLB null group, and the control group, respectively. Apoptosis cells accounted for 33.17%, 27.56%, and 1.46%, and living cells accounted for 63.81%, 70.40%, and 98.41% in the three groups, respectively. Conclusion: PLB can play a role as a virulent factor in triggering fungal invasion in corneas immediately after fungal adherence by decomposing membrane phospholipids and leading to cell lysis. However, its virulent effect does not appear to be as critical as in the hematogenous model of disseminated candidiasis. [ABSTRACT FROM AUTHOR]

A phospholipase B (PLB) precursor was purified from normal human granulocytes using Sephadex G-75, Mono-S cation-exchange and hydroxyapatite columns. The molecular mass of the protein was estimated to be ∼ 130 kDa by gel filtration and 22 and 42 kDa by SDS/PAGE. Tryptic peptide and sequence analyses by MALDI-TOF and tandem mass spectrometry (MS/MS) identified the protein as a FLJ22662 ( Homo sapiens) gene product, a homologue of the amoeba Dictyostelium discoideum PLB. The native protein needed modifications to acquire deacylation activity against phospholipids including phosphatidylcholine, phosphatidylinositol, phosphatidylethanolamine and lysophospholipids. Enzyme activity was associated with fragments derived from the 42 kDa fragment. The enzyme revealed a PLB nature by removing fatty acids from both the sn-1 and sn-2 positions of phospholipids. The enzyme is active at a broad pH range with an optimum of 7.4. Immunoblotting of neutrophil postnuclear supernatant using antibodies against the 42 kDa fragment detected a band at a molecular mass of 42 kDa, indicating a neutrophil origin of the novel PLB precursor. The existence of the PLB precursor in neutrophils and its enzymatic activity against phospholipids suggest a role in the defence against invading microorganisms and in the generation of lipid mediators of inflammation. [ABSTRACT FROM AUTHOR]

Abstract: Moraxella bovis is the causative agent of infectious bovine keratoconjunctivitis (IBK) also known as pinkeye, a highly contagious and painful eye disease that is common in cattle throughout the world. Vaccination appears to be a reasonable and cost-effective means of control of pinkeye. Identification of genes encoding novel secreted antigens have been reported, and these antigens are being assessed for use in a vaccine. One of the genes encodes phospholipase B, which can be expressed with high purity and yield in recombinant Escherichia coli as a secreted, soluble, non-tagged, mature construct (less signal peptide with predicted mass 63kDa). The recombinant phospholipase B exhibited anomalous electrophoretic mobility that was dependent on the temperature of the denaturing process, with bands observed at either 52 or 63kDa. Analysis by in-gel digestion and liquid chromatography–mass spectrometry revealed these two distinct forms most likely had identical sequences. Phospholipase B is a compact, globular protein with a predicted structure typical of a conventional autotransporter. It is suggested that high temperature is required to unfold the protein (to denature the β-barrel-rich transporter domain) and to ensure accessibility of the reducing agent. Interestingly, the two forms of the enzyme, differing in size and isoelectric points, were also detected in cell-free supernatants of M. bovis cultures, indicating that native phospholipase B may exist in two differentially folded states possibly also differing in oxidation status of cysteine residues. [Copyright &y& Elsevier]

Extracellular phospholipase B (PLB) is a virulence determinant of Cryptococcus neoformans and Cryptococcus gattii. In this study, we developed a sensitive enzyme-linked immunosorbent assay (ELISA) for PLB antigen with a detection limit of 3.9 ng mL−1. PLB was detected in culture supernatants of C. neoformans and C. gattii. PLB, however, was not detected in sera of seven human patients and 10 feline patients with active cryptococcosis. Furthermore, none of five rats with extensive pulmonary C. gattii infection had a positive ELISA test result. In conclusion, cryptococcal PLB could not be detected in serum using a PLB antigen-based ELISA. Despite its sensitivity, this ELISA is of limited diagnostic value. Exploration of further extracellular molecules suitable for serodiagnosis of active cryptococcal infection is warranted. [ABSTRACT FROM AUTHOR]

Abstract: Organophosphorus compounds (OP) such as phenyl saligenin phosphate (PSP) and mipafox (MPX) which cause delayed neuropathy, inhibit neuropathy target esterase (NTE), while OPs such as paraoxon (PXN) react more readily with acetylcholinesterase. In yeast and mammalian cell lines, NTE has been shown to have phospholipase B (PLB) activity which deacylates intracellular phosphatidylcholine to glycerophosphocholine (GroPCho) and can be detected by metabolic labeling with [14C]choline. Here we investigated PLB activity in primary cultures of mouse neural cells. In cortical and cerebellar granule neurons and astrocytes, [14C]GroPCho labeling was inhibited by PSP and MPX: phenyl dipentylphosphinate (PDPP), a non-neuropathic NTE inhibitor, was more potent, while PXN, was substantially less so. In all three cell types, conversion of [14C]phosphatidylcholine to [14C]GroPCho over 24 h was relatively small (2.3–14%). Consequently, even with >80% inhibition of [14C]GroPCho production, increased [14C]phosphatidylcholine was not detected. At concentrations of 1–10 μM, only PSP was cytotoxic to cortical and cerebellar granule neurons after 24-h exposure. Moreover, dramatic changes in glial cell morphology were induced by PSP, but not PDPP or MPX, with rapid (2–3 h) rounding up of astrocytes and of Schwann cells in cultures of dissociated mouse dorsal root ganglia. We conclude that PLB activity is present in a variety of cultured mouse neural cell types but that acute loss of this activity is not cytotoxic. Conversely, the rapid toxic effects of PSP in vitro suggest that a serine hydrolase distinct from NTE is required continuously by neurons and glia. [Copyright &y& Elsevier]

Glycerophosphocholine (GPC) is an osmoprotective compatible and counteracting organic osmolyte that accumulates in renal inner medullary cells in response to high NaCI and urea. We previously found that high NaCI increases GPC in renal [Madin-Darby canine kidney (MOCK)] cells. The GPC is derived from phosphatidylcholine, catalyzed by a phospholipase that was not identified at that time. Neuropathy target esterase (NTE) was recently shown to be a phospholipase B that catalyzes production of GPC from phosphatidylcholine. The purpose of the present study was to test whether NTE contributes to the high NaCI-induced increase of GPC synthesis in renal cells. We find that in mouse inner medullary collecting duct cells, high NaCI increases NTE mRNA within 8 h and NTE protein within 16 h. Diisopropyl fluorophosphate, which inhibits NTE esterase activity, reduces GPC accumulation, as does an siRNA that specifically reduces NTE protein abundance. The 20-h half-life of NTE mRNA is unaffected by high NaCI. TonEBP/OREBP is a transcription factor that is activated by high NaCI. Knockdown of TonEBP/OREBP by a specific siRNA inhibits the high NaCl-induced increase of NTE mRNA. Further, the lower renal inner medullary interstitial NaCI concentration that occurs chronically in CICK1-/- mice and acutely in normal mice given furosemide is associated with lower NTE mRNA and protein. We conclude that high NaCI increases transcription of NTE, likely mediated by TonEBP/OREBP, and that the resultant increase of NTE expression contributes to increased production and accumulation of GPC in mammalian renal cells in tissue culture and in vivo. [ABSTRACT FROM AUTHOR]

Abstract: Secreted phospholipase B enzymes (PLB1) with high levels of N-linked glycosylation are proven fungal virulence determinants. We demonstrated that removal of N-linked glycans from secreted cryptococcal PLB1 leads to loss of enzyme activity. To determine if individual N-glycan attachment sites affect secretion of active enzyme, we altered three along the entire length of the protein, by site-directed mutagenesis, namely Asn56, Asn430 and Asn550 to Ala, in wild-type PLB1 (full length) and a glycosylphosphatidylinositol (GPI) anchorless version (PLB1GPI−) that is hypersecreted due to lack of membrane association. Alteration of Asn56 and Asn550 in both PLB1 and PLB1GPI− abolished enzyme secretion while alteration of Asn430 reduced secretion by 60%, following expression in Saccharomyces cerevisiae. Reduced secretion coincided with reduced enzyme in membranes and cell walls confirming a reduction in the rate of PLB1 transport to the cell surface. Deglycosylation of cryptococcal PLB1 increased its susceptibility to proteolysis suggesting that the absence of full glycosylation status leads to degradation of unstable PLB1, resulting in reduced traffic through the secretory pathway. We conclude that individual N-linked glycans are required for optimal transport of PLB1 to the cell surface and optimal secretion of both PLB1 and PLB1GPI−. [Copyright &y& Elsevier]

The article focuses on the refinement and attributes of phospholipase B (PLB) from Candida utilis. In the present study, PLB was purged to homogeneity from the culture filtrate of Candida utilis. PLB appears to be a rare enzyme, because a single protein comprises three catalytic activities and two optimum pH ranges. The unequaled enzymatic properties will become more clear by examining the three-dimensional structure of the protein.

Phospholipase B (PLB) is an enzyme that displays both phospholipase A2 and lysophospholipase activities. Analysis of human epidermis homogenates indicated the presence of a 97 kDa PLB protein, as well as a phospholipase A2 activity, both being enriched in the soluble fraction. Immunolabelling and in situ hybridization experiments showed that this enzyme is expressed in the different layers of epidermis with an accumulation at the dermo-epidermis junction. RT-PCR data indicated that PLB is specifically expressed in natural and reconstructed epidermis. By 3′-RACE-PCR and screening of human genome databases, we obtained a 3600 bp cDNA coding for human PLB highly homologous to already described intestinal brush border PLBs. These data led us to conclude that the soluble PLB corresponds to a proteolytic cleavage of the membrane anchored protein. Altogether, our results provide the first characterization of human PLB which should play an important role in epidermal barrier function. [Copyright &y& Elsevier]