A mammary epithelial cell line is transiently stimulated towards milk lipid synthesis by lactogenic treatments

The effect of moisture sorption at different relative humidities on the tensile strength and the physical stability of compacts of crystalline and partly amorphous lactose, alone and in binary mixtures with PVP, has been studied. Furthermore, the role of moisture as a plasticizer and its effect on the glass transition temperature, Tg, are related to the compactibiltiy. Samples were conditioned for 2 hr using a climate test chamber at different relative humidities. Moisture sorption was determined, the radial crushing strength for compacts was measured immediately and after storage, and the tensile strength was calculated. The glass transition temperature, Tg, was determined using DSC. The tensile strength of the compacts was found to depend on both the conditioning humidity and the humidity during storage. An increase in humidity to a level at which the glass transition temperature, Tg, fell below the operating temperature, T, resulted in transition from a rigid glassy state to a mobile rubbery state. For compacts of partly amorphous lactose, an increase in the tensile strength was observed during storage of tablets, due to recrystallization of the amorphous regions above Tg. Tablets of mixtures of lactose and PVP exhibit a sharp decrease in tensile strength at humidities above 70% RH, due to the glass-to-rubber transition of PVP.     

A novel calcium-independent phospholipase A2, cPLA2-gamma, that is prenylated and contains homology to cPLA2

We report the cloning and characterization of a novel membrane-bound, calcium-independent PLA2, named cPLA2-gamma. The sequence encodes a 541-amino acid protein containing a domain with significant homology to the catalytic domain of the 85-kDa cPLA2 (cPLA2-alpha). cPLA2-gamma does not contain the regulatory calcium-dependent lipid binding (CaLB) domain found in cPLA2-alpha. However, cPLA2-gamma does contain two consensus motifs for lipid modification, a prenylation motif (-CCLA) at the C terminus and a myristoylation site at the N terminus. We present evidence that the isoprenoid precursor [3H]mevalonolactone is incorporated into the prenylation motif of cPLA2-gamma. Interestingly, cPLA2-gamma demonstrates a preference for arachidonic acid at the sn-2 position of phosphatidylcholine as compared with palmitic acid. cPLA2-gamma encodes a 3-kilobase message, which is highly expressed in heart and skeletal muscle, suggesting a specific role in these tissues. Identification of cPLA2-gamma reveals a newly defined family of phospholipases A2 with homology to cPLA2-alpha.     

Differential activation of human neutrophil cytosolic phospholipase A2 and secretory phospholipase A2 during priming by 1,2-diacyl- and 1-O-alkyl-2-acylglycerols

We have shown previously that both 1,2-diacylglycerol (AAG) and 1-O-alkyl-2-acylglycerol (EAG) prime neutrophil release of arachidonic acid via uncharacterized phospholipases A2. Therefore, we investigated the actions of EAG and AAG specifically on neutrophil cytosolic (cPLA2) and secretory (sPLA2) phospholipase A2s. We hypothesized that AAG as a protein kinase activator would activate cPLA2 via phosphorylation events. EAG is antagonistic to the AAG activation of PKC, thus it was not expected to act via phosphorylation of cPLA2. Neutrophils were primed with either AAG or EAG and then stimulated with fMLP. When neutrophils were primed with 5-20 microM 1,2-diacylglycerol, a shift was observed in cPLA2 migration on SDS-PAGE gels, consistent with phosphorylation of the protein. This gel shift was not seen after exposure to EAG. AAG also caused a parallel increase in enzymatic activity of cPLA2 that was not seen with EAG. We also investigated whether either diglyceride would cause similar priming or direct secretion of sPLA2. Both AAG and EAG directly caused significant secretion of neutrophil sPLA2. EAG also increased the release of sPLA2 in cells subsequently stimulated with fMLP. Thus, AAG activated cPLA2 and stimulated secretion of sPLA2. In contrast, EAG did not activate cPLA2, but directly activated secretion of sPLA2. We also demonstrated that human synovial fluid sPLA2 increased AA release from resting and fMLP-stimulated neutrophils. Given that diglycerides prime for release of AA, PAF, and LTB4, these current data support the hypothesis that such priming may be mediated by phosphorylation dependent (cPLA2) or phosphorylation independent (e.g. secretion of sPLA2) events.     

Rat and human pancreatic islet cells contain a calcium ion independent phospholipase A2 activity selective for hydrolysis of arachidonate which is stimulated by adenosine triphosphate and is specifically localized to islet beta-cells

The recent demonstration that myocardial Ca(2+)-independent phospholipase A2 exists as a complex of catalytic and regulatory polypeptides that is modulated by ATP has suggested a novel mechanisms through which alterations in glycolytic flux can be coupled to the generation of eicosanoids which facilitate insulin secretion. To determine the potential relevance of this mechanism, we examined the kinetic characteristics, substrate specificities, and cellular locus of phospholipase A2 activity in pancreatic islets. Rat pancreatic islets contain a Ca(2+)-independent phospholipase A2 activity which is optimal at physiologic pH, preferentially hydrolyzes phospholipid substrates containing a vinyl ether linkage at the sn-1 position, and prefers arachidonic acid compared to oleic acid in the sn-2 position. Rat islet Ca(2+)-independent phospholipase A2 activity is inhibited by the mechanism-based inhibitor (E)-6-(bromomethylene)-3-(1-naphthalenyl)-2H-tetrahydropyran-2-one and is stimulated by ATP. Purification of beta-cells from dispersed pancreatic islet cells by fluorescence-activated cell sorting demonstrated that beta-cells (but not non-beta-cells) contain Ca(2+)-independent, ATP-stimulated phospholipase A2 activity. Remarkably, clonal RIN-m5f insulinoma cells, which possess a defect in glucose-induced insulin secretion, contain a Ca(2+)-independent phospholipase A2 which is not modulated by alterations in ATP concentration. Collectively, these results and those of an accompanying paper [Ramanadham et al. (1993) Biochemistry (following paper in this issue)] implicate Ca(2+)-independent phospholipase A2 as a putative glucose sensor which can couple alterations in glycolytic metabolism to the generation of biologically active eicosanoids and thereby facilitate glucose-induced insulin secretion.     

Barley beta-glucosidase: expression during seed germination and maturation and partial amino acid sequences

Confusion regarding proper use of the terms rate and risk persists in the literature. This has implications for the proper modeling of prognosis and transition between health states in decision analysis and related techniques. The issue is complicated by the plethora of terms related to rate and risk. Although the suggestion to use the terms force and probability as substitutes for rate and risk has some appeal, the change in terminology by itself is unlikely to solve all the confusion or misuse of terms. This paper clarifies the proper definitions and estimations of rates and risks and suggests critical factors for the decision analyst to remember when using, modeling, or interpreting transition rates and risks.     

Fas-induced arachidonic acid release is mediated by Ca2+-independent phospholipase A2 but not cytosolic phospholipase A2, which undergoes proteolytic inactivation

Fas-mediated apoptosis of human leukemic U937 cells was accompanied by increased arachidonic acid (AA) and oleic acid release from membrane glycerophospholipids, indicating phospholipase A2 (PLA2) activation. During apoptosis, type IV cytosolic PLA2 (cPLA2), a PLA2 isozyme with an apparent molecular mass of 110 kDa critical for stimulus-coupled AA release, was converted to a 78-kDa fragment with concomitant loss of catalytic activity. Cleavage of cPLA2 correlated with increased caspase-3-like protease activity in apoptotic cells and was abrogated by a caspase-3 inhibitor. A mutant cPLA2 protein in which Asp522 was replaced by Asn, which aligns with the consensus sequence of the caspase-3 cleavage site (DXXD downward arrowX), was resistant to apo-ptosis-associated proteolysis. Moreover, a COOH-terminal deletion mutant of cPLA2 truncated at Asp522 comigrated with the 78-kDa fragment and exhibited no enzymatic activity. Thus, caspase-3-mediated cPLA2 cleavage eventually leads to destruction of a catalytic triad essential for cPLA2 activity, thereby terminating its AA-releasing function. In contrast, the activity of type VI Ca2+-independent PLA2 (iPLA2), a PLA2 isozyme implicated in phospholipid remodeling, remained intact during apoptosis. Inhibitors of iPLA2, but neither cPLA2 nor secretory PLA2 inhibitors, suppressed AA release markedly and, importantly, delayed cell death induced by Fas. Therefore, we conclude that iPLA2-mediated fatty acid release is facilitated in Fas-stimulated cells and plays a modifying although not essential role in the apoptotic cell death process.     

Positive Darwinian selection in Vipera palaestinae phospholipase A2 genes is unexpectedly limited to the third exon

The venom of Vipera palaestinae contains a two-component toxin, consisting of an acidic phospholipase A2 (PLA2) and a basic protein. Here we report the cloning and sequence analysis of the complete V. palaestinae PLA2 genes. Since in all Viperidae PLA2 multigene families the 5' and 3' flanking regions are highly conserved, we designed oligonucleotide primers that allow amplification of the whole PLA2 multigene family in a single step. The structural organization of both genes is the same as in the Vipera ammodytes PLA2 multigene family, there being five exons separated by four introns. Comparison of V. palaestinae PLA2 genes with other Viperidae PLA2 genes has shown that the structural organization of the genes and the nucleotide sequence of all introns and flanking regions are highly conserved, whereas the third exon clearly shows a higher number of amino acid replacements, an indication of positive Darwinian selection. The positive Darwinian selection is surprisingly limited to the third exon, in contrast to other Viperidae PLA2 genes, where it is present in all mature protein coding exons.     

Resistance to endotoxic shock in phospholipase A2 receptor-deficient mice

Mammals possess various types of secretory phospholipase A2, which differ in the primary structure and tissue distribution. The phosholipase A2 receptor (PLA2R) recognizes group IB phospholipase A2 (PLA2-IB) and mediates the PLA2-IB-induced biological responses in non-digestive organs, including eicosanoid production and contraction of airway smooth muscles. In this study, we generated PLA2R-deficient mice to define its biological roles further. These mice are viable, fertile, and without evident histopathological abnormalities. There was no difference in the clearance of circulating PLA2-IB between wild-type and mutant mice. After challenge with bacterial lipopolysaccharide (LPS), PLA2R-deficient mice exhibited longer survival than wild-type mice. The mutant mice were also resistant to lethal effects of exogenous PLA2-IB after sensitization with sublethal dose of LPS. The plasma levels of tumor necrosis factor-alpha and interleukin-1beta elevated after LPS treatment were significantly reduced in mutant mice compared with wild-type mice. These findings suggest a potential role of PLA2R in the progression of endotoxic shock.     

Prostacyclin formation elicited by endothelin-1 in rat aorta is mediated via phospholipase D activation and not phospholipase C or A2

Endothelin-1 (ET-1) is a potent vasoconstrictor peptide that also stimulates production of prostacyclin (PGI2) from arachidonic acid. The purpose of this study was to determine the contribution of phospholipases (PLs) A2, C, and/or D in ET-1-induced PGI2 formation in the rat aorta, measured as immunoreactive 6-ketoprostaglandin (PG) F1 alpha. ET-1 increased 6-keto-PGF1 alpha formation, which was not affected by a PLA2 inhibitor, 7,7-dimethyl eicosadienoic acid (DEDA). Furthermore, ET-1 failed to stimulate PLA2 activity measured in the cytosol (cPLA2), using phosphatidylcholine, L-a-1-palmitoyl-2-arachidonyl[14C] as a substrate. However, the adrenergic agonist norepinephrine increased 6-keto-PGF1 alpha formation, which was attenuated by DEDA, and enhanced PLA2 activity. ET-1 enhanced PLC activity, as indicated by increased inositol phosphate production, which was prevented by a PLC inhibitor, U-73122. However, ET-1-induced 6-keto-PGF1 alpha production was not altered by U-73122. An inhibitor of PLD activation, C2-ceramide, attenuated ET-1-induced PLD activity, as indicated by the production of phosphatidylethanol. Furthermore, ET-1-induced 6-keto-PGF1 alpha formation was inhibited by C2-ceramide as well as by ethanol treatment. Moreover, inhibitors of phosphatidate phosphohydrolase (propranolol) and diacylglycerol lipase (RHC-80267), attenuated ET-1-induced 6-keto-PGF1 alpha formation. Finally, ET-1-induced activation of PLD was not attenuated by a selective PKC inhibitor, bisindolylmaleimide I. These data suggest a novel pathway for ET-1-induced PGI2 formation in the rat aorta involving activation of PLD but not cPLA2 and independent of PLC or PKC activation.     

Pyrimidinoceptor-mediated activation of phospholipase C and phospholipase A2 in RAW 264.7 macrophages

Electron microscopy of toad (Bufo marinus) muscle fixed without relaxing after a single eccentric contraction at muscle lengths greater than optimum showed over-stretched half-sarcomeres in sufficient numbers to account for more than half of the imposed stretch. Such sarcomeres were absent in another muscle fixed without relaxing after an isometric contraction at the same length and largely absent in a third muscle that underwent eccentric contraction at muscle lengths less than optimum. This provides direct evidence in support of the hypothesis that lengthening of muscles at long length involves lengthening of a few half sarcomeres to beyond filament overlap, while most half sarcomeres are extended much less than in proportion to muscle extension.     

Melittin binds to secretory phospholipase A2 and inhibits its enzymatic activity

Synthetic melittin inhibited the enzymatic activity of secretory phospholipase A2 (PLA2) from various sources, including bee and snake venoms, bovine pancreas, and synovial fluid from rheumatoid arthritis patients, irrespective of substrate (e.g., [14C]-phosphatidylcholine or phosphatidylethanolamine vesicles and [3H]-oleic acid-labeled E.coli). A Lineweaver-Burk analysis showed that melittin was a noncompetitive inhibitor of bee venom PLA2, causing a change in Vmax from 200 to 50 units/min/mg of protein. The Km remained unchanged (0.75 nmole). Melittin inhibited approximately 50% of purified bee venom PLA2 activity in a 30:1 molar ratio (melittin:enzyme). Because the enzyme kinetics indicated a PLA2-melittin interaction, a melittin-sepharose affinity column was used to purify a PLA2 from human serum. Further, an enzyme-linked assay was developed to quantitate PLA2 activity in biological fluids using avidin-peroxidase and ELISA plates coated with biotinylated melittin. These observations may have potential therapeutic significance, as well as provide a convenient basis for the isolation and quantitation of PLA2.     

Raised neutrophil phospholipase A2 activity and defective priming of NADPH oxidase and phospholipase A2 in sickle cell disease

Intermittent painful crises due to vasoocclusion are the major clinical manifestation of sickle cell disease (SCD), but subclinical episodes may also occur. There is sparse evidence for the involvement of neutrophils in the pathophysiology of SCD, but production of cytokines by the damaged endothelium might influence neutrophil function and modulate responses to subsequent cytokine exposure. In addition, the activation of neutrophils in the microcirculation could itself exacerbate vasoocclusion. To test whether neutrophil inflammatory responses were altered in SCD, neutrophil phospholipase A2 and NADPH oxidase activity in response to in vitro priming by granulocyte-macrophage colony-stimulating factor (GM-CSF) and tumor necrosis factor-alpha (TNF-alpha) were measured both during and between painful crises. Resting levels of neutrophil phospholipase A2 activity in steady-state SCD (4.0% +/- 0. 5% of total cell radioactivity) were raised relative to control values (2.0% +/- 0.2%, n = 10, P = .008). There was no defect of agonist-stimulated phospholipase A2 or NADPH oxidase activity in steady-state SCD; however, the ability of phospholipase A2 to respond to priming with GM-CSF was attenuated to 63% +/- 17% of control values (n = 10, P = .04). Similarly, neutrophil NADPH oxidase activity after priming with GM-CSF and TNF-alpha was, respectively, 65% +/- 11% (n = 7, P = .03) and 57% +/- 7% of control (n = 10, P = .007) in steady-state disease, and was further reduced during painful vasoocclusive crises to 34% +/- 9% and 25% +/- 3% of control for GM-CSF and TNF-alpha, respectively. These data were not explained by poor splenic function or any racial factor, as normal cytokine responses were seen in splenectomized patients in remission from Hodgkin's disease and in healthy Afro-Caribbean subjects. Abnormal neutrophil cytokine priming responses were not observed in either patients with rheumatoid arthritis or iron-deficiency anemia. Our findings are indicative of an ongoing inflammatory state in SCD between painful crises involving neutrophil activation and an abnormality of cytokine-regulated neutrophil function, which may compromise the host defenses against certain microorganisms.     

Cerebral cortical phospholipase A2 activity of senescence-accelerated mouse is increased in an age-dependent manner

An immunogenetic examination of 86 cases of stomach cancer established a correlation between predisposition and resistance, on the one hand, and the distribution of allele sets of HLA-genes (classes I and II), on the other. The relationship was found to vary according to sex and age. The most significant relationships with respect to predisposition were identified for HLA-B51 (RR = 19.82) alleles and allele combinations of HLA-DRI-DR7 (RR = 25.52) and HLA-A9-DRI (RR = 33.67). High relative risk of stomach cancer was attributed to the absence of relevant alleles in 91 patients included into the group of comparison. Also, combinations of allele sets were identified in healthy subjects which never occur in stomach cancer patients. The results provide a substantiation for developing an automated system of interpreting HLA-typing data which are instrumental in evaluating the patient's predisposition, resistance and prognosis.     

Evidence that phospholipase A2 activity is required for Golgi complex and trans Golgi network membrane tubulation

Membrane tubules of uniform diameter (60-80 nm) and various lengths (up to several micrometers) emanate from elements of the Golgi stack and trans Golgi network (TGN). These organelle membrane tubules are thought to be involved in membrane trafficking and maintenance of Golgi/TGN architecture. The number of these tubules, and their frequency of formation, can be greatly enhanced by the fungal metabolite brefeldin A (BFA), an inhibitor of Golgi/TGN-associated coated vesicle formation. We show here that BFA stimulation of Golgi and TGN membrane tubulation, and the resultant retrograde transport of resident Golgi enzymes to the endoplasmic reticulum, was potently inhibited by a number of membrane-permeant antagonists of phospholipase A2 (PLA2; EC 3.1.1.4) activity. In addition, PLA2 inhibitors on their own caused a reversible fragmentation of the Golgi complex into juxtanuclear, stacked cisternal elements. We conclude from these observations that tubulation of Golgi complex and TGN membranes requires a PLA2 activity, and that this activity may participate not only in Golgi tubule-mediated retrograde trafficking to the endoplasmic reticulum, but also in the maintenance of Golgi complex architecture.     

Extracellular calcium regulates HeLa cell morphology during adhesion to gelatin: role of translocation and phosphorylation of cytosolic phospholipase A2

Attachment of HeLa cells to gelatin induces the release of arachidonic acid (AA), which is essential for cell spreading. HeLa cells spreading in the presence of extracellular Ca2+ released more AA and formed more distinctive lamellipodia and filopodia than cells spreading in the absence of Ca2+. Addition of exogenous AA to cells spreading in the absence of extracellular Ca2+ restored the formation of lamellipodia and filopodia. To investigate the role of cytosolic phospholipase A2 (cPLA2) in regulating the differential release of AA and subsequent formation of lamellipodia and filopodia during HeLa cell adhesion, cPLA2 phosphorylation and translocation from the cytosol to the membrane were evaluated. During HeLa cell attachment and spreading in the presence of Ca2+, all cPLA2 became phosphorylated within 2 min, which is the earliest time cell attachment could be measured. In the absence of extracellular Ca2+, the time for complete cPLA2 phosphorylation was lengthened to <4 min. Maximal translocation of cPLA2 from cytosol to membrane during adhesion of cells to gelatin was similar in the presence or absence of extracellular Ca2+ and remained membrane associated throughout the duration of cell spreading. The amount of total cellular cPLA2 translocated to the membrane in the presence of extracellular Ca2+ went from <20% for unspread cells to >95% for spread cells. In the absence of Ca2+ only 55-65% of the total cPLA2 was translocated to the membrane during cell spreading. The decrease in the amount translocated could account for the comparable decrease in the amount of AA released by cells during spreading without extracellular Ca2+. Although translocation of cPLA2 from cytosol to membrane was Ca2+ dependent, phosphorylation of cPLA2 was attachment dependent and could occur both on the membrane and in the cytosol. To elucidate potential activators of cPLA2, the extracellular signal-related protein kinase 2 (ERK2) and protein kinase C (PKC) were investigated. ERK2 underwent a rapid phosphorylation upon early attachment followed by a dephosphorylation. Both rates were enhanced during cell spreading in the presence of extracellular Ca2+. Treatment of cells with the ERK kinase inhibitor PD98059 completely inhibited the attachment-dependent ERK2 phosphorylation but did not inhibit cell spreading, cPLA2 phosphorylation, translocation, or AA release. Activation of PKC by phorbol ester (12-O-tetradecanoylphorbol-13-acetate) induced and attachment-dependent phosphorylation of both cPLA2 and ERK2 in suspension cells. However, in cells treated with the PKC inhibitor Calphostin C before attachment, ERK2 phosphorylation was inhibited, whereas cPLA2 translocation and phosphorylation remained unaffected. In conclusion, although cPLA2-mediated release of AA during HeLa cell attachment to a gelatin substrate was essential for cell spreading, neither ERK2 nor PKC appeared to be responsible for the attachment-induced cPLA2 phosphorylation and the release of AA.     

A new method for seed oil body purification and examination of oil body integrity following germination

Plant seeds store triacylglycerols as energy sources for germination and postgerminative growth of seedlings. The triacylglycerols are preserved in small, discrete, intracellular organelles called oil bodies. A new method was developed to purify seed oil bodies. The method included extraction, flotation by centrifugation, detergent washing, ionic elution, treatment with a chaotropic agent, and integrity testing by use of hexane. These processes subsequently removed non-specifically associated or trapped proteins within the oil bodies. Oil bodies purified by this method maintained their integrity and displayed electrostatic repulsion and steric hindrance on their surface. Compared with the previous procedure, this method allowed higher purification of oil bodies, as demonstrated by SDS-PAGE using five species of oilseeds. Oil bodies purified from sesame were further analyzed by two-dimensional gel electrophoresis and revealed two potential oleosin isoforms. The integrity of oil bodies in germinating sesame seedlings was examined by hexane extraction. Our results indicated that consumption of triacylglycerols reduced gradually the total amount of oil bodies in seedlings, whereas no alteration was observed in the integrity of remaining oil bodies. This observation implies that oil bodies in germinating seeds are not degraded simultaneously. It is suggested that glyoxisomes, with the assistance of mitochondria, fuse and digest oil bodies one at a time, while the remaining oil bodies are preserved intact during the whole period of germination.