Pharmacological profile of opicapone, a third-generation nitrocatechol catechol-O-methyl transferase inhibitor, in the rat.

BACKGROUND AND PURPOSE: Catechol-O-methyltransferase (COMT) is an important target in the levodopa treatment of Parkinson's disease; however, the inhibitors available have problems, and not all patients benefit from their efficacy. Opicapone was developed to overcome those limitations. In this study, opicapone's pharmacological properties were evaluated as well as its potential cytotoxic effects. EXPERIMENTAL APPROACH: The pharmacodynamic effects of opicapone were explored by evaluating rat COMT activity and levodopa pharmacokinetics, in the periphery through microdialysis and in whole brain. The potential cytotoxicity risk of opicapone was explored in human hepatocytes by assessing cellular ATP content and mitochondrial membrane potential. KEY RESULTS: Opicapone inhibited rat peripheral COMT with ED50 values below 1.4 mg⋅kg(-1) up to 6 h post-administration. The effect was sustained over the first 8 h and by 24 h COMT had not returned to control values. A single administration of opicapone resulted in increased and sustained plasma levodopa levels with a concomitant reduction in 3-O-methyldopa from 2 h up to 24 h post-administration, while tolcapone produced significant effects only at 2 h post-administration. The effects of opicapone on brain catecholamines after levodopa administration were sustained up to 24 h post-administration. Opicapone was also the least potent compound in decreasing both the mitochondrial membrane potential and the ATP content in human primary hepatocytes after a 24 h incubation period. CONCLUSIONS AND IMPLICATIONS: Opicapone has a prolonged inhibitory effect on peripheral COMT, which extends the bioavailability of levodopa, without inducing toxicity. Thus, it exhibits some improved properties compared to the currently available COMT inhibitors.

Brain and peripheral pharmacokinetics of levodopa in the cynomolgus monkey following administration of opicapone, a third generation nitrocatechol COMT inhibitor.

OBJECTIVE: The present study aimed at evaluating the effect of opicapone, a third generation nitrocatechol catechol-O-methyltransferase (COMT) inhibitor, on the systemic and central bioavailability of 3,4-dihydroxy-l-phenylalanine (levodopa) and related metabolites in the cynomolgus monkey. METHODS: Four monkeys, implanted with guiding cannulas for microdialysis probes, in the substantia nigra, dorsal striatum and prefrontal cortex, were randomized in two groups that received, in a crossover design, vehicle or 100 mg/kg opicapone for 14 days. Twenty-three hours after last administration of vehicle or opicapone, animals were challenged with levodopa/benserazide (12/3 mg/kg). Extracellular dialysate and blood samples were collected over 360 min (at 30 min intervals) for the assays of catecholamine and COMT activity. RESULTS: Opicapone increased levodopa systemic exposure by 2-fold not changing Cmax values and reduced both 3-O-methyldopa (3-OMD) exposure and Cmax values by 5-fold. These changes were accompanied by ∼76-84% reduction in erythrocyte COMT activity. In dorsal striatum and substantia nigra, opicapone increased levodopa exposure by 1.7- and 1.4-fold, respectively, reducing 3-OMD exposure by 5- and 7-fold respectively. DOPAC exposure was increased by 4-fold in the substantia nigra. In the prefrontal cortex, opicapone increased levodopa exposure and reduced 3-OMD levels by 2.3- and 2.4-fold, respectively. CONCLUSIONS: Opicapone behaved as long-acting COMT inhibitor that markedly increased systemic and central levodopa bioavailability. Opicapone is a strong candidate to fill the unmet need for COMT inhibitors that lead to more sustained levodopa levels in Parkinson's disease patients.

Population density and phenology of Tetranychus urticae (Acari: Tetranychidae) in hop is linked to the timing of sulfur applications.

The twospotted spider mite, Tetranychus urticae Koch, is a worldwide pest of numerous agronomic and horticultural plants. Sulfur fungicides are known to induce outbreaks of this pest on several crops, although mechanisms associated with sulfur-induced mite outbreaks are largely unknown. Studies were conducted during 2007-2009 in Oregon and Washington hop yards to evaluate the effect of timing of sulfur applications on T. urticae and key predators. In both regions, applications of sulfur made relatively late in the growing season (mid-June to mid-July) were associated with the greatest exacerbation of spider mite outbreaks, particularly in the upper canopy of the crop. The severity of mite outbreaks was closely associated with sulfur applications made during a relatively narrow time period coincident with the early exponential phase of spider mite increase and rapid host growth. A nonlinear model relating mean cumulative mite days during the time of sulfur sprays to the percent increase in total cumulative mite days (standardized to a nontreated plot) explained 58% of the variability observed in increased spider mite severity related to sulfur spray timing. Spatial patterns of spider mites in the Oregon plots indicated similar dispersal of motile stages of spider mites among leaves treated with sulfur versus nontreated leaves; however, in two of three years, eggs were less aggregated on leaves of sulfur-treated plants, pointing to enhanced dispersal. Apart from one experiment in Washington, relatively few predatory mites were observed during the course of these studies, and sulfur-induced mite outbreaks generally occurred irrespective of predatory mite abundance. Collectively, these studies indicate sulfur induces mite outbreaks through direct or indirect effects on T. urticae, mostly independent of predatory mite abundance or toxicity to these predators. Avoidance of exacerbation of spider mite outbreaks by sulfur sprays was achieved by carefully timing applications to periods of low spider mite abundance and slower host development, which is generally early to mid-spring for hop.

Development and validation of an enantioselective liquid-chromatography/tandem mass spectrometry method for the separation and quantification of eslicarbazepine acetate, eslicarbazepine, R-licarbazepine and oxcarbazepine in human plasma.

The purpose of this study was develop and validate a sensitive and specific enantioselective liquid-chromatography/tandem mass spectrometry (LC-MS/MS) method, for the simultaneous quantification of eslicarbazepine acetate (ESL), eslicarbazepine (S-Lic), oxcarbazepine (OXC) and R-licarbazepine (R-Lic) in human plasma. Analytes were extracted from human plasma using solid phase extraction and the chromatographic separation was achieved using a mobile phase of 80% n-hexane and 20% ethanol/isopropyl alcohol (66.7/33.3, v/v). A Daicel CHIRALCEL OD-H column (5 µm, 50 mm × 4.6 mm) was used with a flow rate of 0.8 mL/min, and a run time of 8 min. ESL, S-Lic, R-Lic, OXC and the internal standard, 10,11-dihydrocarbamazepine, were quantified by positive ion electrospray ionization mass spectrometry. The method was fully validated, demonstrating acceptable accuracy, precision, linearity, and specificity in accordance with FDA regulations for the validation of bioanalytical methods. Linearity was proven over the range of 50.0-1000.0 ng/mL for ESL and OXC and over the range of 50.0-25,000.0 ng/mL for S-Lic and R-Lic. The intra- and inter-day coefficient of variation in plasma was less than 9.7% for ESL, 6.0% for OXC, 7.7% for S-Lic and less than 12.6% for R-Lic. The accuracy was between 98.7% and 107.2% for all the compounds quantified. The lower limit of quantification (LLOQ) was 50.0ng/mL for ESL, S-Lic, OXC and R-Lic in human plasma. The short-term stability in plasma, freeze-thaw stability in plasma, frozen long-term stability in plasma, autosampler stability and stock solution stability all met acceptance criteria. The human plasma samples, collected from 8 volunteers, showed that this method can be used for therapeutic monitoring of ESL and its metabolites in humans treated with ESL.

Effects of powdery mildew fungicide programs on twospotted spider mite (Acari: Tetranychidae), hop aphid (Hemiptera: Aphididae), and their natural enemies in hop yards.

Twospotted spider mite, Tetranychus urticae Koch (Acari: Tetranychidae), and hop aphid, Phorodon humuli (Schrank) (Hemiptera: Aphididae), are the most important arthropod pests of hop (Humulus lupulus L.) in the Northern Hemisphere. A potential barrier for greater adoption of conservation biological control strategies for spider mites and hop aphid is the extensive use of fungicides for management of hop powdery mildew, Podosphaera macularis (Wallr.:Fr.) U. Braun & S. Takamatsu. Field studies conducted in experimental plots in Oregon and Washington in 2005 and 2006 quantified the effects of powdery mildew fungicide programs (i.e., sulfur, paraffinic oil, and synthetic fungicides) on arthropod pests and natural enemies on hop. Fungicide treatment significantly affected spider mite populations in all four studies. Multiple applications of sulfur fungicides applied before burr development resulted in 1.4-3.3-fold greater spider mite populations during summer. Near the cessation of the sulfur applications, or after a lag of 20-30 d, spider mite populations increased significantly faster on sulfur treated plants compared with water-treated plants in three of four experiments. The effect of paraffinic oil on spider mites was varied, leading to exacerbation of spider mites in Oregon and Washington in 2005, suppression of mites in Oregon in 2006, and no significant effect compared with water in Washington in 2006. Significant relative treatment effects for cone damage due to spider mite feeding were detected in Oregon in 2005 in plots treated with sulfur and paraffinic oil compared with water and synthetic fungicides. Mean populations of hop aphids were similar among treatments in Oregon, although sulfur treatment suppressed hop aphid populations in Washington in 2005 and 2006. Populations of individual predacious insect species and cumulative abundance of macropredators were not consistently suppressed or stimulated by treatments in all trials. However, predatory mite abundance in Washington was affected by fungicide treatments, with plots treated with sulfur consistently having 10-fold fewer phytoseiids per leaf compared with the other treatments. Based on the results of these studies, powdery mildew fungicide programs that minimize or eliminate applications of sulfur and paraffinic oil would tend to conserve predatory mites and minimize the severity of spider mite outbreaks. However, mechanisms other than direct or indirect toxicity to phytoseiid mites likely are associated with exacerbation of spider mite outbreaks on hop.

Chitotriosidase and gene therapy for fungal infections.

Chitotriosidase secreted by activated human macrophages has been implicated in the defence against chitin-bearing pathogens. The antifungal properties of human chitotriosidase were investigated here following retroviral vector-mediated gene transfer of the open reading frame of the chitotriosidase gene into Chinese hamster ovary cells. A chitinase assay confirmed that the engineered cells secreted recombinant chitotriosidase constitutively. Two dimensional gel electrophoresis and western blotting indicated that the recombinant protein is the major, chitin-binding, fifty kilodalton isoform. Culture medium conditioned by the transduced cells inhibited growth of isolates of Aspergillus niger, Candida albicans and Cryptococcus neoformans. Furthermore, longevity was significantly increased in a mouse model of cryptococcosis when cells transduced with the chitotriosidase gene and encapsulated in alginate microspheres were implanted subcutaneously in the animals. Engraftment of microcapsules containing cells transduced with the chitotriosidase gene has the potential to combat infections caused by chitinous pathogens through the prolonged delivery of recombinant chitotriosidase.

Human metabolism of nebicapone (BIA 3-202), a novel catechol-o-methyltransferase inhibitor: characterization of in vitro glucuronidation.

Nebicapone (BIA 3-202; 1-[3,4-dihydroxy-5-nitrophenyl]-2-phenylethanone), a novel catechol-O-methyltransferase inhibitor, is mainly metabolized by glucuronidation. The purpose of this study was to characterize the major plasma metabolites of nebicapone following p.o. administration of nebicapone to healthy volunteers, and to determine the human UDP-glucuronosyltransferase (UGT) enzymes involved in nebicapone glucuronidation. Plasma samples were collected as part of a clinical trial at different time points postdose and were analyzed for nebicapone and its metabolites using a validated method consisting of a solid-phase extraction, followed by high-performance liquid chromatography/mass spectrometry detection. The primary metabolic pathways of nebicapone in humans involve mainly 3-O-glucuronidation, the major early metabolite, and 3-O-methylation, the predominant late metabolite. Of the nine commercially available recombinant UGT enzymes studied (UGT1A1, UGT1A3, UGT1A6, UGT1A7, UGT1A8, UGT1A9, UGT1A10, UGT2B7, and UGT2B15), only UGT1A9 exhibited high nebicapone glucuronosyltransferase specific activity (24.3 +/- 1.3 nmol/mg protein/min). UGT1A6, UGT1A7, UGT1A8, UGT1A10, UGT2B7, and UGT2B15 exhibited low activity (0.1-1.1 nmol/mg protein/min), and UGT1A1 and UGT1A3 showed extremely low activities (less than 0.03 nmol/mg protein/min). The results show that nebicapone is mainly glucuronidated in humans and that multiple UGT enzymes are involved in this reaction.

Detection of antibodies to phospholipase B in patients infected with Cryptococcus neoformans by enzyme-linked immunosorbent assay (ELISA).

Secreted phospholipases are virulence factors of several fungi, including Cryptococcus neoformans. We describe for the first time the detection by ELISA of antibodies to cryptococcal phospholipase B in the serum of patients infected with C. neoformans or C. gattii. Sixty-nine sera from 25 patients with cryptococcosis, 23 patients with Candida infections and 26 with bacterial or viral infections were tested. The sensitivity of the ELISA in patients with cryptococcosis was 100% in immunocompetent hosts and 64.3% in immunosuppressed patients with cryptococcal meningitis. Absorbance readings were significantly higher in immunocompetent patients (P<0.001). Titres remained positive for up to 2 years. Positive results were noted in 10 episodes of invasive candidiasis, three patients colonised with Candida, and three cases of bacterial infection. Mean absorbance readings were significantly lower in patients with bacterial infection (P <0.001). We conclude that phospholipase B is produced in vivo during cryptococcal infection and that serum phospholipase B antibodies are a sensitive marker of present or past infection. Cross-reactivity of the ELISA with sera from patients with candidiasis indicates that common epitopes are present on cryptococcal and candidal phospholipase B, hence fungal phospholipase B may constitute a new therapeutic target.

Regulatory roles for the homeodomain and C2H2 zinc finger regions of Cryptococcus neoformans Ste12alphap.

The STE12alpha gene of Cryptococcus neoformans encodes a protein containing both homeodomain and zinc finger regions. As homeodomains and zinc finger regions are important domains for the function of many transcription factors, we used site-specific mutagenesis to delineate the roles of these two domains. The homeodomain and zinc finger regions are each important for the function of Ste12alphap. DNA binding ability, mating frequency, and haploid fruiting capability were reduced in strains with mutations in the homeodomain, whereas virulence and capsule size in the mouse brain were increased. In contrast, mutations in the zinc fingers region resulted in decreased virulence, reduced capsule size in the mouse brain and decreased gene expression of capsule associated genes. In addition, phospholipase activity was increased in the zinc finger mutants. Taken together, most of the phenotypes previously observed in the ste12alpha deletion strains were reproduced in these two types of mutants. However, unlike mutations in the homeodomain/zinc finger region, complete deletion of STE12alpha caused a severe reduction in virulence and a decrease in phospholipase activity. These data suggest that region(s) other than the homeodomain and zinc finger regions of Ste12alphap contribute to the variable influences on the different phenotypes observed in C. neoformans.

Molecular modeling and metabolic studies of the interaction of catechol-O-methyltransferase and a new nitrocatechol inhibitor.

Catechol-O-methyltransferase (COMT, EC 2.1.1.6) plays a central role in the metabolic inactivation of neurotransmitters and neuroactive xenobiotics possessing a catechol motif. 1-(3,4-Dihydroxy-5-nitrophenyl)-2-phenyl-ethanone (BIA 3-202) is a novel nitrocatechol-type inhibitor of COMT, the potential clinical benefit of which is currently being evaluated in the treatment of Parkinson's disease. In the present work we characterize the molecular interactions of BIA 3-202 within the active site of COMT and discuss their implication on the regioselectivity of metabolic O-methylation. Unrestrained flexible-docking simulations suggest that the solution structure of this complex is better described as an ensemble of alternative binding modes, in contrast to the well defined bound configuration revealed by the X-ray structures of related nitrocatechol inhibitors, co-crystallized with COMT. The docking results wherein presented are well supported by experimental evidence, where the pattern of in vitro enzymatic O-methylation and O-demethylation reactions are analyzed. We propose a plausible explanation for the paradoxical in vivo regioselectivity of O-methylation of BIA 3-202, as well as of its related COMT inhibitor tolcapone. Both compounds undergo in vivo O-methylation by COMT at either meta or para catechol hydroxyl groups. However, results herein presented suggest that, in a subsequent step, the p-O-methyl derivatives are selectively demethylated by a microsomal enzyme system. The overall balance is the accumulation of the m-O-methylated metabolites over the para-regioisomers. The implications for the general recognition of nitrocatechol-type inhibitors by COMT and the regioselectivity of their metabolic O-methylation are discussed.

Distribution patterns of entomopathogenic nematodes applied through drip irrigation systems.

The distribution of entomopathogenic nematodes applied by drip irrigation was evaluated by injecting small volumes of Steinernema carpocapsae (Weiser) All strain, Steinernema feltiae (Filipjev) SN strain, Steinernema glaseri Steiner, and Heterorhabditis bacteriophora HP 88 strain Poinar suspensions into drip irrigation lines. Additionally, Steinernema riobrave Cabanillas, Poinar, & Raulston, and S. carpocapsae were injected in a 10-liter volume of water with an injection pump. Overall, the nematodes were evenly distributed along the drip lines. The total number of nematodes recovered from drip emitters was variable ranging from 42 to 92%. However, drip irrigation lines have potential to deliver entomopathogenic nematodes efficiently into pest habitats.

Extracellular phospholipase activity is a virulence factor for Cryptococcus neoformans.

The human pathogenic fungus Cryptococcus neoformans secretes a phospholipase enzyme that demonstrates phospholipase B (PLB), lysophospholipase hydrolase and lysophospholipase transacylase activities. This enzyme has been postulated to be a cryptococcal virulence factor. We cloned a phospholipase-encoding gene (PLB1) from C. neoformans and constructed plb1 mutants using targeted gene disruption. All three enzyme activities were markedly reduced in the mutants compared with the wild-type parent. The plb1 strains did not have any defects in the known cryptococcal virulence phenotypes of growth at 37 degrees C, capsule formation, laccase activity and urease activity. The plb1 strains were reconstituted using the wild-type locus and this resulted in restoration of all extracellular PLB activities. In vivo testing demonstrated that the plb1 strain was significantly less virulent than the control strains in both the mouse inhalational model and the rabbit meningitis model. We also found that the plb1 strain exhibited a growth defect in a macrophage-like cell line. These data demonstrate that secretory phospholipase is a virulence factor for C. neoformans.

Purification and characterization of secretory phospholipase B, lysophospholipase and lysophospholipase/transacylase from a virulent strain of the pathogenic fungus Cryptococcus neoformans.

Infection caused by the fungus Cryptococcus neoformans is potentially fatal. A highly active extracellular phospholipase, demonstrating phospholipase B (PLB), lysophospholipase (LPL) and lysophospholipase/transacylase (LPTA) activities, was purified to homogeneity from C. neoformans using (NH(4))(2)SO(4) fractionation, and hydrophobic-interaction, anion-exchange and gel-filtration chromatography. All three enzyme activities co-purified as a single protein with an apparent molecular mass of 70-90 kDa by SDS/PAGE and 160-180 kDa by gel filtration. The ratio of the three activities remained constant after each purification step. The amino acid composition, as well as the sequences of the N-terminus and of five internal peptide fragments were novel. The protein was an acidic glycoprotein containing N-linked carbohydrate moieties, with pI values of 5.5 and 3.5. The apparent V(max) values for PLB and LPL activities were 12.3 and 870 micromol/min per mg of protein respectively; the corresponding K(m) values were approx. 185.3 and 92.2 microM. The enzyme was active only at acidic pH (pH optimum of 4.0 for PLB and 4.0-5.0 for LPL and LPTA). Enzyme activity did not require added cations, but was inhibited by Fe(3+). LPL and LPTA activities were decreased by 0.1% (v/v) Triton X-100 to 50% of the control value. Palmitoylcarnitine (0.5 mM) inhibited PLB (97% inhibition) and LPL and LPTA activities (35% inhibition) competitively. All phospholipids except phosphatidic acid were degraded by PLB, but dipalmitoyl phosphatidylcholine and dioleoyl phosphatidylcholine were the preferred substrates. This is the first complete description of the purification and properties of a phospholipase, which may be involved in virulence, from a pathogenic fungus.

The origin of 1H NMR-visible triacylglycerol in human neutrophils. Highfatty acid environments result in preferential sequestration of palmitic acid into plasma membrane triacylglycerol.

Human neutrophils incubated for 1 h in vitro with 10% commercial pooled, human serum containing high levels of free fatty acids (1141 microM) displayed a distinct lipid signal, typical of triacylglycerol, in the 1H NMR spectrum. Concurrently their plasma membrane triacylglycerol mass increased 4.6-fold with a selective rise in the content of palmitic and linoleic acids. Although qualitatively similar, these effects were much greater than those observed after incubating neutrophils with 50 microg.mL-1 of lipopolysaccharide in the presence of 10% AB serum with normal free fatty acid content (345 microM, LPS/S). Incubation of neutrophils with an artificial mixture of free fatty acids at concentrations found in commercial serum, or with the fatty acid fraction isolated from commercial serum increased the 1H NMR-detectable triacylglycerol. The signal intensity of the 1H NMR-detectable triacylglycerol depended on the triacylglycerol composition, and correlated with increased membrane triacylglycerol mass. Cellular uptake of 3H-labelled palmitic or oleic acids increased in the presence of commercial serum but not with LPS/S, with little contribution in either case to the triacylglycerol pool that increased in mass. Pulse-chase experiments demonstrated that with LPS/S and commercial serum, radiolabelled palmitic acid was preferentially incorporated into triacylglycerol located in the plasma membrane. This process could occur at the plasma membrane, as cytoplasts efficiently convert exogenous fatty acids into triacylglycerol. We propose that LPS/S and serum containing high levels of free fatty acid, important in conditions of sepsis and inflammation, may facilitate the sequestration of palmitic acid into triacylglycerol by different pathways. This triacylglycerol originates from exogenous and endogenous free fatty acids, is 1H NMR-visible, and may have a role in regulating apoptosis.

Heteronuclear NMR studies of metabolites produced by Cryptococcus neoformans in culture media: identification of possible virulence factors.

The yeast, Cryptococcus neoformans var. neoformans is a major contributor to the morbidity and mortality experienced by the immunosuppressed population. With a view to providing better treatment, identification of cryptococcal virulence factors is an important goal, with most effort to date directed toward the significance of structural variations in the polysaccharide capsule. The present work describes the characterization of supernatants obtained from cryptococcal cultures. This was achieved by thorough identification of the spin systems of individual metabolites through both homonuclear and heteronuclear NMR experiments that circumvented the difficulties imposed by limited dispersion and a range of concentrations in different cultures covering more than 3 orders of magnitude. More than 30 metabolites, including amino acids, alditols, nucleosides, acetate, and ethanol, were identified by their (1)H and (13)C chemical shifts and observed long-range correlations. The possible contribution of some detected substances to the pathogenicity of Cryptococcus neoformans is discussed. Magn Reson Med 42:442-453, 1999.

Phosphodiesterase expression in human epithelial cells.

Epithelial cells play a critical role in airway inflammation and have the capacity to produce many inflammatory mediators, including bioactive lipids and proinflammatory cytokines. Intracellular levels of cAMP and cGMP are important in the control of inflammatory cell function. These cyclic nucleotides are inactivated via a family of phosphodiesterase (PDE) enzymes, providing a possible site for drug intervention in chronic inflammatory conditions. We studied the expression of PDE activity in an epithelial cell line (A549) and in primary human airway epithelial cells (HAECs). We measured PDE function using specific inhibitors to identify the PDE families present and used RT-PCR to elucidate the expression of PDE isogenes. Both A549 cells and HAECs predominantly expressed PDE4 activity, with lesser PDE1, PDE3, and PDE5 activity. RT-PCR identified HSPDE4A5 and HSPDE4D3 together with HSPDE7. Inhibition of PDE4 and PDE3 reduced secretion by these cells. Epithelial PDE may be an important target for PDE4 inhibitors in the development of the control of asthmatic inflammation, particularly when delivered via the inhaled route.

Increased saturated triacylglycerol levels in plasma membranes of human neutrophils stimulated by lipopolysaccharide.

Neutrophils isolated from patients with bacterial infections or stimulated in vitro with lipopolysaccharide (LPS) produce a high resolution, lipid-dominated spectrum on 1H-NMR spectroscopy (May et al, 1993. J. Infect. Dis. 168: 386-392). We have investigated the origin of this lipid signal using NMR and chemical analyses of both whole neutrophils and purified plasma membranes. Plasma membranes from neutrophils that had been stimulated with 50 microg/ml LPS exhibited the high resolution 1H-NMR signal, and contained double the triacylglycerol (TAG) content of plasma membranes isolated from resting cells. Chemical analysis of the whole cells indicated that the TAG also increased at the cellular level (1.7-fold) after stimulation with LPS. Diradylglycerol increased 2- to 3-fold in both whole cells and plasma membranes after stimulation, but was only a minor component compared with TAG. The plasma membrane protein/phospholipid ratio increased 2.6-fold, whereas cholesterol (free and esterified) was unchanged. The membranes from LPS-stimulated neutrophils exhibited increased fluidity, as judged by increased merocyanine 540 binding, consistent with a 2-fold reduction in cholesterol/phospholipid ratio. LPS induced a shift in fatty acid content of whole cell polar lipids towards more oleic acid and less palmitic acid, whereas the neutral lipid fraction contained increased amounts of palmitic and stearic acids. The TAG fraction of plasma membrane lipids contained increased amounts of palmitic acid when prepared from cells stimulated with LPS. We conclude that the 1H-NMR signal in LPS-stimulated neutrophils arises from increased amounts of plasma membrane TAG with an elevated content of palmitic acid.

Phospholipase activity in Cryptococcus neoformans: a new virulence factor?

Fifty isolates of Cryptococcus neoformans were examined for extracellular phospholipase production after inoculation onto egg yolk agar; 49 produced a pericolonial precipitate indicative of phospholipase activity. Phospholipase B (PLB), lysophospholipase, and lysophospholipase-transacylase activities were identified by radiometric analysis in supernatants from 4 clinical isolates. The ratio of colony diameter to colony plus precipitate on agar (Pz) correlated with PLB activity. Phospholipase production was similar in 12 environmental and 13 clinical isolates of C. neoformans var. gattii. Environmental strains of C. neoformans var. neoformans (n = 8) produced more phospholipase at 72 h than did 17 clinical isolates (mean Pz, 0.57 vs. 0.72; P < .01); however, Pz values were similar at 96 h. Quantitation of cryptococci in the lungs and brains of BALB/c mice inoculated intravenously with 4 strains expressing high, intermediate, or low phospholipase activity revealed a correlation between phospholipase activity and virulence. Phospholipases secreted by C. neoformans may be implicated in virulence.

Identification of extracellular phospholipase B, lysophospholipase, and acyltransferase produced by Cryptococcus neoformans.

We recently identified phospholipase activity as a potential virulence factor of Cryptococcus neoformans. We have now defined the nature of the phospholipase activity produced by a clinical isolate of C. neoformans var. neoformans, under native conditions, by 1H and 31P nuclear magnetic resonance (NMR) spectroscopy and thin-layer chromatography (TLC) of radiolabelled substrates. Glycerophosphocholine was identified by NMR spectroscopy as the sole phospholipid degradation product of the reaction between substrate phosphatidylcholine (PC) and cryptococcal culture supernatants indicating the presence of phospholipase B (PLB). No lysophosphatidylcholine (lyso-PC) or products indicative of phospholipase C, phospholipase D, or other lipase activity were identified. Use of PC and lyso-PC containing radiolabelled acyl chains and separation of products by TLC confirmed the PLB and lysophospholipase (LPL) activities. Lysophospholipase transacylase (LPTA) activity was identified by the formation of radioactive PC from lyso-PC. Extracellular enzyme production was maximal after 6 to 10 h in fresh medium. Assay conditions were optimized for pH, linearity with time, enzyme concentration, and saturation by substrates to allow comparison with phospholipases from other organisms. LPL activity was 10- to 20-fold greater than PLB activity, with mean (+/- standard deviation) specific activities of 34.9 +/- 7.9 and 3.18 +/- 0.2 micromol of substrate hydrolyzed per min per mg of protein, respectively. The response of PLB to increasing substrate concentrations was bimodal, whereas inhibition of LPL and LPTA activities occurred at concentrations of substrate lyso-PC greater than 200 microM. Enzyme activities were stable at acid pH (3.8), with pH optima of 3.5 to 4.5. Activities were unchanged in the presence of exogenous serine protease inhibitors, divalent cations, and EDTA. We conclude that C. neoformans produces highly active extracellular PLB, LPL, and LPTA under native conditions.

Changes in cellular and plasma membrane phospholipid composition after lipopolysaccharide stimulation of human neutrophils, studied by 31P NMR.

Lipopolysaccharide (endotoxin, LPS) exerts potent proinflammatory effects on neutrophils which may involve membrane phospholipid metabolism. The cellular and plasma membrane phospholipid composition of resting neutrophils and those stimulated with 50 microg ml(-1) LPS were studied by 31P NMR and chemical analysis. A rapid new method for plasma membrane purification was employed, involving the direct lysis of cytoplasts. Chemical analyses showed that, although total cellular phospholipid content did not change with LPS stimulation, there was twice the amount of phospholipid present in plasma membranes isolated from stimulated cells, resulting in a lowered cholesterol/phospholipid ratio. Since internal membranes have lower cholesterol content this result is consistent with an origin from insertion of these membranes (most probably from the endoplasmic reticulum) into the plasma membrane, thereby increasing its fluidity. The individual phospholipid classes of both cells and membranes were quantified by 31P-NMR spectroscopy after dissolution in sodium cholate without prior extraction of lipids, allowing partial resolution of the major phospholipid classes and ether-linked phospholipids. Ether-linked lipids were distinguished from diacyl phospholipids by hydrolysis of lipid extracts with HCl and phospholipase A1, There was a significant increase in phosphatidylserine in both cells and plasma membranes after stimulation, with a decrease in the phosphatidylethanolamine (diacyl and plasmalogen) content in the cells. Plasma membranes from stimulated cells exhibited a significant decrease in a phospholipid tentatively identified as 2-arachidonoyl-1-alkyl-sn-glycero-3-phosphocholine, a precursor of the lipid inflammatory mediator, platelet-activating factor. This report is the first to elaborate the changes in phospholipid composition in human neutrophils as a whole, and in plasma membranes separated from them, before and after stimulation by the physiological activator, LPS.