Use of [1-14C]oleate labelled autoclaved Escherichia coli as a membranous substrate for measurement of in vitro phospholipase D activity.

Autoclaved Escherichia coli labelled with [1-14C]oleate in the 2-acyl position have been used extensively to measure phospholipase A2 activity in vitro. The present study demonstrates that this membranous substrate is also useful for the measurement of in vitro phospholipase D activity. Phospholipase D from Streptomyces chromofuscus catalyzed the hydrolysis of [1-14C]oleate labelled, autoclaved E. coli optimally at pH 7.0-8.0 to generate [14C]phosphatidic acid in the presence of 5 mM added Ca2+. Other divalent cations would not substitute for Ca2+. Activity was linear with time and protein up to 30% of the hydrolysis of substrate. Phospholipase D activity was stimulated in a dose-dependent manner by the addition of Triton X-100. The activity was increased 5.5-fold with 0.05% Triton, a concentration that totally inhibited hydrolysis of E. coli by human synovial fluid phospholipase A2. Accumulation of [14C]diglyceride was observed after 10 min of incubation. This accumulation was inhibited by NaF (IC50 = 18 microM) or propanolol (IC50 = 180 microM) suggesting the S. chromofuscus phospholipase D was contaminated with phosphatidate phosphohydrolase. Phosphatidic acid released by the action of cabbage phospholipase D was converted to phosphatidylethanol in an ethanol concentration dependent manner. These results demonstrate that [1-14C]oleate labelled, autoclaved E. coli can be used to measure phospholipase D activity by monitoring accumulation of either [14C]phosphatidic acid or [14C]phosphatidylethanol.     

Endogenous suppression of neutral-active and calcium-dependent phospholipase A2 in human polymorphonuclear leukocytes

Phospholipase A2 activity was measured in homogenized and acid-extracted human polymorphonuclear leukocytes using [1-14C]oleate-labelled autoclaved Escherichia coli as substrate. In whole homogenate and in the supernatant and particular fractions separated by centrifugation at 150,000 X g, phospholipase activity was barely detectable (1-4 pmol/h per 10(6) cell equivalents). By contrast, acid extracts of these fractions contained over 10-times as much phospholipase activity in the dialyzed supernatants (20-300 pmol/h per 10(6) cell equivalents), whereas phospholipase inhibitor(s) were found in the sediment. The acid-solubilized phospholipase A2 activity was absolutely Ca2+-dependent and optimal at pH 7.0-7.5 with 1.0 mM added Ca2+. Addition of the resuspended sediment of the acid extract dose-dependently suppressed phospholipase activity in the supernatant; less than equivalent amounts were sufficient to inhibit 95%. Suppressor activity was lipid-extractable. After thin layer chromatography of lipid extracts, the bulk of inhibitory activity was recovered from the free fatty acid region. Analysis of the fatty acids by gas liquid chromatography showed that 63% were unsaturated. All unsaturated fatty acids tested were potent inhibitors of phospholipase A2 activity (IC50 3-10 microM). Oleoyl-CoA, hydroxyeicosatetraenoic acids and leukotriene D4 were also inhibitory, while methyl oleate, saturated fatty acids and the prostaglandins E2 and F2 alpha had no effect. These in vitro data indicate that neutral-active and calcium-dependent phospholipase A2 in human polymorphonuclear leukocytes is largely suppressed by endogenous inhibitors and suggest that unsaturated fatty acids and some of their metabolites may partly account for this suppressor activity.     

Characterization and localization of neutral sphingomyelinase in bovine adrenal medulla

Homogenates of bovine adrenal medullae hydrolyzed exogenous sphingomyelin at 4.3 +/- 1.6 nmol X mg-1 X min-1 and 97% of this sphingomyelinase activity was sedimentable at 110,000 g. The sphingomyelinase had a broad pH optimum centered at pH 7. Enzymatic activity was maximal with 80 microM added Mn2+; Mg2+ supported less than half maximal activity and both Ca2+ and EDTA inhibited activity. No activity was detected in the absence of Triton X-100. Response to detergent was biphasic with dose-dependent stimulation from 0.02% to 0.05% Triton X-100 followed by inhibition with increasing concentrations of detergent. Activity in response to detergent was also modulated by protein concentration. Sphingomyelinase activity was associated with a plasma membrane-microsomal fraction. Phosphatidylcholine was not hydrolyzed under optimal conditions for sphingomyelin hydrolysis and a variety of other conditions. Neutral-active sphingomyelinase activity in adrenal medulla was similar in magnitude to that observed in other non-neural bovine tissues. This study demonstrates the presence of a potent neutral-active sphingomyelinase in a plasma membrane-microsomal fraction of bovine adrenal medulla. This enzyme may be involved in membrane fusion and lysis during catecholamine secretion through its ability to alter membrane composition.     

Phosphatidylinositol-specific phospholipase C activity of chromaffin granule-binding proteins

Using [U-14C]phosphatidylinositol as substrate, Ca2+-dependent phospholipase C activity was detected in a group of bovine adrenal medullary proteins that bind to chromaffin granule membranes in the presence of Ca2+ ("chromobindins," Creutz, C. E., Dowling, L. G., Sando, J. J., Villar-Palasi, C., Whipple, J. H., and Zaks, W. J. (1983) J. Biol. Chem. 258, 14664-14674). The activity was maximal at neutral pH and represented an 80- to 240-fold enrichment of adrenal medullary cytosol phospholipase C activity measured at pH 7.3. The stimulation of activity by Ca2+ was complex; no activity was present in the absence of Ca2+, 25% activation occurred at 1 microM Ca2+, and full activation at 5 mM Ca2+. The enzyme bound to chromaffin granule membranes in the presence of 2 mM Ca2+ but was released at 40 microM Ca2+, suggesting that intrinsic enzyme activity may be regulated by [Ca2+] at 1 microM, but additional activation at higher concentrations of Ca2+ is seen in vitro as a result of Ca2+-dependent binding of the active enzyme to substrate-containing membranes. This enzyme may generate diacylglycerol and phosphorylated inositol to act as intracellular messengers in the vicinity of the chromaffin granule membrane during the process of exocytosis.     

Surface-active phospholipase A2 in mouse spermatozoa

The partial characterization of a calcium-dependent phospholipase A2 associated with membranes of mouse sperm is described. Intact and sonicated sperm had comparable phospholipase A2 activity which was maximal at pH 8.0 using [1-14C]oleate-labeled autoclaved Escherichia coli or 1-[1-14C]stearoyl-2-acyl-3-sn-glycerophosphorylethanolamine as substrates. More than 90% of the activity was sedimented when the sperm sonicate was centrifuged at 100 000 X g, indicating that the enzyme is almost totally membrane-associated. The activity is stimulated 200% during the ionophore-induced acrosome reaction and is almost equally distributed between plasma/outer acrosomal and inner acrosomal membrane fractions. The membrane-associated phospholipase A2 had an absolute requirement for low concentrations of Ca2+; Sr2+, Mg2+ and other divalent and monovalent cations would not substitute for Ca2+. In the presence of optimal Ca2+, zinc and gold ions inhibited the activity while Cu2+ and Cd2+ were without effect. Incubation of sperm sonicates with 1-[1-14C]stearoyl-2-acyl-3-sn-glycerophosphorylethanolamine in the presence and absence of sodium deoxycholate demonstrated the presence of phospholipase A2 and lysophospholipase activities. No phospholipase A1 activity was detectable. Indomethacin, sodium meclofenamate and mepacrine, but not dexamethasone or aspirin, inhibited the sperm phospholipase A2 activity. Preincubation with p-bromophenacyl bromide inhibited phospholipase A2, suggesting the presence of histidine at the active site. The enzyme may play an important role in the membrane fusion events in fertilization.     

Hemorrhagic enteritis in captive American kestrels (Falco sparverius)

Hemorrhagic enteritis and hepatitis of suspected adenovirus etiology were the apparent cause of death of nine captive American kestrels. Cloacal hemorrhage was the only prominent gross lesion: disseminated hepatocellular necrosis, and intranuclear inclusion bodies were evident microscopically. Electron microscopy revealed numerous adenovirus-like particles associated with the hepatic lesions. Attempts to serologically identify the agent were unsuccessful.     

Acid-active neuraminidases in the growth media from cultures of pathogenic Naegleria fowleri and in sonicates of rabbit alveolar macrophages

Using bovine mucin and isolated human myelin as sources of sialic acid, we demonstrate the presence of neuraminidase activities in the growth media of pathogenic, but not nonpathogenic, Naegleria sp. and in sonicates of rabbit alveolar macrophages. Neuraminidase activity was maximal at pH 4.5 and 5.0, and the specific activity for sialic acid release was up to 13-fold greater with mucin than with human myelin. Activity in the growth media from cultures of pathogenic Naegleria fowleri was ion-independent, while that of macrophage sonicates required divalent cation; optimal activity was noted with 2.5 mM Zn2+, while Mg2+ and Mn2+ supported activity to a lesser extent. Such acid-active neuraminidases may contribute to the reported glycolipid alterations associated with demyelinating diseases.     

High Seroprevalence of Antibodies to Avian Influenza Viruses among Wild Waterfowl in Alaska: Implications for Surveillance

We examined seroprevalence (presence of detectable antibodies in serum) for avian influenza viruses (AIV) among 4,485 birds, from 11 species of wild waterfowl in Alaska (1998–2010), sampled during breeding/molting periods. Seroprevalence varied among species (highest in eiders (Somateria and Polysticta species), and emperor geese (Chen canagica)), ages (adults higher than juveniles), across geographic locations (highest in the Arctic and Alaska Peninsula) and among years in tundra swans (Cygnus columbianus). All seroprevalence rates in excess of 60% were found in marine-dependent species. Seroprevalence was much higher than AIV infection based on rRT-PCR or virus isolation alone. Because pre-existing AIV antibodies can infer some protection against highly pathogenic AIV (HPAI H5N1), our results imply that some wild waterfowl in Alaska could be protected from lethal HPAIV infections. Seroprevalence should be considered in deciphering patterns of exposure, differential infection, and rates of AIV transmission. Our results suggest surveillance programs include species and populations with high AIV seroprevalences, in addition to those with high infection rates. Serologic testing, including examination of serotype-specific antibodies throughout the annual cycle, would help to better assess spatial and temporal patterns of AIV transmission and overall disease dynamics.     

Use of serum biochemistry to evaluate nutritional status and health of incubating common eiders (Somateria mollissima) in Finland

During 1997-1999, we collected serum samples from 156 common eider (Somateria mollissima) females incubating eggs in the Finnish archipelago of the Baltic Sea. We used serum chemistry profiles to evaluate metabolic changes in eiders during incubation and to compare the health and nutritional status of birds nesting at a breeding area where the eider population has declined by over 50% during the past decade, with birds nesting at two areas with stable populations. Several changes in serum chemistries were observed during incubation, including (1) decreases in serum glucose, total protein, albumin, beta-globulin, and gamma-globulin concentrations and (2) increases in serum uric acid, creatine kinase, and beta-hydroxybutyrate concentrations. However, these changes were not consistent throughout the 3-yr period, suggesting differences among years in the rate of carbohydrate, lipid, and protein utilization during incubation. The mean serum concentrations of free fatty acids, glycerol, and albumin were lowest and the serum alpha- and gamma-globulin levels were highest in the area where the eider population has declined, suggesting a role for nutrition and diseases in the population dynamics of Baltic eiders.