Molecular forms of acetyl- and butyrylcholinesterase in human glioma

Specimens of astrocytoma, oligodendroglioma and medulloblastoma were sequentially extracted with saline and saline-Triton X-100 buffers. Acetyl- (AChE) and butyrylcholinesterase (BuChE) activities were assayed in the soluble fractions, these being further analyzed to establish the distribution of molecular forms. All the tumors tested showed AChE and BuChE activities, the measured AChE/BuChE ratios being unrelated to the malignant grading. Hydrophilic and amphiphilic AChE and BuChE tetramers, amphiphilic AChE dimers and monomers, and hydrophilic BuChE monomers were identified in all the tumors analyzed. The amphiphilic behavior of the enzyme forms was assessed by sedimentation analysis and hydrophobic chromatography on phenyl-Agarose. A small fraction of glioma AChE monomers was released as, or transformed into, hydrophilic forms by incubation with phosphatidylinositol-specific phospholipase C (PIPLC). These data suggest that AChE monomers bearing distinct hydrophobic domains coexist in human glioma.     

cis-4-Methylsphingosine decreases sphingolipid biosynthesis by specifically interfering with serine palmitoyltransferase activity in primary cultured neurons

The effect of six different structurally modified sphingosine analogues on biosynthesis of sphingolipids was studied in primary cultured murine cerebellar neurons. Treatment of cells with cis-4-methylsphingosine at micromolar levels resulted in a markedly decreased sphingolipid biosynthesis, whereas the other compounds examined, trans-4-methylsphingosine, cis-5-methylsphingosine, trans-5-methylsphingosine, cis-sphingosine, and 1-deoxysphingosine, inhibited sphingolipid biosynthesis less efficiently. The inhibition of sphingolipid biosynthesis by the various compounds was paralleled by a decrease of serine palmitoyltransferase activity in situ. For cis-4-methylsphingosine the inhibitory effect on serine palmitoyltransferase activity was shown to be concentration- and time-dependent. Half-maximal reduction of enzyme activity occurred after 24 h of treatment with 10 microM of the compound. The activity of other enzymes of sphingolipid biosynthesis as well as phospholipid and protein biosynthesis was not affected. Analysis of the sphingoid moiety of cellular sphingolipids suggests that the sphingosine analogues listed above were subject to degradation rather than being utilized as precursors for sphingolipid biosynthesis by cultured neurons. Except of 1-deoxysphingosine, the other five sphingosine analogues were shown to be substrates for sphingosine kinase in vitro. After 24 h of treatment of primary cerebellar neurons with the various sphingosine analogues the relative percentage of the respective intracellular 1-phosphate derivatives paralleled exactly the inhibitory effect on serine palmitoyltransferase activity observed when cells were treated with the unphosphorylated compounds. In contrast to the respective 1-phosphate derivatives of the other methyl-branched sphingosine analogues examined, cis-4-methylsphingosine 1-phosphate showed an intracellular accumulation suggesting a delayed turnover rate in cultured murine neurons for this compound. These results suggest that the inhibitory effect of the sphingosine analogues on serine palmitoyltransferase is mediated by their respective 1-phosphate derivatives and that the pronounced effect of cis-4-methylsphingosine is caused by a high intracellular concentration of cis-4-methylsphingosine 1-phosphate. cis-4-Methylsphingosine, in addition, caused drastic changes in cell morphology of primary cerebellar neurons, which were not observed when these cells were treated with one of the other sphingosine analogues examined.     

Possible involvement of the base exchange enzymes in the phospholipid metabolism in LAN-2 cells

The incorporation of [14C] choline, [14C] ethanolamine, and [14C] serine by LAN-2 cells into their corresponding phospholipids was investigated in the presence or absence of TPA. The presence of TPA increased the amount of radioactivity incorporated into the phospholipids with a corresponding decrease in the amount of radioactivity in the cytosolic compartment compared to control cultures. There were no differences between TPA-exposed and control cells in the distribution of radioactivity in free choline, phosphorylcholine or CDP-choline of [14C] choline labeled cells. This indicates that the increased lipid labeling was not accompanied by enhanced labeling of the intermediates of the de novo pathway. These results suggest that a choline base exchange enzyme was stimulated in TPA exposed cells. In addition, the enhanced incorporation of serine by TPA into its corresponding phospholipid implies the stimulation of the serine base exchange enzyme which is responsible for phosphatidylserine synthesis in mammals. These observations suggest a     

A novel sphingosine-dependent protein kinase (SDK1) specifically phosphorylates certain isoforms of 14-3-3 protein

Protein kinases activated by sphingosine or N,N'-dimethylsphingosine, but not by other lipids, have been detected and are termed sphingosine-dependent protein kinases (SDKs). These SDKs were previously shown to phosphorylate endogenous 14-3-3 proteins (Megidish, T., White, T., Takio, K., Titani, K., Igarashi, Y., and Hakomori, S. (1995) Biochem. Biophys. Res. Commun. 216, 739-747). We have now partially purified one SDK, termed SDK1, from cytosol of mouse Balb/c 3T3(A31) fibroblasts. SDK1 is a serine kinase with molecular mass 50-60 kDa that is strongly activated by N, N'-dimethylsphingosine and sphingosine, but not by ceramide, sphingosine 1-phosphate, or other sphingo-, phospho-, or glycerolipids tested. Its activity is inhibited by the protein kinase C activator phosphatidylserine. Activity of SDK1 is clearly distinct from other types of serine kinases tested, including casein kinase II, the alpha and zeta isoforms of protein kinase C, extracellular signal-regulated mitogene-activated protein kinase 1 (Erk-1), Erk-2, and Raf-1. SDK1 specifically phosphorylates certain isoforms of 14-3-3 (eta, beta, zeta) but not others (sigma, tau). The phosphorylation site was identified as Ser* in the sequence Arg-Arg-Ser-Ser*-Trp-Arg in 14-3-3 beta. The sigma and tau isoforms of 14-3-3 lack serine at this position, potentially explaining their lack of phosphorylation by SDK1. Interestingly, the phosphorylation site is located on the dimer interface of 14-3-3. Phosphorylation of this site by SDK1 was studied in 14-3-3 mutants. Mutation of a lysine residue, located 9 amino acids N-terminal to the phosphorylation site, abolished 14-3-3 phosphorylation. Furthermore, co-immunoprecipitation experiments demonstrate an association between an SDK and 14-3-3 in situ. Exogenous N, N'-dimethylsphingosine stimulates 14-3-3 phosphorylation in Balb/c 3T3 fibroblasts, suggesting that SDK1 may phosphorylate 14-3-3 in situ. These data support a biological role of SDK1 activation and consequent phosphorylation of specific 14-3-3 isoforms that regulate signal transduction. In view of the three-dimensional structure of 14-3-3, it is likely that phosphorylation by SDK1 would alter dimerization of 14-3-3, and/or induce conformational changes that alter 14-3-3 association with other kinases involved in signal transduction.     

Analysis of the developing Xenopus tail bud reveals separate phases of gene expression during determination and outgrowth

Fourier-transform infrared spectroscopy (FT-IR) has been applied to the quantitative study of the dehydration of the phosphatidylserine phosphate group in the presence of Ca2+ exerted by different molecules, such as diacylglycerol, sphingosine and stearylarnine, by using a partial least-squares statistical procedure. By using this method it was observed that diacylglycerol enhanced the dehydration of this PO2- group produced by Ca2+ whereas the amino-bases sphingosine and stearylamine protected the phosphate group from the dehydration produced by Ca2+ due to the very strong electrostatic interaction established. The apparent pKa of lipid carboxyl groups can also be estimated by using FTIR. The method consisted in quantifying the absorbance intensities due to the protonated and the unprotonated forms of the specific group being studied. The pKa of the carboxyl group of [1-13C]-palmitic acid included in dipalmitoylphosphatidylcholine membranes was found to be 8.7, a value much higher than that estimated from a molecular solution of the fatty acid. It was observed using the same method that the pKa of free fatty acids in model stratum corneum lipid mixtures was in the range 6.2-7.3 increasing with the preponderance of oleic acid over palmitic acid. Finally the pKa of the carboxyl group of phosphatidylserine was shifted from 4.6 in the pure phospholipid to 2.1 and 2.2 in the presence of equimolar sphingosine and stearylamine, respectively, as a consequence of electrostatic interactions.     

The effect of various treatments in vitro and in vivo on the binding of 125I-labeled anti-rat serum albumin Fab'' to rat tissue polyribosomes

With 125I-labeled Fab' specific for rat liver serine dehydratase it has been possible to localize polyribosomes synthesizing the enzyme under several different environmental conditions. Evidence is presented to show that, following the administration of amino acids in vivo, the relative synthetic capabilities of free and membrane-bound polyribosomes synthesizing serine dehydratase vary with time. Early during the period of induction of the enzyme by administration of amino acids or by feeding a high protein diet the majority of the newly synthesized enzyme is derived from membrane-bound polyribosomes. Later in the induction process an increasing proportion of the enzyme is synthesized by the free polyribosomes. Subcellular localization studies clearly show that serine dehydratase is synthesized by both subclasses of hepatic membrane-bound polyribosomes, the loose and tight membrane-bound polyribosomes, as well as by the free polyribosomes. It was found that the membrane-bound polyribosomes are the preferential sites of synthesis of the majority of serine dehydratase molecules in the Morris hepatomas 5123C and 7800. It is concluded that the synthesis of the enzyme, serine dehydratase, in rat liver is not discretely compartmentalized in either class of free or membrane-bound polyribosomes. Rather, the relative proportions of the serine dehydratase synthesizing polyribosomes within these two classes of polyribosomes can vary depending on the metabolic and physiologic state of the liver cell.     

Chest radiographic staging in allergic bronchopulmonary aspergillosis: relationship with immunological findings

Considering the structural similarity between gabexate mesylate (FOY), a drug for serine proteinase-mediated diseases, and L-arginine, the effect of gabexate mesylate on the nitric oxide (NO) pathway has been investigated. Gabexate mesylate inhibits competitively constitutive and inducible NO synthase (cNOS and iNOS, respectively), with Ki values of 1.0 x 10(-4) M and 5.0 x 10(-3) M, respectively, at pH 7.4 and 37.0 degrees C. However, gabexate mesylate is not an NO precursor. Moreover, like other NOS inhibitors, gabexate mesylate increases iNOS mRNA expression in rat C6 glioma cells, as induced by E. coli lipopolysaccharide plus interferon-gamma. Finally, gabexate mesylate inhibits dose-dependently nitrite production (i.e. NO release) in rat C6 glioma cells, as induced by E. coli lipopolysaccharide plus interferon-gamma. Thus, this drug should be administered under careful control, since enzyme inhibition may occur also in vivo.     

Formyl peptide receptor signaling in HL-60 cells through sphingosine kinase

Sphingosine-1-phosphate (SPP) produced from sphingosine by sphingosine kinase has recently been reported to act as intracellular second messenger for a number of plasma membrane receptors. In the present study, we investigated whether the sphingosine kinase/SPP pathway is involved in cellular signaling of the Gi protein-coupled formyl peptide receptor in myeloid differentiated human leukemia (HL-60) cells. Receptor activation resulted in rapid and transient production of SPP by sphingosine kinase, which was abolished after pertussis toxin treatment. Direct activation of heterotrimeric G proteins by AlF4- also rapidly increased SPP formation in intact HL-60 cells. In cytosolic preparations of HL-60 cells, sphingosine kinase activity was stimulated by the stable GTP analog, guanosine 5'-O-(3-thiotriphosphate). Inhibition of sphingosine kinase by DL-threo-dihydrosphingosine and N,N-dimethylsphingosine did not affect phospholipase C stimulation and superoxide production but markedly inhibited receptor-stimulated Ca2+ mobilization and enzyme release. We conclude that the formyl peptide receptor stimulates through Gi-type G proteins SPP production by sphingosine kinase, that the enzyme is also stimulated by direct G protein activation, and that the sphingosine kinase/SPP pathway apparently plays an important role in chemoattractant signaling in myeloid differentiated HL-60 cells.     

Phosphatidylserine specific binding protein in rat brain: purification and characterization

A calcium-independent phosphatidylserine specific binding protein detected on liposome blotting analysis was purified from rat brain and revealed to be identical to myristoylated, alanine-rich C kinase substrate (MARCKS). MARCKS specifically binds to phosphatidylserine but not phosphatidylcholine. The binding of MARCKS to phosphatidylserine was abolished on protein kinase C-dependent phosphorylation. Since bacterially expressed MARCKS also specifically binds to phosphatidylserine, myristoylation of the N-terminal glycine seems not to be essential for the binding of MARCKS to phosphatidylserine. These data suggest that phosphatidylserine is a membranous target molecule of MARCKS.     

Sphingosine-mediated phosphatidylinositol metabolism and calcium mobilization

The cytokine-mediated stimulation of sphingomyelin (SM) metabolism is emerging as an important signal transduction pathway via the generation of ceramide and sphingosine, products which have been shown to affect a wide variety of biological processes. Because SM-mediated signal transduction is initiated via the hydrolysis of an integral membrane phospholipid by a phospholipase C-like enzyme (sphingomyelinase) to yield lipids which modulate protein kinase C activity, the SM and phosphatidylinositol (PI) signaling pathways share certain similarities. The present study was undertaken to examine the potential for interplay between SM and PI turnover by testing the effects of sphingosine, sphingosine-1-phosphate, and ceramide on PI turnover. In dermal fibroblasts, sphingosine stimulated a rapid dose-dependent hydrolysis of PI, yielding inositol 1,4,5-triphosphate, followed by increased levels of intracellular calcium. Sphingosine-induced inositol phosphate (IP) accumulation was observed between 5 and 30 microM sphingosine with a maximal accumulation of 2.7-fold over control levels. Enhanced IP formation was measured as early as 5 s following sphingosine treatment and IP levels remained elevated for more than 60 min. Intracellular calcium mobilization accompanied the dose-dependent accumulation of IPs in response to sphingosine, although this effect was not apparent until after a 30-40-s lag period. Interestingly, sphingosine-1-phosphate stimulated a more rapid release of intracellular Ca2+ than sphingosine, but it had no effect on PI turnover. DL-threo-Dihydrosphingosine, a competitive inhibitor of sphingosine kinase, stimulates both PI turnover and Ca2+ flux, but does not block the action of sphingosine relative to those two processes. Ceramide (added as C2-ceramide), N-stearylamine, and stearoyl-D-sphingosine did not affect PI turnover or Ca2+ mobilization. Pretreatment of intact cells with pertussis toxin partially inhibited sphingosine-mediated IP accumulation, suggesting a role for guanine nucleotide binding protein(s) (G protein) in sphingosine-stimulated PI turnover. Furthermore, guanosine 5'-O-(3-thiotriphosphate) stimulated, whereas guanosine 5'-O-(2-thiodiphosphate) inhibited, sphingosine-induced IP accumulation in permeabilized cells. Collectively, these data suggest that sphingosine enhances PI turnover by stimulating phospholipase C activity, and the activation of this process may be modulated by G protein interactions. Thus, the regulation of PI turnover and Ca2+ mobilization by sphingosine may represent another mechanism by which sphingosine modulates cell function and that these effects can be distinguished from those of ceramide.     

Preferential binding of Plasmodium falciparum SERA and rhoptry proteins to erythrocyte membrane inner leaflet phospholipids

Proteins of an apical organelle, the rhoptry, of Plasmodium falciparum are secreted into the host erythrocyte membrane during merozoite invasion. To identify the membrane-binding site for rhoptry proteins, we examined the binding of parasite proteins to phospholipid vesicles. A specific interaction between the rhoptry proteins of 140, 130, and 110 kDa to vesicles containing phosphatidylserine and phosphatidylinositol was observed. Both phospholipids are preferentially localized on the inner leaflet of the bilayer. Binding to other phospholipids, including sphingomyelin, was considerably less. In addition, the 120-kDa serine repeat antigen known as SERA, which was determined to be present on the merozoite, bound to phosphatidylserine vesicles and much less to vesicles of other phospholipids. Both the rhoptry and SERA proteins exhibited a preference for phosphatidylserine with short acyl side chains. Specific binding of SERA and the rhoptry proteins to phospholipids of the inner leaflet of membranes suggests a possible mechanism by which the protein facilitate invasion into host cells.     

Cations affect [3H]mazindol and [3H]WIN 35,428 binding to the human dopamine transporter in a similar fashion

The present study addresses the possibility that there are different cocaine-related and mazindol-related binding domains on the dopamine transporter (DAT) that show differential sensitivity to cations. The effects of Zn2+, Mg2+, Hg2+, Li+, K+, and Na+ were assessed on the binding of [3H]mazindol and [3H]WIN 35,428 to the human (h) DAT expressed in C6 glioma cells under identical conditions for intact cell and membrane assays. The latter were performed at both 0 and 21 degrees C. Zn2+ (30-100 microM) stimulated binding of both radioligands to membranes, with a relatively smaller effect for [3H]mazindol; Mg2+ (0.1-100 microM) had no effect; Hg2+ at approximately 3 microM stimulated binding to membranes, with a relatively smaller effect for [3H]mazindol than [3H]WIN 35,428 at 0 degrees C, and at 30-100 microM inhibited both intact cell and membrane binding; Li+ and K+ substitution (30-100 mM) inhibited binding to membranes more severely than to intact cells; and Na+ substitution was strongly stimulatory. With only a few exceptions, the patterns of ion effects were remarkably similar for both radioligands at both 0 and 21 degrees C, suggesting the involvement of common binding domains on the hDAT impacted similarly by cations. Therefore, if there are different binding domains for WIN 35,428 and mazindol, these are not affected differentially by the cations studied in the present experiments, except for the stimulatory effect of Zn2+ at 0 and 21 degrees C and Hg2+ at 0 degrees C.     

Molecular cloning and functional characterization of murine sphingosine kinase

Sphingosine-1-phosphate (SPP) is a novel lipid messenger that has dual function. Intracellularly it regulates proliferation and survival, and extracellularly, it is a ligand for the G protein-coupled receptor Edg-1. Based on peptide sequences obtained from purified rat kidney sphingosine kinase, the enzyme that regulates SPP levels, we report here the cloning, identification, and characterization of the first mammalian sphingosine kinases (murine SPHK1a and SPHK1b). Sequence analysis indicates that these are novel kinases, which are not similar to other known kinases, and that they are evolutionarily conserved. Comparison with Saccharomyces cerevisiae and Caenorhabditis elegans sphingosine kinase sequences shows that several blocks are highly conserved in all of these sequences. One of these blocks contains an invariant, positively charged motif, GGKGK, which may be part of the ATP binding site. From Northern blot analysis of multiple mouse tissues, we observed that expression was highest in adult lung and spleen, with barely detectable levels in skeletal muscle and liver. Human embryonic kidney cells and NIH 3T3 fibroblasts transiently transfected with either sphingosine kinase expression vectors had marked increases (more than 100-fold) in sphingosine kinase activity. The enzyme specifically phosphorylated D-erythro-sphingosine and did not catalyze the phosphorylation of phosphatidylinositol, diacylglycerol, ceramide, D,L-threo-dihydrosphingosine or N, N-dimethylsphingosine. The latter two sphingolipids were competitive inhibitors of sphingosine kinase in the transfected cells as was previously found with the purified rat kidney enzyme. Transfected cells also had a marked increase in mass levels of SPP with a concomitant decrease in levels of sphingosine and, to a lesser extent, in ceramide levels. Our data suggest that sphingosine kinase is a prototypical member of a new class of lipid kinases. Cloning of sphingosine kinase is an important step in corroborating the intracellular role of SPP as a second messenger.     

Targeted gene disruption shows that knobs enable malaria-infected red cells to cytoadhere under physiological shear stress

Sphingosine, which is on the pathway of sphingomyelin degradation, activates phospholipase C (PLC) delta1 moderately. In the liposome assay effect of sphingosine on PLC delta1 activity depends on KCl concentration. Stimulation of PLC delta1 by sphingosine increased as the KCl concentration is increased from 0 to 100 mM, and then diminished with the increasing KCl. In the liposome assay sphingosine diminishes inhibition of PLC delta1 by sphingomyelin. To determine the domain of PLC delta1 which interacts with sphingosine active proteolytic fragments of PLC delta1 were generated by trypsin digestion of the native enzyme. Sphingosine affects the activity of PLC delta1 fragment which lacked the amino-terminal domain (first 60 amino acids) but not the active fragment that has cleaved the domain spanning the X and Y region of PLC delta1. These observations indicate that for interaction of sphingosine with PLC delta1 intact domain that span regions of conservation, designated as X and Y is necessary. When the activity of PLC delta1 was assayed with PIP2 in the erythrocyte membrane as substrate, sphingosine strongly inhibited PLC delta1. The other homolog of sphingosine 4-hydroxysphinganine (phytosphingosine) inhibited PLC delta1 to much lesser extent. The activity of PLC delta1 was inhibited by 68% and 22% in the presence of 20 microM sphingosine and phytosphingosine, respectively. This inhibition was completely abolished by deoxycholate at a concentration of 1.5 mM. These observations suggest that sphingosine may regulate activity of PLC delta1 in the cell.     

Signal transduction for proliferation of glioma cells in vitro occurs predominantly through a protein kinase C-mediated pathway

Previous work has demonstrated that glioma cells have very high protein kinase C (PKC) enzyme activity when compared to non-malignant glia, and that their PKC activity correlates with their proliferation rate. The purpose of this study was to determine whether the elevated PKC activity in glioma is secondary to an autonomously active PKC isoform implying oncogenic transformation, or whether this activity is driven by upstream ligand-receptor tyrosine kinase interactions. We treated established human glioma cell lines A172, U563 or U251 with either the highly selective PKC inhibitor CGP 41 251, or with genistein, a tyrosine kinase inhibitor. The proliferation rate and PKC activity of all the glioma lines was reduced by CGP 41 251; the IC50 values for inhibiting cell proliferation corresponded to the IC50v values for inhibition of PKC activity. Genistein also inhibited cell proliferation, with IC50 proliferation values approximating those for inhibition of tyrosine kinase activity in cell free protein extracts. Importantly, in genistein-treated cells, downstream PKC enzyme activity was dose dependently reduced such that the correlation coefficient for effects of genistein on proliferation rate and PKC activity was 0.92. These findings suggest that upstream tyrosine kinase linked events, rather than an autonomously functioning PKC, result in the high PKC activity observed in glioma. Finally, fetal calf serum (FCS) evoked a strong mitogenic effect on glioma cell lines. This mitogenic activity was completely blocked by CGP 41 251, suggesting that although the many mitogens in FCS for glioma cells signal initially through genistein-inhibitable tyrosine kinases, they ultimately channel through a PKC-dependent pathway. We conclude that proliferative signal transduction in glioma cells occurs through a predominantly PKC-dependent pathway and that selectively targeting this enzyme provides an approach to glioma therapy.     

The C2 domain of the Ca(2+)-independent protein kinase C Apl II inhibits phorbol ester binding to the C1 domain in a phosphatidic acid-sensitive manner

There are two protein kinase Cs (PKCs) in the Aplysia nervous system, PKC Apl I, which is homologous to the Ca(2+)-activated PKC family, and PKC Apl II, which is homologous to the Ca(2+)-independent PKCs epsilon and eta. Purified PKC Apl I requires much less phosphatidylserine for activation than does purified PKC Apl II, and this may explain why the neurotransmitter serotonin activates PKC Apl I but not PKC Apl II in the intact nervous system [Sossin, W. S., Fan, X., and Baseri, F. (1996) J. Neurosci. 16, 10-18]. PKC Apl II's requirement for high levels of phosphatidylserine may be mediated by its C2 domain, since removal of this domain allows PKC Apl II to be activated at lower concentrations of phosphatidylserine. To begin to understand how this inhibition is mediated, we generated fusion proteins containing the C1 and C2 domains from PKC Apl II and determined their lipid dependence for phorbol ester binding. Our results indicate that the presence of the C2 domain lowers the affinity of protein kinase C activators for the C1 domains and this inhibition can be removed by phosphatidylserine. Phosphatidic acid, however, is much more potent than phosphatidylserine in reducing C2 domain-mediated inhibition, suggesting that phosphatidic acid may be a required cofactor for the activation of PKC Apl II.     

Substrate specificity and some other enzymatic properties of dihydroceramide desaturase (ceramide synthase) in fetal rat skin

Dihydroceramide desaturase, which catalyzes the introduction of a double bond at the 4,5-position of the sphingosine base in a dihydroceramide, was assayed in vitro using radiolabeled D-erythro-C18-dihydroceramide (N-stearoyl sphinganine) and homogenates of fetal rat skin, and some enzymatic properties, including substrate specificity, were determined. The ceramide structure, as the enzymatic product, was confirmed by (i) oxidation of the product with 2,3-dicyano-5,6-dichlorobenzoquinone, which revealed the conversion to 3-ketoceramide (3,3'-didehydroceramide), indicating that a double bond was introduced at the adjacent to the C-3 hydroxyl residue of sphinganine, and (ii) mass spectrometry of a long chain base released from the enzymatic product, which revealed a spectrum identical to that of authentic sphingenine. A short chain dihydroceramide, which was radiolabeled at sphinganine through a newly established method, having a C2- or C6-fatty acid was not desaturated by the skin enzyme, whereas that having a C10-, C14-, or C18-acid was desaturated, maximal reactivity being observed for the C14-dihydroceramide. Other enzymatic properties were confirmed: NAD(H) or NADP(H) and a detergent were required for elevation of the activity; the optimum pH was approximately 6.7; and metal cations were not essential, but Zn2+, Cu2+, and Fe2+ were rather inhibitory. These properties of rat skin desaturase were partly similar to those of rat liver microsomes, as reported recently, however, their substrate specificities were different.     

Differences in prostate size between patients from University and Veterans Affairs Medical Center populations

We have previously determined that Nippostrongylus brasiliensis secretes three monomeric nonamphiphilic (G1na) variants of acetylcholinesterase (AChE) with broadly similar properties. In this study we have examined AChE expression in somatic extracts of N. brasiliensis and report the identification of an additional enzyme which is not secreted. The enzyme was resolved by sucrose density gradient centrifugation with a sedimentation coefficient of 10.2 S which was shifted to 9.4 S in the presence of Triton X-100, identifying the enzyme as a tetrameric amphiphilic (G4a) form. The amphiphilic properties of this enzyme were confirmed by charge-shift electrophoresis, in which migration was accelerated by interaction with sodium deoxycholate. The enzyme showed low activity with butyrylthiocholine, and a Michaelis constant of 91 +/- 13 microM for acetylthiocholine was determined. It was highly sensitive to the AChE-specific inhibitor bis (4-allyldimethylammoniumphenyl)pentan-3-one dibromide, with an IC50 of 6.5 +/- 0.4 microM, but was also inhibited by the butyrylcholinesterase-specific inhibitor tetramonoisopropylpyrophosphortetramide, albeit with a higher IC50 of 46.5 +/- 6.1 microM. This enzyme can therefore be distinguished from the secreted AChEs by its amphiphilic properties, sedimentation in sucrose gradients, and sensitivity to cholinesterase inhibitors.     

Phosphatidate phosphohydrolase and signal transduction

A Mg(2+)-independent and N-ethylmaleimide-insensitive phosphatidate phosphohydrolase (PAP-2) has been identified in the plasma membrane of cells and it has been purified. The enzyme is a multi-functional phosphohydrolase that can dephosphorylate phosphatidate, lysophosphatidate, sphingosine 1-phosphate and ceramide 1-phosphate and these substrates are competitive inhibitors of the reaction. The action of PAP-2 could terminate signalling by these bioactive lipids and at the same time generates compounds such as diacylglycerol, sphingosine and ceramide which are also potent signalling molecules. In relation to phosphatidate metabolism, sphingosine (or sphingosine 1-phosphate) stimulates phospholipase D and thus the formation of phosphatidate. At the same time sphingosine inhibits PAP-2 activity thus further increasing phosphatidate concentrations. By contrast, ceramides inhibit the activation of phospholipase D by a wide variety of agonists and increase the dephosphorylation of phosphatidate, lysophosphatidate, sphingosine 1-phosphate and ceramide 1-phosphate. These actions demonstrate "cross-talk' between the glycerolipid and sphingolipid signalling pathways and the involvement of PAP-2 in modifying the balance of the bioactive lipids generated by these pathways during cell activation.