Developmental regulation of neural cell adhesion molecule in human prefrontal cortex.

Neural cell adhesion molecule (NCAM) is a membrane-bound cell recognition molecule that exerts important functions in normal neurodevelopment including cell migration, neurite outgrowth, axon fasciculation, and synaptic plasticity. Alternative splicing of NCAM mRNA generates three main protein isoforms: NCAM-180, -140, and -120. Ectodomain shedding of NCAM isoforms can produce an extracellular 105-115 kilodalton soluble neural cell adhesion molecule fragment (NCAM-EC) and a smaller intracellular cytoplasmic fragment (NCAM-IC). NCAM also undergoes a unique post-translational modification in brain by the addition of polysialic acid (PSA)-NCAM. Interestingly, both PSA-NCAM and NCAM-EC have been implicated in the pathophysiology of schizophrenia. The developmental expression patterns of the main NCAM isoforms and PSA-NCAM have been described in rodent brain, but no studies have examined NCAM expression across human cortical development. Western blotting was used to quantify NCAM in human postmortem prefrontal cortex in 42 individuals ranging in age from mid-gestation to early adulthood. Each NCAM isoform (NCAM-180, -140, and -120), post-translational modification (PSA-NCAM) and cleavage fragment (NCAM-EC and NCAM-IC) demonstrated developmental regulation in frontal cortex. NCAM-180, -140, and -120, as well as PSA-NCAM, and NCAM-IC all showed strong developmental regulation during fetal and early postnatal ages, consistent with their identified roles in axon growth and plasticity. NCAM-EC demonstrated a more gradual increase from the early postnatal period to reach a plateau by early adolescence, potentially implicating involvement in later developmental processes. In summary, this study implicates the major NCAM isoforms, PSA-NCAM and proteolytically cleaved NCAM in pre- and postnatal development of the human prefrontal cortex. These data provide new insights on human cortical development and also provide a basis for how altered NCAM signaling during specific developmental intervals could affect synaptic connectivity and circuit formation, and thereby contribute to neurodevelopmental disorders.

Tsc13p is required for fatty acid elongation and localizes to a novel structure at the nuclear-vacuolar interface in Saccharomyces cerevisiae.

The TSC13/YDL015c gene was identified in a screen for suppressors of the calcium sensitivity of csg2Delta mutants that are defective in sphingolipid synthesis. The fatty acid moiety of sphingolipids in Saccharomyces cerevisiae is a very long chain fatty acid (VLCFA) that is synthesized by a microsomal enzyme system that lengthens the palmitate produced by cytosolic fatty acid synthase by two carbon units in each cycle of elongation. The TSC13 gene encodes a protein required for elongation, possibly the enoyl reductase that catalyzes the last step in each cycle of elongation. The tsc13 mutant accumulates high levels of long-chain bases as well as ceramides that harbor fatty acids with chain lengths shorter than 26 carbons. These phenotypes are exacerbated by the deletion of either the ELO2 or ELO3 gene, both of which have previously been shown to be required for VLCFA synthesis. Compromising the synthesis of malonyl coenzyme A (malonyl-CoA) by inactivating acetyl-CoA carboxylase in a tsc13 mutant is lethal, further supporting a role of Tsc13p in VLCFA synthesis. Tsc13p coimmunoprecipitates with Elo2p and Elo3p, suggesting that the elongating proteins are organized in a complex. Tsc13p localizes to the endoplasmic reticulum and is highly enriched in a novel structure marking nuclear-vacuolar junctions.

Tsc3p is an 80-amino acid protein associated with serine palmitoyltransferase and required for optimal enzyme activity.

Serine palmitoyltransferase catalyzes the first step of sphingolipid synthesis, condensation of serine and palmitoyl CoA to form the long chain base 3-ketosphinganine. The LCB1/TSC2 and LCB2/TSC1 genes encode homologous proteins of the alpha-oxoamine synthase family required for serine palmitoyltransferase activity. The other alpha-oxoamine synthases are soluble homodimers, but serine palmitoyltransferase is a membrane-associated enzyme composed of at least two subunits, Lcb1p and Lcb2p. Here, we report the characterization of a third gene, TSC3, required for optimal 3-ketosphinganine synthesis in Saccharomyces cerevisiae. S. cerevisiae cells lacking the TSC3 gene have a temperature-sensitive lethal phenotype that is reversed by supplying 3-ketosphinganine, dihydrosphingosine, or phytosphingosine in the growth medium. The tsc3 mutant cells have severely reduced serine palmitoyltransferase activity. The TSC3 gene encodes a novel 80-amino acid protein with a predominantly hydrophilic amino-terminal half and a hydrophobic carboxyl terminus that is membrane-associated. Tsc3p coimmunoprecipitates with Lcb1p and/or Lcb2p but does not bind as tightly as Lcb1p and Lcb2p bind to each other. Lcb1p and Lcb2p remain tightly associated with each other and localize to the membrane in cells lacking Tsc3p. However, Lcb2p is unstable in cells lacking Lcb1p and vice versa.

The Saccharomyces cerevisiae TSC10/YBR265w gene encoding 3-ketosphinganine reductase is identified in a screen for temperature-sensitive suppressors of the Ca2+-sensitive csg2Delta mutant.

Saccharomyces cerevisiae csg2Delta mutants accumulate the sphingolipid inositolphosphorylceramide, which renders the cells Ca2+-sensitive. Temperature-sensitive mutations that suppress the Ca2+ sensitivity of csg2Delta mutants were isolated and characterized to identify genes that encode sphingolipid synthesis enzymes. These temperature-sensitive csg2Delta suppressors (tsc) fall into 15 complementation groups. The TSC10/YBR265w gene was found to encode 3-ketosphinganine reductase, the enzyme that catalyzes the second step in the synthesis of phytosphingosine, the long chain base found in yeast sphingolipids. 3-Ketosphinganine reductase (Tsc10p) is essential for growth in the absence of exogenous dihydrosphingosine or phytosphingosine. Tsc10p is a member of the short chain dehydrogenase/reductase protein family. The tsc10 mutants accumulate 3-ketosphinganine and microsomal membranes isolated from tsc10 mutants have low 3-ketosphinganine reductase activity. His6-tagged Tsc10p was expressed in Escherichia coli and isolated by nickel-nitrilotriacetic acid column chromatography. The recombinant protein catalyzes the NADPH-dependent reduction of 3-ketosphinganine. These data indicate that Tsc10p is necessary and sufficient for catalyzing the NADPH-dependent reduction of 3-ketosphinganine to dihydrosphingosine.

Hydroxylation of Saccharomyces cerevisiae ceramides requires Sur2p and Scs7p.

The Saccharomyces cerevisiae SCS7 and SUR2 genes are members of a gene family that encodes enzymes that desaturate or hydroxylate lipids. Sur2p is required for the hydroxylation of C-4 of the sphingoid moiety of ceramide, and Scs7p is required for the hydroxylation of the very long chain fatty acid. Neither SCS7 nor SUR2 are essential for growth, and lack of the Scs7p- or Sur2p-dependent hydroxylation does not prevent the synthesis of mannosyldiinositolphosphorylceramide, the mature sphingolipid found in yeast. Deletion of either gene suppresses the Ca2+-sensitive phenotype of csg2Delta mutants, which arises from overaccumulation of inositolphosphorylceramide due to a defect in sphingolipid mannosylation. Characterization of scs7 and sur2 mutants is expected to provide insight into the function of ceramide hydroxylation.

Activation of divalent cation influx into S. cerevisiae cells by hypotonic downshift.

Subjecting Saccharomyces cerevisiae cells to a hypotonic downshift by transferring cells form YPD medium containing 0.8 M sorbitol to YPD medium without sorbitol induces a transient rapid influx of Ca2+ and other divalent cations into the cell. For cells grown in YPD at 37 degrees C, this hypotonic downshift increases Ca2+ accumulation 6.7-fold. Hypotonic downshift-induced Ca2+ accumulation and steady-state Ca2+ accumulation in isotonic YPD medium are differentially affected by dodecylamine and Mg2+. The Ca(2+)-influx pathway responsible for hypotonic-induced Ca2+ influx may account for about 10-35% of Ca2+ accumulation by cells growing in YPD. Ca2+ influx is not required for cells to survive a hypotonic downshift. Hypotonic downshift greatly reduces the ability of S. cerevisiae cells to survive a 5-min exposure to 10 mM Cd2+ suggesting that mutants resistant to acute Cd2+ exposure may help identify genes required for hypotonic downshift-induced divalent cation influx.

SUR1 (CSG1/BCL21), a gene necessary for growth of Saccharomyces cerevisiae in the presence of high Ca2+ concentrations at 37 degrees C, is required for mannosylation of inositolphosphorylceramide.

Saccharomyces cerevisiae cells require two genes, CSG1/SUR1 and CSG2, for growth in 50 mM Ca2+, but not 50 mM Sr2+. CSG2 was previously shown to be required for the mannosylation of inositolphosphorylceramide (IPC) to form mannosylinositolphosphorylceramide (MIPC). Here we demonstrate that SUR1/CSG1 is both genetically and biochemically related to CSG2. Like CSG2, SUR1/CSG1 is required for IPC mannosylation. A 93-amino acid stretch of Csg1p shows 29% identity with the alpha-1, 6-mannosyltransferase encoded by OCH1. The SUR1/CSG1 gene is a dose-dependent suppressor of the Ca(2+)-sensitive phenotype of the csg2 mutant, but overexpression of CSG2 does not suppress the Ca2+ sensitivity of the csg1 mutant. The csg1 and csg2 mutants display normal growth in YPD, indicating that mannosylation of sphingolipids is not essential. Increased osmolarity of the growth medium increases the Ca2+ tolerance of csg1 and csg2 mutant cells, suggesting that altered cell wall synthesis causes Ca(2+)-induced death. Hydroxylation of IPC-C to form IPC-D requires CCC2, a gene encoding an intracellular Cu2+ transporter. Increased expression of CCC2 or increased Cu2+ concentration in the growth medium enhances the Ca2+ tolerance of csg1 mutants, suggesting that accumulation of IPC-C renders csg1 cells Ca2+ sensitive.

Regulation of cellular Ca2+ by yeast vacuoles.

The role of vacuolar Ca2+ transport systems in regulating cellular Ca2+ was investigated by measuring the vacuolar Ca2+ transport rate, the free energy available to drive vacuolar Ca2+ transport, the ability of the vacuole to buffer lumenal Ca2+, and the vacuolar Ca2+ efflux rate. The magnitude of the Ca2+ gradient generated by the vacuolar H+ gradient best supports a 1 Ca2+:2 H+ coupling ratio for the vacuolar Ca2+/H+ exchanger. This coupling ratio along with a cytosolic Ca2+ concentration of 125 nM would give a vacuolar free Ca2+ concentration of approximately 30 microM. The total vacuolar Ca2+ concentration is approximately 2 mM due to Ca2+ binding to vacuolar polyphosphate. The Ca2+ efflux rate from the vacuole is less than the growth rate indicating that the steady-state Ca2+ loading level of the vacuole is dependent mainly on the Ca2+ transport rate and the rate that vacuolar Ca2+ is diluted by growth. Based on the kinetic parameters of vacuolar Ca2+ accumulation in vitro, the maximum rate of Ca2+ accumulation in vivo is expected to be approximately 0.2 nmol of Ca2+ min-1 mg protein-1, a rate that is similar to the cellular Ca2+ accumulation rate. The cytosolic Ca2+ concentration increases from 0.1 microM to 1-2 microM as the extracellular Ca2+ concentration is raised from 0.3 mM to 50 mM. The rise in cytosolic Ca2+ concentration increases cellular Ca2+ from 10 to 300 nmol Ca2+/mg by increasing the rate of vacuolar Ca2+ accumulation but does not significantly alter the cellular growth rate.

A novel protein, CSG2p, is required for Ca2+ regulation in Saccharomyces cerevisiae.

Nineteen mutants that lost the ability to grow in 100 mM Ca2+ (but remained insensitive to 50 mM Sr2+) were identified in a screen of approximately 60,000 mutagenized yeast colonies. Cells carrying mutations in the CSG2 gene grow normally in low Ca2+ medium but have decreased growth rates when the Ca2+ concentration is above 10 mM. The csg2 mutant cells accumulate much higher levels of Ca2+ in a compartment that is exchangeable with extracellular Ca2+ but the nonexchangeable Ca2+ pool which predominates in wild-type cells is not influenced. Sr2+ influx is not increased in the csg2 mutant cells. Mg2+ decreases the amount of Ca2+ in the non-exchangeable pool without influencing the csg2-induced exchangeable Ca2+ pool. The data indicate that the csg2 mutation causes a selective increase in Ca2+ accumulation into a pool which is distinct from the vacuolar pool. The CSG2 protein consists of 410 amino acids, contains nine putative transmembrane segments, four potential sites for N-linked glycosylation, and a sequence with homology to the EF-hand Ca(2+)-binding site.

Phosphate, nitrendipine and valinomycin increase the Ca2+/ATP coupling ratio of rat skeletal muscle sarcoplasmic reticulum Ca(2+)-ATPase.

Nitrendipine and valinomycin act synergistically to stimulate ATP-dependent Ca2+ accumulation by rat skeletal muscle sarcoplasmic reticulum vesicles 3-fold. The stimulation is not caused by activation of the Ca(2+)-ATPase or by inhibition of the sarcoplasmic reticulum Ca2+ channel, but is due to an increased efficiency of transport by Ca(2+)-loaded vesicles. At low Ca2+ concentrations, nitrendipine+valinomycin inhibits Ca2+ uptake by increasing the Ca2+ KM but does not effect equilibrium Ca2+ binding to the Ca(2+)-ATPase (Kd = 0.75 microM). In the presence of 50 mM phosphate, nitrendipine+valinomycin increases the steady-state coupling ratio (Ca2+ accumulated per ATP hydrolyzed) from 0.6 to 1.9 by decreasing the rate of ATP hydrolysis by 72%, while reducing the Ca2+ accumulation rate by only 13%. The rates of both passive and Ca(2+)-ATPase-mediated Ca2+ release are reduced by nitrendipine+valinomycin. The data indicate that nitrendipine and valinomycin act directly on the Ca(2+)-ATPase to decrease the ATP hydrolysis rate, increase the Ca2+ KM, decrease Ca2+ efflux, and increase the Ca2+/ATP coupling ratio of Ca(2+)-loaded vesicles.

Effect of the general anesthetic halothane on the activity of the transverse tubule Ca(2+)-activated K+ channel.

The effect of the general anesthetic halothane on the activity of the rat skeletal muscle Ca(2+)-activated K+ channel in planar lipid bilayers was investigated. Halothane concentrations in the clinical range (1.0-0.2 mM) alter the regulation of the channel by both Ca2+ and membrane potential. At Ca2+ concentrations between 10 and 250 microM and membrane potentials between 0 and -30 mV, halothane significantly decreases the open state probability without changing the channel conductance. The results demonstrate that halothane can act directly on the Ca(2+)-activated K+ channel or its lipid environment to alter the channel gating kinetics.

Activation and inhibition of the sarcoplasmic reticulum Ca2+ channel by the polycationic dyes Hoechst 33342 and Hoechst 33258.

The polycationic dyes, Hoechst 33342 (Bisbenzimide,2'-(4-ethoxyphenyl)-5-(4-methyl-1-piperazinyl)-2,5'-bi-1H- benzimidazole) and Hoechst 33258 (Bisbenzimide,2'-(4-hydroxyphenyl)-5-(4-methyl-1-piperazinyl)- 2,5'-bi-1H-benzimidazole) alter the activity of the sarcoplasmic reticulum Ca2+ channel. Although they act competitively, Hoechst 33342 decreases, while Hoechst 33258 increases, the rate of channel-mediated Ca2+ efflux from junctional sarcoplasmic reticulum vesicles. Unlike other cationic sarcoplasmic reticulum Ca2+ channel antagonists, Hoechst 33342 blocks the ryanodine-activated Ca2+ channel. Both Hoechst 33342 and Hoechst 33258 inhibit the channel incorporated into the planar lipid bilayer. Since the only structural difference between the two dyes is that the agonist Hoechst 33258 has a hydroxy group where the antagonist Hoechst 33342 has an ethoxy group, it is possible that the more hydrophobic, bulky ethoxy group blocks Ca2+ movement through the channel, whereas the hydroxy group only reduces the rate of Ca2+ movement.

Activation of Ca2+ release from sarcoplasmic reticulum vesicles by 4-alkylphenols.

4-Alkylphenols induce Ca2+ release from junctional (terminal cisternae) sarcoplasmic reticulum vesicles, but not from nonjunctional sarcoplasmic reticulum vesicles. The 4-alkylphenol concentration required to induce Ca2+ release decreases about threefold for every methylene carbon increase in the alkyl chain length, indicating that the Ca(2+)-releasing potency of 4-alkylphenols is related to their ability to partition into the membrane. The rate and amount of Ca2+ release induced by relatively low 4-octylphenol concentrations (25 nmol/mg protein) are altered by the sarcoplasmic reticulum Ca2+ channel activators, Ca2+ and ATP, and the Ca2+ channel inhibitors, Mg2+ and ruthenium red. Ca2+ release induced by 250 nmol 4-octylphenol/mg protein is much less influenced by Ca2+ channel activators and inhibitors; however, even at this high 4-octylphenol concentration, Ca2+ release is not induced from nonjunctional sarcoplasmic reticulum vesicles. The data indicate that 4-alkylphenols induce Ca2+ release by activating the sarcoplasmic reticulum Ca2+ channel.

Mechanism of magainin 2a induced permeabilization of phospholipid vesicles.

The magainins, peptide antibiotics secreted by the frog Xenopus laevis, have previously been shown to permeabilize phospholipid vesicles. To elucidate the mechanism of permeabilization, we have conducted detailed kinetic studies of magainin 2 amide (mgn2a)-induced release of 6-carboxyfluorescein from vesicles of phosphatidylserine. The results show that dye release occurs in (at least) two stages--an initial rapid phase, with t1/2 approximately 3 s, followed by a much slower phase that approaches zero leakage rate before all the dye is released. Light-scattering studies showed that mgn2a does not cause gross changes in vesicle structure. The peptide was found to rapidly equilibrate between vesicles; this was demonstrated by determining a binding isotherm for the peptide-lipid interaction, and by showing that addition of unloaded vesicles rapidly quenches peptide-induced leakage from loaded vesicles. Transient dye release in the presence of an equilibrating peptide can be explained in two ways: (1) the peptide exists only transiently in an active form; (2) the vesicles are only transiently leaky. Preincubation of mgn2a at assay concentrations in buffer alone or with unloaded vesicles did not inactivate the peptide. Therefore, rapid leakage is probably due to transient destabilization of the vesicle upon addition of mgn2a.

Raman spectroscopy of synthetic antimicrobial frog peptides magainin 2a and PGLa.

Magainin and PGLa are 23- and 21-residue peptides isolated from the skin of the African clawed frog Xenopus laevis. They protect the frog from infection and exhibit a broad-spectrum antimicrobial activity in vitro. The mechanism of this activity involves the interaction of magainin with microbial membranes. We have measured the secondary structure and membrane-perturbing ability of these peptides to obtain information about this mechanism. Our results show that mgn2a forms a helix with an average length of less than 20 A upon binding to liposomes. At high concentrations (50 mg/mL) mgn2a spontaneously solubilizes phosphatidylcholine liposomes at temperatures above the gel-liquid-crystalline phase transition. Mgn2a appears to bind to the surface of liposomes made of negatively charged lipids without spontaneously penetrating the bilayer. Finally, mgn2a and PGLa interact together with liposomes in a synergistic way that enhances the helix content of one or both of the peptides and allows the peptides to more easily penetrate the bilayer. PGLa mixed with a small nonperturbing amount of magainin 2 amide is 25-43 times as potent as PGLa alone at inducing the release of carboxyfluorescein from liposomes. The results suggest that the mechanism of antimicrobial activity does not involve a channel formed by transmembrane helical peptides.

Comparison of the rat microsomal Mg-ATPase of various tissues.

The microsomal Mg-ATPase from various rat tissues was compared. After fractionating the microsomal vesicles by sucrose gradient centrifugation, the highest specific activity of the Mg-ATPase was found in the low-density vesicles which contained plasma membrane. A large fraction (25-90%) of the microsomal Ca-independent Mg-ATPase found in each tissue had the following properties: (1) the Km for ATP was 0.2 mM; (2) the rate of ATP hydrolysis by the Mg-ATPase was nonlinear due to an ATP-stimulated inactivation of the enzyme; (3) wheat germ agglutinin, concanavalin A, glutaraldehyde, and antiserum prevented inactivation induced by ATP or AdoPP[NH]P; (4) detergents at relatively low detergent:protein ratios increased the rate of inactivation with little change in the initial rate of ATP hydrolysis; (5) the Mg-ATPase was inactivated by irradiation in the presence of 8-azido ATP. (6) in addition to ATP, the Mg-ATPase was able to hydrolyze CTP, GTP, UTP, ITP, and GTP but was unable to hydrolyze any of the 10 nonnucleotide phosphocompounds which were tested; (7) the bivalent cation requirement of the Mg-ATPase could be provided by Mg2+, Ca2+, Mn2+, Zn2+, or Co2+ but the enzyme was inactive in the presence of Cu2+, Sr2+, Ba2+, or Be2+; (8) the Mg-ATPase activity was not altered by ionophores or inhibitors of the Na,K-ATPase, the Ca,Mg-ATPase or the mitochondrial F1ATPase. These data suggest that a major portion of the microsomal, basal Mg-ATPase activity is due to one unique enzyme found in most if not all tissues.

Effect of halothane on Ca2+-induced Ca2+ release from sarcoplasmic reticulum vesicles isolated from rat skeletal muscle.

Halothane induces the release of Ca2+ from a subpopulation of sarcoplasmic reticulum vesicles that are derived from the terminal cisternae of rat skeletal muscle. Halothane-induced Ca2+ release appears to be an enhancement of Ca2+-induced Ca2+ release. The low-density sarcoplasmic reticulum vesicles which are believed to be derived from nonjunctional sarcoplasmic reticulum lack the capability of both Ca2+-induced and halothane-induced Ca2+ release. Ca2+ release from terminal cisternae vesicles induced by halothane is inhibited by Ruthenium red and Mg2+, and require ATP (or an ATP analogue), KCl (or similar salt) and extravesicular Ca2+. Ca2+-induced Ca2+ release has similar characteristics.

Characterization of the membrane bound Mg2+-ATPase of rat skeletal muscle.

A procedure was developed to isolate a membrane fraction of rat skeletal muscle which contains a highly active Mg2+-ATPase (5-25 mumol Pi/mg min). The rate of ATP hydrolysis by the Mg2+-ATPase was nonlinear but decayed exponentially (first-order rate constant greater than or equal to 0.2 s-1 at 37 degrees C). The rapid decline in the ATPase activity depended on the presence of ATP or its nonhydrolyzable analog 5'-adenylyl imidodiphosphate (AdoPP[NH]P). Once inactivated, removal of ATP from the medium did not immediately restore the original activity. ATP- or AdoPP[NH]P-dependent inactivation could be blocked by concanavalin A, wheat germ agglutinin or rabbit antiserum against the membrane. Additions of these proteins after ATP addition prevented further inactivation but did not restore the original activity. Low concentrations of ionic and nonionic detergents increased the rate of ATP-dependent inactivation. Higher concentrations of detergents, which solubilize the membrane completely, inactivated the Mg2+-ATPase. Cross-linking the membrane components with glutaraldehyde prevented ATP-dependent inactivation and decreased the sensitivity of the Mg2+-ATPase to detergents. It is proposed that the regulation of the Mg2+-ATPase by ATP requires the mobility of proteins within the membrane. Cross-linking the membrane proteins with lectins, antiserum or glutaraldehyde prevents inactivation; increasing the mobility with detergents accelerates ATP-dependent inactivation.