Human embryonic stem cell derived astrocytes mediate non-cell-autonomous neuroprotection through endogenous and drug-induced mechanisms.

The glial environment is an important determinant of neuronal health in experimental models of neurodegeneration. Specifically, astrocytes have been shown, dependent on context, to be both injurious and protective. Human pluripotent stem cells offer a powerful new system to improve our understanding of the mechanisms underlying astrocyte-mediated neuroprotection. Here, we describe a human embryonic stem cell (HESC)-based system to assess the scope and mechanism of human astrocyte-mediated neuroprotection. We first report the generation of enriched and functional HESC-derived astrocytes, by combining BMP-mediated Smad and LIF-mediated JAK-STAT signalling. These astrocytes promote the protection of HESC-derived neurons against oxidative insults. Moreover, their neuroprotective capacity can be greatly enhanced by treatment with the nuclear factor-erythroid 2-related factor 2 (Nrf2)-activating triterpenoid 1[2-Cyano-3,12-dioxool-eana-1,9(11)-dien-28-oyl] trifluoroethylamide (CDDO(TFEA)). Activation of the transcription factor Nrf2 in human astrocytes by CDDO(TFEA) treatment induced expression of the glutamate-cysteine ligase (GCL) catalytic subunit, leading to enhanced GCL activity and glutathione production, and strong neuroprotection against H(2)O(2). This enhanced neuroprotection was found to be dependent on astrocytic GCL activity, unlike the basal neuroprotection afforded by untreated astrocytes. Direct treatment of HESC-derived neurons with CDDO(TFEA) elicited no induction of Nrf2 target genes, nor any neuroprotection. Thus, human astrocytes can mediate neuroprotection through glutathione-dependent and glutathione-independent mechanisms, and represent a therapeutic target for human disorders associated with neuronal oxidative stress.

Relationships between lipolysis induced by various lipolytic agents and hormone-sensitive lipase in rat fat cells.

Norepinephrine induced lipolysis in rat fat cells, in vitro, in a time- and concentration-dependent manner, without concomitantly increasing hormone-sensitive lipase (HSL) activity. It also induced, time and concentration dependently, HSL translocation from the cytosol to the lipid droplets in fat cells. Isoproterenol, forskolin, dibutyryl cyclic AMP, and theophylline also induced lipolysis in fat cells, but did not stimulate HSL activity. These agents also induced HSL translocation from the cytosol to the lipid droplets in fat cells: about 80% to 90% of all HSL was located in lipid droplets after incubation for 1 h. These results suggest that the critical event in lipolytic activation of fat cells induced by lipolytic agents is not an increase in the catalytic activity of HSL but translocation of HSL to its substrate on the surfaces of lipid droplets in fat cells.-Morimoto, C., K. Kameda, T. Tsujita, and H. Okuda. Relationships between lipolysis induced by various lipolytic agents and hormone-sensitive lipase in rat fat cells. J. Lipid Res. 2001. 42: 120;-127.

Lack of association between the hKCa3 gene and Japanese schizophrenia patients.

Several researchers have suggested an association between large numbers of CAG repeats in the hKCa3 gene and schizophrenia. However, these reports remain inconclusive and require further investigation. We tried to replicate these results in 112 Japanese schizophrenia patients and 102 control subjects of highly matched age and sex by applying an allele dichotomization model. No association was found. The overall distributions of allele frequencies were not significantly different between schizophrenic patients and normal control subjects. In addition, we tested the association between the size of the CAG repeats and the scores on three dimensions (positive and negative symptoms, and disorganization), but no significant results were obtained. Our results do not support the involvement of the hKCa3 gene in schizophrenia, at least in the Japanese population.

Reductive dimerization of alkylidenemalonates using samarium(II) diiodide and 1H-NMR behavior of the dimers, 2,3-diaryl-1,1,4,4-butanetetracarboxylates.

Alkylidenemalonates were readily dimerized in the presence of SmI2 to give 2,3-disubstituted 1,1,4,4-butanetetracarboxylates as mixtures of meso and racemic isomers in moderate to good yields. The structure of the less polar isomer of tetraethyl 2,3-diphenyl-1,1,4,4-butanetetracarboxylate was determined by X-ray crystallographic analysis to be the meso form. Characteristic 1H-NMR behavior of the meso and racemic isomers is also discussed.

Inhibitory effects of chondroitin sulfate prepared from salmon nasal cartilage on fat storage in mice fed a high-fat diet.

OBJECTIVE: Chondroitin sulfate is an acidic polymer consisting of repeating D-glucuronic acid and D-N-acetylgalactosamine units, and the N-acetylgalactosamine is substituted with the sulfate at either the 4' or 6' position, with approximately one sulfate being present per disaccharide unit. The present study assessed the effects of chondroitin sulfate on the activity of pancreatic lipase and lipid uptake into brush border membrane vesicles of the rat small intestine in vitro, and on the degree of fat storage induced in mice by the oral administration of a high-fat diet for 8 weeks. DESIGN AND MEASUREMENTS: Experiments were carried out to clarify whether or not chondroitin sulfate inhibited pancreatic lipase activity in assay systems using triolein emulsified with phosphatidylcholine or gum arabic. In addition, the effects of chondroitin sulfate on lipid absorption by brush border membrane vesicles were examined. Moreover, mice were fed a high-fat diet and treated with chondroitin sulfate for 8 weeks. RESULTS: Chondroitin sulfate dose-dependently inhibited the pancreatic lipase activity in an assay system using triolein emulsified with phosphatidylcholine. In addition, chondroitin sulfate inhibited the palmitic acid uptake into the brush border membrane vesicles of the rat jejunum. Chondroitin sulfate caused the reduction of body weight and parametrial adipose tissue weight, and prevention of fatty liver and hyperlipidemia in mice fed a high-fat diet. CONCLUSION: The reduction of fat storage and the antihyperlipidemic action of chondroitin sulfate might be due to the inhibition of small intestinal absorption of dietary fat through the inhibition of pancreatic lipase activity and fatty acid uptake through brush border membrane.

Substrate-dependent lipolysis induced by isoproterenol.

The relationship between isoproterenol-induced lipolysis and the phosphorylation of perilipin and hormone-sensitive lipase (HSL) was examined using cell-free systems consisting of lipid droplets isolated from rat fat cells and HSL, and/or trioleoylglycerol emulsified with gum arabic and HSL. Isoproterenol was found to stimulate lipolysis in the cell-free system with the lipid droplets without an increase in the phosphorylation of either perilipin or HSL. On the other hand, no stimulation of lipolysis was found in the cell-free system containing lipid droplets despite increases in the phosphorylation of perilipin and HSL. In the cell-free system consisting of trioleoylglycerol emulsified with gum arabic and HSL, neither isoproterenol nor increases in the phosphorylation of perilipin and HSL accelerated lipolysis. These results suggest that isoproterenol-induced lipolysis may not be mediated through the phosphorylation of perilipin and HSL, and may rather be dependent on the substrate of HSL.

Molecular characterization of the S-adenosyl-L-methionine:3'-hydroxy-N-methylcoclaurine 4'-O-methyltransferase involved in isoquinoline alkaloid biosynthesis in Coptis japonica.

S-adenosyl-L-methionine:3'-hydroxy-N-methylcoclaurine 4'-O-methyltransferase (4'-OMT) catalyzes the conversion of 3'-hydroxy-N-methylcoclaurine to reticuline, an important intermediate in synthesizing isoquinoline alkaloids. In an earlier step in the biosynthetic pathway to reticuline, another O-methyltransferase, S-adenosyl-L-methionine:norcoclaurine 6-O-methyltransferase (6-OMT), catalyzes methylation of the 6-hydroxyl group of norcoclaurine. We isolated two kinds of cDNA clones that correspond to the internal amino acid sequences of a 6-OMT/4'-OMT preparation from cultured Coptis japonica cells. Heterologously expressed proteins had 6-OMT or 4'-OMT activities, indicative that each cDNA encodes a different enzyme. 4'-OMT was purified using recombinant protein, and its enzymological properties were characterized. It had enzymological characteristics similar to those of 6-OMT; the active enzyme was the dimer of the subunit, no divalent cations were required for activity, and there was inhibition by Fe(2+), Cu(2+), Co(2+), Zn(2+), or Ni(2+), but none by the SH reagent. 4'-OMT clearly had different substrate specificity. It methylated (R,S)-6-O-methylnorlaudanosoline, as well as (R, S)-laudanosoline and (R,S)-norlaudanosoline. Laudanosoline, an N-methylated substrate, was a much better substrate for 4'-OMT than norlaudanosoline. 6-OMT methylated norlaudanosoline and laudanosoline equally. Further characterization of the substrate saturation and product inhibition kinetics indicated that 4'-OMT follows an ordered Bi Bi mechanism, whereas 6-OMT follows a Ping-Pong Bi Bi mechanism. The molecular evolution of these two related O-methyltransferases is discussed.

Association of carboxyl esterase with facilitative glucose transporter isoform 4 (GLUT4) intracellular compartments in rat adipocytes and its possible role in insulin-induced GLUT4 recruitment.

Facilitative glucose transporter isoform 4 (GLUT4) in rat adipocytes is largely sequestered in intracellular sites, and insulin recruits GLUT4 from these sites to the cell surface. The process is known to involve multiple intracellular compartments and associated proteins, many of which are yet to be identified. Recently, we purified three distinct insulin-sensitive intracellular GLUT4 compartments (G4T(L), G4H, and G4L) in rat adipocytes and unraveled several new resident proteins in these compartments. Here, we describe one of them, a 62-kDa protein, purified and identified as rat adipose tissue carboxyl esterase (p62/CE) by matrix-assisted laser desorption/ionization time of flight mass spectroscopy, reverse transcription-polymerase chain reaction, gene cloning, and immunological and enzymatic activity measurements. p62/CE in rat adipocytes was 80% cytosolic and 20% microsome-associated. It was found in all of the three insulin-sensitive intracellular GLUT4 compartments, and particularly enriched in G4T(L,) a compartment thought to represent GLUT4 endocytic vesicles. Significantly, an antibody against p62/CE introduced into rat adipocytes completely abolished the insulin-induced GLUT4 recruitment to the plasma membrane in host cells without affecting the basal GLUT4 distribution. Together, these findings suggest that p62/CE plays a key role in insulin-induced GLUT4 recruitment in rat adipocytes, probably by hydrolyzing acylglycerols or acyl-CoA esters to the respective free acids that are required for GLUT4 transport vesicle budding and/or fusion.

Mechanism of the stimulatory action of okadaic acid on lipolysis in rat fat cells.

Okadaic acid was found to induce concentration- and time-dependent lipolysis in rat fat cells in the absence of lipolytic hormones, but it did not significantly increase the total hormone-sensitive lipase (HSL) activity in these fat cells, the activity of HSL extracted from fat layer and that of HSL in the supernatant of homogenized fat cells. Western blotting of fat cell homogenate fractions with an antiserum raised against synthetic peptide derived from rat HSL showed that HSL protein shifted from the supernatant to the fat layer in response to okadaic acid, which increased the HSL protein content on the fat layer and concomitantly reduced that of the supernatant, concentration- and time-dependently. Sonication of the fat cells abolished their responsiveness to okadaic acid. The lipolytic action of okadaic acid was examined and its site was identified using a cell-free system comprising lipid droplets isolated from rat fat cells and HSL. Okadaic acid induced lipolysis in this cell-free system and sonication of the lipid droplets caused disappearance of lipolytic action of okadaic acid. Okadaic acid failed to stimulate lipolysis in a cell-free system comprising HSL and artificial lipid droplets (trioleoylglycerol emulsified with gum arabic) instead of lipid droplets isolated from rat fat cells. These results suggest that okadaic acid does not increase the catalytic activity of HSL but induces translocation of HSL to the lipid droplets isolated from rat fat cells. The site of the lipolytic action of okadaic acid in relation to the interaction between HSL and lipid droplet is discussed.

Wax ester-synthesizing activity of lipases.

The synthesis/hydrolysis of wax esters was studied in an aqueous solution using purified rat pancreatic lipase, porcine pancreatic carboxylester lipase, and Pseudomonas fluorescens lipase. The equilibrium between wax ester synthesis and hydrolysis favored ester formation at neutral pH. The synthesizing activities were measured using free fatty acid or triacylglycerol as the acyl donor and an equimolar amount of long-chain alcohol as the acyl acceptor. When oleic acid and hexadecanol emulsified with gum arabic were incubated with these lipases, wax ester was synthesized, in a dose- and time-dependent manner, and the apparent equilibrium ratio of palmityl oleate/free oleic acid was about 0.9/0.1. These lipases catalyzed the hydrolysis of palmityl oleate emulsified with gum arabic, and the apparent equilibrium ratio of palmityl oleate/free oleic acid was also about 0.9/0.1. The apparent equilibrium ratio of wax ester/free fatty acid catalyzed by lipase depended on incubation pH and fatty alcohol chain length. When equimolar amounts of trioleoylglycerol and fatty acyl alcohol were incubated with pancreatic lipase, carboxylester lipase, or P. fluorescens lipase, wax esters were synthesized dose-dependently. These results suggest that lipases can catalyze the synthesis of wax esters from free fatty acids or through degradation of triacylglycerol in an aqueous medium.

Paroxysmal kinesigenic choreoathetosis locus maps to chromosome 16p11.2-q12.1.

Paroxysmal kinesigenic choreoathetosis (PKC), the most frequently described type of paroxysmal dyskinesia, is characterized by recurrent, brief attacks of involuntary movements induced by sudden voluntary movements. Some patients with PKC have a history of infantile afebrile convulsions with a favorable outcome. To localize the PKC locus, we performed genomewide linkage analysis on eight Japanese families with autosomal dominant PKC. Two-point linkage analysis provided a maximum LOD score of 10.27 (recombination fraction [theta] =.00; penetrance [p] =.7) at marker D16S3081, and a maximum multipoint LOD score for a subset of markers was calculated to be 11.51 (p = 0.8) at D16S3080. Haplotype analysis defined the disease locus within a region of approximately 12.4 cM between D16S3093 and D16S416. P1-derived artificial chromosome clones containing loci D16S3093 and D16S416 were mapped, by use of FISH, to 16p11.2 and 16q12.1, respectively. Thus, in the eight families studied, the chromosomal localization of the PKC critical region (PKCR) is 16p11.2-q12.1. The PKCR overlaps with a region responsible for "infantile convulsions and paroxysmal choreoathetosis" (MIM 602066), a recently recognized clinical entity with benign infantile convulsions and nonkinesigenic paroxysmal dyskinesias.

Fatty acid alcohol ester-synthesizing activity of lipoprotein lipase.

The fatty acid alcohol ester-synthesizing activity of lipoprotein lipase (LPL) was characterized using bovine milk LPL. Synthesizing activities were determined in an aqueous medium using oleic acid or trioleylglycerol as the acyl donor and equimolar amounts of long-chain alcohols as the acyl acceptor. When oleic acid and hexadecanol emulsified with gum arabic were incubated with LPL, palmityl oleate was synthesized, in a time- and dose-dependent manner. Apo-very low density lipoprotein (apoVLDL) stimulated LPL-catalyzed palmityl oleate synthesis. The apparent equilibrium ratio of fatty acid alcohol ester/oleic acid was estimated using a high concentration of LPL and a long (20 h) incubation period. The equilibrium ratio was affected by the incubation pH and the alcohol chain length. When the incubation pH was below pH 7.0 and long chain fatty acyl alcohols were used as substrates, the fatty acid alcohol ester/free fatty acid equilibrium ratio favored ester formation, with an apparent equilibrium ratio of fatty acid alcohol ester/fatty acid of about 0.9/0.1. The equilibrium ratio decreased sharply at alkaline pH (above pH 8.0). The ratio also decreased when fatty alcohols with acyl chains shorter than dodecanol were used. When a trioleoylglycerol/fatty acyl alcohol emulsion was incubated with LPL, fatty acid alcohol esters were synthesized in a dose- and time-dependent fashion. Fatty acid alcohol esters were easily synthesized from trioleoylglycerol when fatty alcohols with acyl chains longer than dodecanol were used, but synthesis was decreased with fatty alcohols with acyl chain lengths shorter than decanol, and little synthesizing activity was detected with shorter-chain fatty alcohols such as butanol or ethanol.

Screening of lipase inhibitors from marine algae.

The possible presence of an inhibitor of pancreatic lipase (triacylglycerol acylhydrolase, EC 3.1.1.3) was screened in 54 marine algae. An active inhibitor, caulerpenyne, was purified from an extract of Caulerpa taxifolia, using ethyl acetate extraction, followed by successive chromatographies on ODS and silica gel columns. The purified inhibitor was identified by thin-layer chromatography, infrared and nuclear magnetic resonance spectroscopy. Caulerpenyne competitively inhibited lipase activities using emulsified triolein and dispersed 4-methylumbelliferyl oleate (4-MU oleate) as substrates. The concentrations producing 50% inhibition against triolein and 4-MU oleate hydrolysis were 2 mM and 13 microM, respectively. In vivo, oral administration of corn oil with or without caulerpenyne to rats demonstrated a reduced and delayed peak plasma triacylglycerol concentration with caulerpenyne.

Relationship between hormone-sensitive lipolysis and lipase activity in rat fat cells.

An assay for total hormone-sensitive lipase (HSL) in rat fat cells was devised in which fat-associated HSL was solubilized with ether, and triolein or cholesteryloleate was used as substrate. Norepinephrine (NE) caused marked release of glycerol from fat cells but did not activate HSL as estimated using triolein or cholesteryloleate as substrate. Propranolol, a beta-blocker, inhibited NE-induced lipolysis in fat cells without a concomitant reduction in HSL activity. The antilipolytic action of insulin on NE-induced lipolysis could not be explained by a decrease in HSL activity. Neither ACTH-induced lipolysis in fat cells nor its inhibition by insulin was accompanied by matching fluctuations in HSL activity. These results indicate that neither NE and ACTH-induced lipolysis in fat cells, nor the antilipolytic actions of propranolol and insulin, involve fluctuations in HSL activity.

Antilipolytic actions of insulin on basal and hormone-induced lipolysis in rat adipocytes.

The levels of insulin, free fatty acids (FFA), and triglycerides in rat sera increase with age. The increase in serum FFA levels accompanied the stimulation of basal lipolysis (i.e., lipolysis in the absence of lipolytic agents) in fat cells and enlargement of the diameter of the cells. An overnight fast resulted in a significant increase in basal lipolysis in fat cells from 6- and 8-week-old rats. Although insulin inhibited lipolysis induced by norepinephrine and ACTH at a concentration of 10(-10) M, it failed to inhibit basal lipolysis even at a concentration of 10(-6) M. Propranolol, another antilipolytic agent like insulin, also did not affect basal lipolysis. Insulin did not inhibit the accelerated basal lipolysis in enlarged fat cells, fasted fat cells, and sonicated cells. These results indicate that insulin inhibits only the lipolysis induced by lipolytic agents such as norepinephrine and ACTH but not the basal lipolysis found in the absence of lipolytic agents. The possibility that free fatty acids produced by enlarged fat cells initiate insulin resistance and diabetes mellitus, is discussed.

Alkaline lipase from brain: is it the same enzyme as pancreatic lipase from pancreas?

A new alkaline lipase was detected in rat brain and its properties were compared with those of the well-characterized pancreatic lipase and pancreatic lipase-related protein 2. The activity of the alkaline lipase was determined using trioleoylglycerol emulsion at pH 8.0. Subcellular fractions were prepared from brain homogenates by differential centrifugation. Lipase activities of the cytosolic fraction (the supernatant obtained by differential centrifugation of 100,000g) were stimulated by addition of colipase and bile salts and inhibited by addition of an antibody against rat pancreatic lipase. The partially purified enzyme had an isoelectric point of pH 6.8, which was identical to that found for rat pancreatic lipase. The enzyme had interfacial activation and dependence on colipase in the presence of bile salts. The enzyme had no measurable phospholipase A activity. The band produced by the enzyme on SDS-polyacrylamide gel electrophoresis was identical to that of the rat pancreatic lipase when detected by immunoblotting analysis using an antibody against pancreatic lipase. These results show that pancreatic lipase such as alkaline lipase is in rat brain.

Genomic discordance between monozygotic twins discordant for schizophrenia.

OBJECTIVE: Genomic DNA of monozygotic twins discordant for schizophrenia was analyzed to determine whether their genomes were truly identical. METHOD: The subjects were monozygotic male twins, one of whom had DSM-III-R schizophrenia, undifferentiated type. Genomic DNA was extracted from leukocytes and was applied to restriction landmark genome scanning analysis, which was developed for a high-speed survey of restriction sites throughout a genome and measurement of their copy number in each locus. RESULTS: After comparisons of patterns with approximately 2,000 spots, the authors detected at least two spots with autoradiographic intensities that obviously differed in the two twins. CONCLUSIONS: The discrepancies likely were generated either by differences in the methylation status at NotI sites between the twins or by submicroscopic changes occurring at NotI-flanking sites in one twin after (or simultaneous with) twinning. In either case, the difference may influence the transcription level of one or more genes.