G1 phase accumulation induced by UCN-01 is associated with dephosphorylation of Rb and CDK2 proteins as well as induction of CDK inhibitor p21/Cip1/WAF1/Sdi1 in p53-mutated human epidermoid carcinoma A431 cells.

UCN-01 (7-hydroxyl-staurosporine) was originally isolated as a Ca2+- and phospholipid-dependent protein kinase C selective inhibitor and now is being developed as an anticancer agent. Results from our and other laboratories have suggested that UCN-01 induces preferential G1-phase accumulation in several human tumor cell lines tested. To elucidate this mechanism, we examined the effects of UCN-01 on several cell cycle-regulatory proteins critical for G1-S-phase transition in p53-mutated human epidermoid carcinoma A431 cells. After 24 h exposure at around 50% growth-inhibitory concentrations (IC50s), 260 and 520 nM, UCN-01 induced the accumulation of pRb (the dephosphorylated retinoblastoma protein form). The protein expression of cyclin A but not cyclin E was markedly reduced and that of cyclin D1 was partially reduced under the same condition. UCN-01 also showed the concentration-dependent inhibitions of the activity of cyclin-dependent kinase 2 (CDK2) using histone H1 and pRb as substrates in vitro (IC50, 530 and 640 nM, respectively). In addition, CDK2 activities of the cells pretreated with UCN-01 for 24 h at 260 and 520 nM were markedly inhibited, giving IC50s of far less than 260 nM. When the same cell lysates were analyzed by Western blotting for CDK2, the lower band (e.g., active and phosphorylated CDK2) was remarkably reduced, in accordance with the reduced activity. Furthermore, UCN-01 induced the expression of the CDK inhibitor p21 protein and its complex formation with CDK2 after 24 h exposure at 260 and 520 nM, whereas the expression level was very low or undetectable in untreated or DNA-damaged cells. The increase of p21 mRNA levels was also induced under the same condition. UCN-01 further increased luciferase activities in A431 cells transiently transfected with p21 promoter-luciferase reporter plasmid after 24 h exposure at 260 and 520 nM. UCN-01 also increased the expression of the CDK inhibitor p27 protein after 24 h exposure at 260 and 520 nM. These results suggest that G1-phase accumulation induced by UCN-01 is associated with dephosphorylation of Rb and CDK2 proteins as well as induction of CDK inhibitors p21 and p27.

Purification and properties of human serum esterase.

Human serum esterase was purified by affinity column chromatography on a column of covalently linked p-trimethylammoniumanilinium dichloride to Sepharose 4B. The purified preparation hydrolysed both benzoylcholine and tributyrin. p-Trimethylammoniumanilinium dichloride inhibited non-competitively the hydrolysis of tributyrin and inhibited competitively the splitting of benzoylcholine. The Km value was 0.62 . 10(-3) M for tributyrin and 0.4 . 10(-3) M for benzoylcholine. Antiserum to this purified esterase was prepared in rabbit and it was found that the antiserum did not inhibit esterase activities of human liver, muscle and adipose tissue, although it could inhibit completely the esterase activities of human serum.

Purification and some properties of carboxylesterase of rat adipose tissue.

Carboxyl ester hydrolase was obtained from rat epididymal adipose tissue in an electrophoretically homogeneous form. Purification was achieved by acetone precipitation, followed by successive chromatographies on DEAE-cellulose and hydroxyapatite and then isoelectric focusing. The monomeric molecular weight of the enzyme was 65,000 and the enzyme associated to form trimers. The enzyme had an isoelectric point at pH 5.9 and contained 2.1% carbohydrate moiety per protein with a molecular weight of 65,000. The amino terminal residue of the enzyme was glycine. The enzyme catalyzed the hydrolysis of short chain triacylglycerols such as tributyrin and medium chain monoacylglycerols such as monocaprin, but not the hydrolysis of cholesterol ester. The optimum pH for the enzymatic function of this enzyme for methyl butylate was 8.0. An antibody against the highly purified enzyme preparation induced in rabbits strongly inhibited the esterase of rat adipose tissue, but did not inhibit the esterase of rat liver, intestinal mucosa and serum.

Characterization of the promoter region, first ten exons and nine intron-exon boundaries of the DNA-dependent protein kinase catalytic subunit gene, DNA-PKcs (XRCC7).

The gene, DNAPKcs (XRCC7), for the human DNA-dependent protein kinase catalytic subunit (DNA-PKcs) is a strong candidate that complements a severe combined immunodeficiency (scid) and hypersensitivity to ionizing radiation in mice. We constructed a cosmid library from a previously identified, X RCC7-covering YAC clone (943G4). From the library, we isolated three cosmid clones containing the 5'-region of XRCC7. Sequence analysis with primer walking on a 6.3-kb segment of these cosmids identified the promoter region, the first ten exons and nine intron-exon boundaries of XRCC7. The promoter region contains several potential Sp1 protein-binding sites and a high G+C content but no TATA or CCAAT boxes. These findings are consistent with the TATA-less housekeeping gene promoter and provides the basis for transcriptional regulatory studies. Since nine other exons spanning an 8-kb segment are already known, a total of 19 exons in the gene have been identified. The cosmids isolated and the primer sets designed in the present study are useful for mutation analysis in patients with a SCID phenotype.

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.

Studies on lipase and esterase in human post heparin plasma.

Lipase and esterase in post heparin plasma from patients with various diseases were well correlated, r=0.77. Lipase and esterase hydrolyzed both tri and monoacylglycerol. The chain lengths of fatty acids in esters susceptible to the enzymes became longer by conversion of the substrate from triacylglycerol to monacylglycerol.

Effect of diolein on hydrolysis of phosphatidylcholine by phospholipase C from Clostridium perfringens.

The activity of phospholipase C from Clostridium perfringens on 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) as a monolayer at an air/water interface was examined. With a pure POPC monolayer, sharp cut-off of the enzyme activity was observed on increase in surface pressure. However, this cut-off disappeared on addition of a 0.3 molar fraction of 1,2-dioleoylglycerol (1,2-DO) to the monolayer. An abrupt change in the enzyme activity was observed with molar fractions of between 0.2 and 0.3 1,2-DO in the POPC monolayer at an initial surface pressure of 35 mN/m. For examination of the effect of 1,2-DO on the phospholipase C activity, the quantity of [125I]phospholipase C adsorbed to the surface was determined. The enzyme was found to be adsorbed nonspecifically to all lipid films except that of POPC only. The adsorption of enzyme was not affected by the presence or absence of Ca2+ and Zn2+. The rate constant for enzyme adsorption to a 1,2-DO film was 4.5 times that for its adsorption to a POPC film. The adsorption decreased linearly with increase in the surface concentration of POPC, and increased with increase in the surface concentration of 1,2-DO. These data suggest that 1,2-DO (a reaction product) regulates the interaction of phospholipase C with films containing substrate and may also regulate the enzyme activity.

Hydrolysis of cholesteryl oleate liquid crystals by hormone-sensitive lipase.

Cholesteryl oleate liquid crystals were prepared by sonication as a model of lipid droplets accumulated in foam cells derived from macrophages. These liquid crystals were spherical and showed an anisotropic cross image. Hormone-sensitive lipase from bovine adipose tissue hydrolyzed these liquid crystals optimally at pH 6.8. The Km for the liquid crystals was about 8 times that for vesicles or emulsified cholesteryl oleate. The Vmax for the liquid crystals was the same as that for the emulsified substrate and 6 times that for the vesicle substrate. Phospholipid inhibited cholesteryl oleate hydrolysis in a concentration-dependent fashion, phosphatidylserine being especially inhibitory. The effect of phospholipids on the activity changed upon their incorporation into the cholesteryl oleate liquid crystals, phosphatidylethanolamine increasing the activity to about twice that of the control. These results suggest that hormone-sensitive lipase hydrolyzes cholesteryl oleate liquid crystals as a model of endogenous lipid droplets and its activities are affected by phospholipids.

Human liver carboxylesterase. Properties and comparison with human serum carboxylesterase.

Carboxylesterase was obtained from human liver in an electrophoretically homogeneous form. The monomeric molecular weight of the enzyme was 60,000 and the enzyme associated to form trimers. Purified human liver carboxylesterase was compared with human serum carboxylesterase, purified earlier. Serum carboxylesterase hydrolyzed a typical cholinesterase substrate and aryl acylamide, whereas liver carboxylesterase did not hydrolyze these compounds. Both carboxylesterases catalyzed the hydrolysis of short-chain triacylglycerols, such as tributyrin, and medium-chain monoacylglycerols, such as monocaprin, but not the hydrolysis of long-chain triacylglycerols. Serum carboxylesterase activity was inhibited by p-trimethylammoniumanilinium dichloride and neostigmine, whereas liver carboxylesterase activity was not affected by these compounds. Liver and serum carboxylesterase activities were both strongly inhibited by phenylmethylsulfonyl fluoride.

Purification of rat kidney carboxylesterase and its comparison with other tissue esterases.

Carboxylesterase was purified from rat kidney in an electrophoretically homogeneous form by acetone precipitation, followed by successive chromatographies on DEAE-cellulose and hydroxyapatite and then isoelectric focusing. The purified enzyme catalyzed the hydrolyses of monoacylglycerols and short-chain triacylglycerols, such as tributyrin, but not the hydrolysis of long-chain triacylglycerol. Its optimum pH with methyl butyrate as a substrate was 8.0. The relation of its activity to the methyl butyrate concentration differed from those for pancreatic lipase and liver esterase, and also from those for lipolytic enzymes from various other tissues. The relations of methyl butyrate-hydrolyzing activity with methyl butyrate concentration were compared among various carboxylester hydrolyzing enzymes. Based on the results, these enzymes were classified into four classes.

Purification and some properties of intracellular esterase from Pseudomonas fluorescens.

A novel esterase was found in Pseudomonas fluorescens cells and purified to homogeneity as determined by polyacrylamide gel electrophoresis. The esterase was extracted from the cells by freeze-thawing and hypotonic treatment. Purification was achieved by ammonium sulfate precipitation, followed by successive chromatographies on DEAE-cellulose and benzylamine-agarose and then electrophoresis. The enzyme catalyzed the hydrolysis of methyl esters, such as methyl butyrate, but its hydrolyzing activity decreased with increase in the chain length of the alcohol moiety, and it did not catalyze the hydrolysis of triacylglycerols, such as triacetin. In contrast, the enzyme acted on various acyl residues in a series of methyl esters, such as dimethyl succinate, methyl methacrylate, and dimethyl malate. The optimum pH for activity of this enzyme with methyl butyrate was 7.0-8.5. The enzyme was inhibited by phenylmethylsulfonylfluoride. Its molecular weight was estimated as 48,000 by molecular sieve electrophoresis and gel filtration on Sephadex G-150.

Effect of Brij 58 on the hydrolysis of methyl butyrate by lipase from Pseudomonas fluorescens.

Purified Pseudomonas fluorescens lipase [EC 3.1.1.3] exhibited slight activity on water-soluble esters such as methyl butyrate, and this activity was increased on addition of Brij 58 (20 oxyethylene hexadecyl ether) to the solution. This stimulating effect of Brij 58 on hydrolysis of various esters (dimethyl succinate, butyl n-acetate, and tributyrin) in aqueous solution was unspecific. Hydrolysis of methyl butyrate depended on the molecular ratio of Brij 58 to lipase, being maximal (about 8 times the basal level at 37 degrees C with 80 mM substrate in 0.1 M NaCl solution) with 30 mol of Brij 58 per mol of lipase. Comparative studies showed that all polyoxyethylene (POE) alkyl ethers tested, stimulated the methyl butyrate hydrolyzing activity and that the Adekatol SO series (dihydric normal alcohol ethoxylate) also stimulated the appreciably active, whereas Triton X-100, sodium cholate, sodium deoxycholate, sodium dodecylsulfate, POE, and fatty acids had no effect. Comparison of the effects of Brij 58 on the methyl butyrate hydrolyzing activities of various lipolytic enzymes indicated that its effect was specific for this lipase. Brij 58 had no detectable effect with emulsified esters, such as supersaturated methyl butyrate and triolein.

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.

Beta-Adrenergic receptors in rat fat cells and their relationship with lipolysis.

Norepinephrine stimulated lipolysis in rat fat cells while (-)-alprenolol completely inhibited this lipolysis. (-)-Alprenolol competed for (-)-[3H] dihydroalprenolol (DHA) binding sites on rat fat cells. The specific (-)-[3H]DHA binding sites identified by competition with (-)-alprenolol were found to be transferred to the solubilized supernatant during preparation of endogenous lipid droplets from the fat cells. Although the lipid droplets did not exhibit specific (-)-[3H]DHA binding, norepinephrine induced lipolysis in a cell-free system consisting of the lipid droplets and hormone-sensitive lipase (HSL). Norepinephrine-induced lipolysis in the cell-free system was inhibited by propranolol and (-)-alprenolol, but not by phenoxybenzamine. The lipolytic action of norepinephrine and the anti-lipolytic actions of propranolol and (-)-alprenolol disappeared after sonication of the lipid droplets in the cell-free system. These results suggest that the adrenergic receptor concerned with lipolysis in fat cells may not be a specific (-)-[3H]DHA binding site, but may be closely related to the lipid droplets.

Preferential acid-catalyzed hydrolysis of the formamide linkage of N'-formylkynurenine in frozen solution.

Acid-catalyzed hydrolysis of the formamide linkage of N-acetyl-N'-formyl-L-kynurenineamide in frozen dilute hydrochloric acid solution followed first-order kinetics, yielding N-acetyl-L-kynurenineamide as the sole reaction product. The maximum rate of reaction in the frozen solution was found at around -7.5 degrees and approximated that of the reaction in liquid solution at 40 degrees. By freezing the dilute acid solution at -8 degrees the reaction was accelerated by 60 times compared with that in super-cooled liquid solution at the same temperature.

Enrichment of enzyme activity on deformylation of 1-NFK-lysozyme.

The formamide linkage of an inactive lysozyme derivative (1-NFK-lysozyme), formed by selective ozonization of tryptophan 62 in hen egg-white lysozyme [EC 3.2.1.17] was hydrolyzed with dilute acid faster in the frozen state at about --10 degrees than at 20 degrees. On hydrolysis of 1-NFK-lysozyme the low lytic activity increased to approximately 80% of that of native lysozyme. It is suggested that the binding ability associated with kynurenine 62 in the lysozyme derivative formed by this hydrolysis may be responsible for increase in enzymatic activity.

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.

Preparation and characterization of an active lysozyme derivative: Kyn 62-lysozyme.

A novel method for the preparation of Kyn 62-lysozyme, in which tryptophan 62 is replaced by kynurenine, is reported. Hen egg-white lysozyme was ozonized in aqueous solution to yield one N'-formylkynurenine residue and deformylated with hydrochloric acid in frozen solution at -10 degrees C. Crude Kyn 62-lysozyme was purified by affinity and Bio Rex 70 chromatography successively. Kyn 62-lysozyme retains affinity for chitin and is essentially an active enzyme with a slightly weakened but distinct catalytic activity. After this modification, the enzyme activity was changed differently depending on the kind of substrate. At the individual optimum pH's, lytic activity was largely retained (80% active), but the catalytic efficiency for hydrolyzing glycol chitin was relatively low (30% active). Lysis of M. lysodeikticus cell suspensions was optimally catalyzed by Kyn 62-lysozyme at pH 6.2 and at 0.088 ionic strength. These values are lower by 1.3 pH unit and 0.04 ionic strength, respectively, than those of intact lysozyme. The optimum pH and ionic strength for the hydrolysis of neutral substrates were scarcely affected. These results suggest the significance of electrostatic interaction in the lysis of lysozyme. Relatively limited loss of activity induced by modification of the 62nd residue, which is thought to participate directly in the binding of the substrate at subsite C, is discussed on the basis of the similarity of side chain structure in tryptophan and kynurenine.

Role of endogenous lipid droplets of fat cells in epinephrine-induced lipolysis.

Endogenous lipid droplets were prepared by subjecting fat cells to hypotonic shock and to Triton X-100 treatment. The structure of the endogenous lipid droplet fraction was examined by scanning and transmission electron microscopies. Neither intact fat cells nor disrupted cell membranes were detectable in the endogenous lipid droplet fraction. With this endogenous substrate, epinephrine elicited lipolysis with either hormone-sensitive lipase or lipoprotein lipase, but no cyclic AMP-protein kinase mediated stimulation of lipolysis was observed. On the other hand, epinephrine did not stimulate lipolysis when triolein emulsified with arabic gum was used as substrate. With the latter exogenous substrate, however, cyclic AMP-protein kinase was found to stimulate lipolysis with hormone-sensitive lipase as enzyme. These results agree with the proposal of Wise and Jungas that the epinephrine-stimulated increase of hydrolysis of endogenous fat is not mediated through cyclic AMP-protein kinase. A possible mechanism of hydrolysis of endogenous fat by induction of lipolysis by epinephrine in fat cells is discussed.

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.