Assembly of Staphylococcal Leukocidin into a Pore-Forming Oligomer on Detergent-Resistant Membrane Microdomains,Lipid Rafts,in Human Polymorphonuclear Leukocytes

Staphylococcal leukocidin (Luk) consists of LukS and LukF, which cooperatively lyse human polymorphonuclear leukocytes (HPMNLs), monocytes, and macrophages. Here we found that LukS and LukF assembles into hetero-oligomeric pore complexes on the detergent-resistant membrane microdomains, lipid rafts of HPMNLs. When HPMNLs were treated with LukS alone, 24% of the added LukS was localized in lipid rafts. Furthermore, in HPMNLs treated with both LukS and LukF simultaneously, about 90% of high molecular-mass complexes of 100 kDa, which consists of LukS and LukF, were detected in the lipid raft fractions. In contrast, in HPMNLs treated with LukF alone, LukF was not localized in lipid rafts despite binding to the target cell membranes. Ten mM methyl-β-cyclodextrin, a dysfunctioning agent of lipid rafts, completely inhibited assembly of Luk on lipid rafts, and resulted in null leukocytolytic activity of Luk. Hence, we concluded that assembly of LukS and LukF into the pore-complex occurs in lipid rafts in HPMNLs and that LukF can bind to LukS, which had already bound to lipid rafts, to assemble into hetero-oligomers.     

Assembly of staphylococcal leukocidin into a pore-forming oligomer on detergent-resistant membrane microdomains, lipid rafts, in human polymorphonuclear leukocytes

Staphylococcal leukocidin (Luk) consists of LukS and LukF, which cooperatively lyse human polymorphonuclear leukocytes (HPMNLs), monocytes, and macrophages. Here we found that LukS and LukF assembles into hetero-oligomeric pore complexes on the detergent-resistant membrane microdomains, lipid rafts of HPMNLs. When HPMNLs were treated with LukS alone, 24% of the added LukS was localized in lipid rafts. Furthermore, in HPMNLs treated with both LukS and LukF simultaneously, about 90% of high molecular-mass complexes of 100 kDa, which consists of LukS and LukF, were detected in the lipid raft fractions. In contrast, in HPMNLs treated with LukF alone, LukF was not localized in lipid rafts despite binding to the target cell membranes. Ten mM methyl-beta-cyclodextrin, a dysfunctioning agent of lipid rafts, completely inhibited assembly of Luk on lipid rafts, and resulted in null leukocytolytic activity of Luk. Hence, we concluded that assembly of LukS and LukF into the pore-complex occurs in lipid rafts in HPMNLs and that LukF can bind to LukS, which had already bound to lipid rafts, to assemble into hetero-oligomers.     

Mutual Binding Inhibition of Tetrodotoxin and Saxitoxin to Their Binding Protein from the Plasma of the Puffer Fish,Fugu pardalis

The mutual binding inhibition of tetrodotoxin and saxitoxin to their binding protein from the plasma of Fugu pardalis was investigated by HPLC. The values for the half inhibitory concentration of tetrodotoxin (1.6 μM) binding to this protein (1.2 μM) for saxitoxin, and of saxitoxin (0.47 μM) binding to that (0.30 μM) for tetrodotoxin were 0.35±0.057 μM and 81±16 μM (n=2), respectively.     

Functional Characterization of D-Galacturonic Acid Reductase,a Key Enzyme of the Ascorbate Biosynthesis Pathway,from Euglena gracilis

D-Galacturonic acid reductase, a key enzyme in ascorbate biosynthesis, was purified to homogeneity from Euglena gracilis. The enzyme was a monomer with a molecular mass of 38–39 kDa, as judged by SDS–PAGE and gel filtration. Apparently it utilized NADPH with a Km value of 62.5±4.5 μM and uronic acids, such as D-galacturonic acid (Km=3.79±0.5 mM) and D-glucuronic acid (Km=4.67±0.6 mM). It failed to catalyze the reverse reaction with L-galactonic acid and NADP⁺. The optimal pH for the reduction of D-galacturonic acid was 7.2. The enzyme was activated 45.6% by 0.1 mM H₂O₂, suggesting that enzyme activity is regulated by cellular redox status. No feedback regulation of the enzyme activity by L-galactono-1,4-lactone or ascorbate was observed. N-terminal amino acid sequence analysis revealed that the enzyme is closely related to the malate dehydrogenase families.     

Suppressive Effects of Genistein on Oxidative Stress and NFκB Activation in RAW 264.7 Macrophages

This study was designed to investigate whether genistein may ameliorate oxidative stress and nuclear factor κB (NFκB) activation in the lipopolysaccharide (LPS)-stimulated RAW 264.7 murine macrophage cell line. Treatment of RAW 264.7 cells with genistein significantly reduced lipopolysaccharide (LPS)-stimulated nitric oxide (NO) production in a dose-dependent manner with an IC₅₀ of 69.4 μM. Genistein at 50 μM and 100 μM concentrations reduced thiobarbituric acid-reactive substances (TBARS) accumulation, increasing the GSH level and antioxidant enzyme activities, such as superoxide dismutase (SOD) and catalase. The specific DNA-binding activities of nuclear factor κB (NFκB) on nuclear extracts from 50 μM and 100 μM genistein treatments were significanly suppressed. These results suggest that genistein has mild antioxidant activity to suppress intracellular oxidative stress and NFκB activation.     

NADP-Specific Glutamate Dehydrogenase from Alkaliphilic Bacillus sp. KSM-635: Purification and Enzymatic Properties

An NADP-specific glutamate dehydrogenase [L-glutamate: NADP⁺ oxidoreductase (deaminating), EC 1.4.1.4] from alkaliphilic Bacillus sp. KSM-635 was purified 5840-fold to homogeneity by a several-step procedure involving Red-Toyopearl affinity chromatography. The native protein, with an isoelectric point of pH 4.87, had a molecular mass of approximately 315 kDa consisting of six identical summits each with a molecular mass of 52 kDa. The pH optima for the aminating and deaminating reactions were 7.5 and 8.5, respectively. The optimum temperature was around 60°C for both. The purified enzyme had a specific activity of 416units/mg protein for the aminating reaction, being over 20-fold greater than that for deaminating reaction, at the respective pH optima and at 30°C. The enzyme was specific for NADPH (K_m 44 μM), 2-oxoglutarate (K_m 3.13 mM), NADP⁺ (K_m 29 μM), and L-glutamate (K_m 6.06 mM). The K_m for NH₄Cl was 5.96 mM. The enzyme could be stored without appreciable loss of enzyme activity at 5°C for half a year in phosphate buffer (pH 7.0) containing 2 mM 2-mercaptoethanol, although the enzyme activity was abolished within 20 h by freezing at ?20°C.     

Development of an Enzyme-Linked Immunosorbent Assay System for the Determination of Asymmetric Dimethylarginine Using a Specific Monoclonal Antibody

We produced a monoclonal antibody (mAb) against N ^G,N ^G-dimethyl-L-arginine (asymmetric dimethylarginine: ADMA), an endogenous competitive inhibitor of nitric oxide synthase (NOS), and developed an enzyme-linked immunosorbent assay (ELISA). The competitive ELISA method using the mAb determined 5 nM–100 nM ADMA, and ADMA levels in human plasma and urine were found to be 0.78 μM and 51.3 μmol/g of creatinine respectively.     

Isolation of a Novel Carotenoid,OH-chlorobactene Glucoside Hexadecanoate,and Related Rare Carotenoids from Rhodococcus sp. CIP and Their Antioxidative Activities

In the course of screening for antioxidative carotenoids from bacteria, we isolated and identified a novel carotenoid, OH-chlorobactene glucoside hexadecanoate (4), and rare carotenoids, OH-chlorobactene glucoside (1), OH-γ-carotene glucoside (2) and OH-4-keto-γ-carotene glucoside hexadecanoate (3) from Rhodococcus sp. CIP. The singlet oxygen (¹O₂) quenching model of these carotenoids showed potent antioxidative activities IC₅₀ 14.6 μM for OH-chlorobactene glucoside hexadecanoate (4), 6.5 μM for OH-chlorobactene glucoside (1), 9.9 μM for OH-γ-carotene glucoside (2) and 7.3 μM for OH-4-keto-γ-carotene glucoside hexadecanoate (3).     

Dihydrosterigmatocystin and Dihydrodemethylsterigmatocystin,New Metabolites from Aspergillus versicolor

L-Arabinose isomerase (L-arabinose ketol-isomerase, EC 5.3.1.4) was demonstrated from the L-arabinose-grown cells of Streptomyces sp. which was isolated from sea water. The enzyme was purified by MnCl₂ treatment, fractionation by polyethylene glycol and by column chromatographies on Sephadex G-150 and DEAE-cellulose. The purified enzyme was specific only for L-arabinose and the Michaelis constant for L-arabinose was 40 mM at pH 7.5. Manganese or cobalt ions were effective for the enzyme activity after dialysis against EDTA. The enzyme activity was inhibited competitively by L-arabitoI, ribitol and xylitol, of which inhibition constants were 1.1, 1.0, and 15 mM, respectively.     

Antitumor Activities and Immunochemical Properties of the Cell-wall Polysaccharides from Aureobasidium pullulans

Delipidated cell walls from Aureobasidium pullulans were fractionated systematically.

The cell surface heteropolysaccharide contains D-mannose, D-galactose, D-glucose, and D-glucuronic acid (ratio, 8.5:3.9:1.0:1.0). It consists of a backbone of (1→6)-α-linked D-mannose residues, some of which are substituted at O-3 with single or β-(1→6)-linked D-galactofuranosyl side chains, some terminated with a D-glucuronic acid residue, and also with single residues of D-glucopyranose, D-galactopyranose, and D-mannopyranose.

This glucurono-gluco-galactomannan interacted with antiserum against Elsinoe leucospila, which also reacted with its galactomannan, indicating that both polysaccharides contain a common epitope, i.e., at least terminal β-galactofuranosyl groups and also possibly internal β-(1→6)-linked galactofuranose residues.

It was further separated by DEAE-Sephacel column chromatography to gluco-galactomannan and glucurono-gluco-galactomannan.

The alkali-extracted β-D-glucan was purified by DEAE-cellulose chromatography to afford two antitumor-active (1→3)-β-D-glucans. One of the glucans (M_r, 1–2 × 10⁵) was a O-6-branched (1→3)-β-D-glucan with a single β-D-glucosyl residue, d.b., 1/7, and the other (M_r, 3.5–4.5 × 10⁵) had similar branched structure, but having d.b., 1/5. Side chains of both glucans contain small proportions of β-(1→6)-and β-(1→4)-D-glucosidic linkages.     

Conversion of the Extracellular Dextransucrase Isoenzymes from Leuconostoc mesenteroides NRRL B-1299

Effect of oxygen tension on l-lysine, l-threonine and l-isoleucine accumulation was investigated. Sufficient supply of oxygen to satisfy the cell’s oxygen demand was essential for the maximum production in each fermentation. The dissolved oxygen level must be controlled at greater than 0.01 atm in every fermentation, and the optimum redox potentials of culture media were above ?170 mV in l-lysine and l-threonine and above ?180 mV in l-isoleucine fermentations. The maximum concentrations of the products were 45.5 mg/ml for l-lysine, 10.3 mg/ml for l-threonine and 15.1 mg/ml for l-isoleucine. The degree of the inhibition due to oxygen limitation was slight in the fermentative production of l-lysine, l-threonine and l-isoleucine, whose biosynthesis is initiated with l-aspartic acid, in contrast to the accumulation of l-proline, l-glutamine and l-arginine, which is biosynthesized by way of l-glutamic acid.     

Establishment of an in Vitro Model of the Human Placental Barrier by Placenta Slice Culture and Ussing Chamber

Aggregation occurs through hydrophobic interactions when a polypeptide chain refolds in non-native states or when genetic variants of biologically active proteins assume inappropriate conformations, as observed in the case of dysfunctional serpins. Here, using the molecular chaperone BiP from bovine liver microsomes, we characterized the hydrophobic nature of the peptide segment which is considered to be a site required for aggregation among a non-inhibitory serpin ovalbumin in a heat-denatured state. Screening of the peptide scan for binding of BiP showed that BiP-binding sites are mostly buried in the folded ovalbumin. When ovalbumin was heat-denatured, the denatured protein was recognized by the antibody that reacts with the hydrophobic surface of the amino-terminal segment of ovalbumin. This antibody significantly suppressed the binding of BiP to denatured ovalbumin. BiP also bound the immobilized peptide in an ATP-dependent manner and the peptide stimulated the ATPase activity of BiP with a K _m of 165 μM and a V _max of 0.4 nmol/min per milligram. Measurement of surface plasmon resonance showed that the peptide had a K _d of 0.52 μM by BiP, lower than that for RCMLA (K _d=1.1 μM) and even lower than that of the peptide P10K, PLSRTLSVAAKK, (K _d=21 μM). These results demonstrate that the aggregation-prone site on heat-denatured ovalbumin has almost the same hydrophobic nature of interacting with the molecular chaperone BiP as the conventionally known peptides that bind to the Escherichia coli chaperone DnaK.     

Enzymatic Quantification of L-Tartrate in Wines and Grapes by Using the Secondary Activity of D-Malate Dehydrogenase

L-Tartrate in wines and grapes was enzymatically quantified by using the secondary activity of D-malate dehydrogenase (D-MDH). NADH formed by the D-MDH reaction was monitored spectrophotometrically. Under the optimal conditions, L-tartrate (a 1.0 mM sample solution) was fully oxidized by D-MDH in 30 min. A linear relationship was obtained between the absorbance difference and the L-tartrate concentration in the range of a 0.02-1.0 mM sample solution with a correlation coefficient of 0.9991. The relative standard deviation from ten measurements was 1.71% at the 1.0 mM sample solution level. The proposed method was compared with HPLC, and the values determined by both methods were in good agreement.     

Purification and Characterization of Novel N-Acyl-D-aspartate Amidohydrolase from Alcaligenes xylosoxydans subsp. xylosoxydans A-6

Alcaligenes xylosoxydans subsp. xylosoxydans A-6 (Alcaligenes A-6) produced N-acyl-D-aspartate amidohydrolase (D-AAase) in the presence of N-acetyl-D-aspartate as an inducer. The enzyme was purified to homogeneity. The enzyme had a molecular mass of 56 kDa and was shown by sodium dodecyl sulfate (SDS)–polyacrylamide gel electrophoresis (PAGE) to be a monomer. The isoelectric point was 4.8. The enzyme had maximal activity at pH 7.5 to 8.0 and 50°C, and was stable at pH 8.0 and up to 45°C. N-Formyl (K_m=12.5 mM), N-acetyl (K_m=2.52 mM), N-propionyl (K_m=0.194 mM), N-butyryl (K_m=0.033 mM), and N-glycyl (K_m =1.11 mM) derivatives of D-aspartate were hydrolyzed, but N-carbobenzoyl-D-aspartate, N-acetyl-L-aspartate, and N-acetyl-D-glutamate were not substrates. The enzyme was inhibited by both divalent cations (Hg²⁺, Ni²⁺, Cu²⁺) and thiol reagents (N-ethylmaleimide, iodoacetic acid, dithiothreitol, and p-chloromercuribenzoic acid). The N-terminal amino acid sequence and amino acid composition were analyzed.     

Structure of Rugulovasine A,B and their Derivatives

Culture conditions for the preparation of cells containing high tyrosine phenol lyase activity were studied with Erwinia herbicola ATCC 21434. Adding pyridoxine to the medium enhanced enzyme formation, suggesting that it was utilized as a precursor of the coenzyme, pyridoxal phosphate. Glycerol plus succinic acid; amino acids, such as, DL-methionine, DL-alanine and glycine; and metallic ion, ferrous ion promoted enzyme formation as well as cell growth. Adding L-tyrosine, as inducer, to the culture medium was essential for enzyme formation. However, when large amounts of L-tyrosine were added, the enzyme formation was repressed by the phenol liberated from L-tyrosine. In fact, formation of the enzyme was enhanced by removing phenol during cultivation. L(D)-Phenylalanine or phenylpyruvic acid had a synergistic effect on the induction of enzyme by L-tyrosine.

Cells with high enzyme activity were prepared by growing cells at 28°C for 28 hr in a medium containing 0.2% L-tyrosine, 0.2% KH₂PO₄, 0.1% MgSO₄7H₂O, 0.001% FeSO_4·7H₂O, 0.01% pyridoxine-HC1, 0.6% glycerol, 0.5% succinic acid, 0.1% DL-methionine, 0.2% DL-alanine, 0.05% glycine, 0.1% L-phenylalanine and 120 ml/liter hydrolyzed soybean protein in tap water with the pH controlled at 7.5 throughout cultivation.     

Effect of Isopentenyladenine,a Cytokinin,on Proliferation and Protein Synthesis in Cultured Myoblasts

The effects of cytokinins, a class of plant hormones, on cell proliferation and protein synthesis were studied in rat-derived L₆ myoblasts cultured in a serum-free medium. Of the three cytokinins tested, isopentenyladenine, zeatin and ribosylzeatin, isopentenyladenine (5 μm) most stimulated the growth and DNA synthesis of the myoblasts, and it dose-dependently (0 ~ 10μm) enhanced the proliferation and DNA synthesis of the cells. Isopentenyladenine (5 and 10 μm) increased protein synthesis to twice that of control (0μm). These results suggest that isopentenyladenine, a trace component in plant food and a plant hormone, can affect the metabolism of an animal cell line of myoblasts.     

S-Carboxymethylcysteine Synthase from Escherichia coli

An enzyme that catalyzes the synthesis of S-carboxymethyl- l-cysteine from 3-chloro- l-alanine (3-Cl-Ala) and thioglycolic acid was found in Escherichia coli W3110 and was designated as S- carboxymethyl-l-cysteine synthase. It was purified from the cell-free extract to electrophoretic homogeneity and was crystallized. The enzyme has a molecular weight of 84,000 and gave one band corresponding to a molecular weight of 37,000 on SDS-polyacrylamide gel electrophoresis. The purified enzyme catalyzed the β-replacement reactions between 3-CI-AIa and various thiol compounds. The apparent Km values for 3-Cl-Ala and thioglycolic acid were 40 mM and 15.4 mM. The enzyme showed very low activity as to the α,β-elimination reaction with 3-Cl-Ala and l-serine. It was not inactivated on the incubation with 3-Cl-Ala. The absorption spectrum of the enzyme shows a maximum at 412 nm, indicating that it contains pyridoxal phosphate as a cofactor. The N-terminal amino acid sequence was determined and the corresponding sequence was detected in the protein sequence data bank, but no homogeneous sequence was found.     

Purification and Some Properties of Acid Invertase of Persimmon Fruits

Single cells were prepared from mesocarp tissue of ripe persimmon (Diospyros kaki cv. Fuyu) fruits, and inter- or intracellular localization of acid invertase (AI, EC 3.2.1.26) was studied. AI was localized in the intercellular fraction (cell wall fraction). AI was isolated and purified from the cell wall fraction of ripe persimmon fruits by column chromatography on SE-53 cellulose and Toyopearl HW 55F. The specific activity of purified AI was 570 units per mg protein at 30°C. The molecular mass of AI was estimated to be 44 kDa by gel filtration over Sephacryl S-200 and 70 kDa by SDS–PAGE. The optimum pH of the activity for sucrose was 4.25. The purified enzyme hydrolyzed sucrose and raffinose but not melibiose. The enzyme had a K_m of 3.2 mM for sucrose and a K_m of 2.6 mM for raffinose. Silver nitrate (5 μM), HgCI₂ (2 μM), p-chloromercuribenzoate (100mM), pyridoxamine (10mM), and pyridoxine (2.5mM) inhibited AI activity by 95, 85, 100, 41, and 300%, respectively.     

Characteristics of Novel Insect Defensin-Based Membrane-Disrupting Trypanocidal Peptides

Synthetic D- and L-amino acid type cationic 9-mer peptides (all sequences were synthesized as D- or L-amino acids) derived from the active sites of insect defensins were tested for their ability to modify the growth of blood-stream form African trypanosomes in vitro. One of them, the D-type peptide A (RLYLRIGRR-NH₂), irreversibly suppressed proliferation of the Trypanosoma brucei brucei GUTat3.1 parasite. The presence of negatively charged phosphatidylserine on the surface of the parasites was demonstrated, suggesting electrostatic interaction between the peptide and the phospholipids. Furthermore, this peptide was found to alter trypanosome membrane-potentials significantly, an effect apparently due to the removal of the parasite’s plasma membrane. The potential toxic effects of D-peptide A on mammalian cells was assessed using human brain microvascular endothelial cells. Only minor effects were found when the endothelial cells were exposed for 16 h to peptide concentrations of less than 200 μM. These findings suggest that insect defensin-based peptides represent a potentially new class of membrane-disrupting trypanocidal drugs.