Cloning and sequence analysis of adrenodoxin reductase cDNA from bovine adrenal cortex

cDNA clones for bovine adrenodoxin reductase were isolated, and the primary structure of the enzyme precursor was deduced from their nucleotide sequences. The precursor consists of 492 amino acids including an extrapeptide of 32 amino acids at the amino terminus. The extrapeptide is hydrophilic [corrected] and rich in arginine. The amino terminal sequence of the precursor is homologous with that of the adrenodoxin precursor. A possible FAD- or NADPH-binding site is present near the amino terminus of the mature enzyme.     

Automated system for high-throughput protein production using the dialysis cell-free method

High-throughput protein production systems have become an important issue, because protein production is one of the bottleneck steps in large-scale structural and functional analyses of proteins. We have developed a dialysis reactor and a fully automated system for protein production using the dialysis cell-free synthesis method, which we previously established to produce protein samples on a milligram scale in a high-throughput manner. The dialysis reactor was designed to be suitable for an automated system and has six dialysis cups attached to a flat dialysis membrane. The automated system is based on a Tecan Freedom EVO 200 workstation in a three-arm configuration, and is equipped with shaking incubators, a vacuum module, a robotic centrifuge, a plate heat sealer, and a custom-made tilting carrier for collection of reaction solutions from the flat-bottom cups with dialysis membranes. The consecutive process, from the dialysis cell-free protein synthesis to the partial purification by immobilized metal affinity chromatography on a 96-well filtration plate, was performed within ca. 14 h, including 8 h of cell-free protein synthesis. The proteins were eluted stepwise in a high concentration using EDTA by centrifugation, while the resin in the filtration plate was washed on the vacuum manifold. The system was validated to be able to simultaneously and automatically produce up to 96 proteins in yields of several milligrams with high well-to-well reliability, sufficient for structural and functional analyses of proteins. The protein samples produced by the automated system have been utilized for NMR screening to judge the protein foldedness and for structure determinations using heteronuclear multi-dimensional NMR spectroscopy. The automated high-throughput protein production system represents an important breakthrough in the structural and functional studies of proteins and has already contributed a massive amount of results in the structural genomics project at the RIKEN Structural Genomics/Proteomics Initiative (RSGI).     

Properties of a DNA-binding protein in a culture medium of thymus cells

A DNA-binding protein, which migrated as one major protein band, with a molecular weight of 14,000, on sodium dodecylsulfate polyacrylamide gel, was purified from a culture medium of mouse thymus cells. The interaction of the isolated protein with DNA in vitro was assayed by a nitrocellulose filter binding technique. Equilibrium competition experiments demonstrated that the DNA-binding protein had the ability to differentiate among sequences of polynucleotides, indicating that the DNA-binding protein-DNA interaction was at least partially specific. This protein increased the helix melting temperature of DNA and inhibited the incorporation of [3H]dTMP into DNA by the DNA polymerase of calf thymus in vitro.     

Treatment with β2‐Adrenoceptor Agonist in Vivo Induces Human Clock Gene,Per1, mRNA Expression in Peripheral Blood

This study examined whether in vivo exposure to a β₂‐adrenoceptor agonist, tulobuterol, induces human Period1 (hPer1) mRNA expression in cells from peripheral whole blood. In one experiment, oral tulobuterol was administered to five healthy volunteers at 22:00 h, while in another, a transdermally tulobuterol patch was applied to the same five subjects at 20:00 h. In each experiment, serum tulobuterol concentrations were measured at four time points, and total RNA was isolated from peripheral blood cells for determinations of hPer1 mRNA expression by real‐time polymerase chain reaction. Both the tulobuterol tablet and the transdermal patch increased hPer1 mRNA expression, suggesting that analyses of human peripheral blood cells could reliably represent peripheral clock gene mRNA expression in vivo.     

Serratia Protease

Protease from a strain of Serratia contained one gram atom of zinc ion per mole and the zinc ion was essential for the activity. Also zinc-free apoenzyme was isolated as a crystalline form from the native-enzyme. Several metalloenzymes were prepared by the addition of corresponding metal ions to the apoenzyme. Studies on activities toward the hydrolysis of casein showed that relative activities of native- (zinc), cobalt- and manganese-enzyme were 1.0, 1.2 and 0.8, respectively. Toward the hydrolysis of hippurylleucinamide, however, specific activity of cobalt-enzyme was about 10 times that of the native- (zinc-) enzyme. Spectroscopic studies did not reveal any significant differences in conformations among native-enzyme, apoenzyme and the other metalloenzymes.     

Clastogenicity of 1-nitropyrene, dinitropyrenes, fluorene and mononitrofluorenes in cultured Chinese hamster cells

The chromosomal aberration test using a Chinese hamster lung cell line (CHL) was carried out on 1-nitropyrene (NP), 3 dinitropyrenes (DNPs), fluorene and 4 mononitrofluorenes with and without metabolic activation (rat S9 mix). The 3 DNPs (1,3-, 1,6- and 1,8-DNP) induced chromosomal aberrations in the absence of S9 mix. The frequencies of cells with aberrations after treatment for 48 h were 43% at 2 micrograms/ml of 1,3-DNP, 55% at 0.1 microgram/ml of 1,6-DNP and 45% at 0.025 microgram/ml of 1,8-DNP, indicating the order of clastogenic potency as 1,8- greater than 1,6- greater than 1,3-DNP. On the other hand, 1-NP, which is known to be a direct-acting mutagen in bacteria, was negative in the chromosomal aberration test without S9 mix, but clearly positive with S9 mix. This effect was dependent on the concentration of the S9 fraction in the reaction mixture. High-pressure liquid chromatography analysis showed that 1-NP was converted by S9 mix to several metabolites, including 1-aminopyrene (AP). The clastogenic activity of 1-AP, however, was equivocal without S9 mix, suggesting that active clastogens other than 1-AP exist. Fluorene induced chromosomal aberrations only in the presence of S9 mix (61.8% at 25 micrograms/ml). 1-, 2-, 3- and 4-nitrofluorene (NF) were more clastogenic in the presence of S9 mix than in the absence of S9 mix, suggesting that NFs were converted to more active clastogens by S9 mix.     

Gliding motility of Mycoplasma mobile can occur by repeated binding to N-acetylneuraminyllactose (sialyllactose) fixed on solid surfaces

Mycoplasma mobile relies on an unknown mechanism to glide across solid surfaces including glass, animal cells, and plastics. To identify the direct binding target, we examined the factors that affect the binding of Mycoplasma pneumoniae to solid surfaces and concluded that N-acetylneuraminyllactose (sialyllactose) attached to a protein can mediate glass binding on the basis of the following four lines of evidence: (i) glass binding was inhibited by N-acetylneuraminidase, (ii) glass binding was inhibited by N-acetylneuraminyllactose in a structure-dependent manner, (iii) binding occurred on glass pretreated with bovine serum albumin attached to N-acetylneuraminyllactose, and (iv) gliding speed depended on the density of N-acetylneuraminyllactose on glass.     

Dexamethasone influences human clock gene expression in bronchial epithelium and peripheral blood mononuclear cells in vitro

We determined whether human peripheral blood mononuclear cells (PBMCs) could be used to analyze clock genes by studying their mRNA expressions in human bronchial epithelium (BEAS-2B) and PBMCs following stimulation by the glucocorticoid homologue dexamethasone (DEX) in vitro. PBMCs were obtained at 10:00 h from two diurnally active (∼07:00 to 23:00 h) healthy volunteers and were evaluated for hPer1 mRNA expression following DEX stimulation in vitro using real time-PCR analysis. DEX stimulation of human BEAS-2B cells and PBMCs in vitro led to a remarkable increase of hPer1 mRNA. The glucocorticoid rapidly affected the expression of hPer1 mRNA in PBMCs, suggesting that human PBMCs may be a useful surrogate marker for the investigation of drug effects on clock genes.     

Redescription of a rare deep-sea batfish, <Emphasis Type="Italic">Halieutopsis bathyoreos</Emphasis> (Lophiiformes: Ogcocephalidae)

Halieutopsis bathyoreos Bradbury, 1988 (Lophiiformes: Ogcocephalidae), previously described only on the basis of the holotype (62.6mm in standard length) from the central North Pacific, is redescribed on the basis of the holotype and six additional specimens (41.2–68.7mm in standard length) collected from the western South Pacific, off Papua New Guinea, and the western North Pacific, including the Japanese Archipelago. Halieutopsis bathyoreos is distinguished from its congeners by having a shelflike rostrum extending anteriorly well beyond the mouth, a dorsal escal lobe slightly bisected ventrally, an illicial cavity square in outline and completely visible in ventral view, and lacking tubercles on the ventral surface of the disk. The following characters are newly added to the diagnoses of this species: rostrum width 21–29% of head length, tubercles on the dorsal surface of the disk about half the diameter of those on the lateral margin, and 13–15 large lateral-line scales on the tail.