Osamu Asami

Effect of negative air ions on computer operation, anxiety and salivary chromogranin A-like immunoreactivity

The effects of negative air ions on computer operation were examined using a biochemical index of the activity of the sympathetic/adrenomedullary system (i.e. salivary chromogranin A-like immunoreactivity (CgA-like IR)) and a self-report questionnaire (State-Trait Anxiety Inventory, Anxiety State--STAI-S). Twelve female students carried out a word processing task for 40 min. The salivary CgA-like IR increased more than three times on the task, but the salivary cortisol did not change. The increase in the CgA-like IR level was attenuated by the exposure to negative air ions during the task. The exposure to the ions during the recovery period following the task was effective for rapidly decreasing the CgA-like IR level that had increased after the task. These effects by negative air ions were also observed using STAI-S. Task performance was slightly but significantly improved by the presence of negative air ions. These results suggest that negative air ions are effective for the reduction of and the prompt recovery from stress caused by computer operation.

The Multi-layered Structure of Dps with a Novel Di-nuclear Ferroxidase Center

The crystallization of cellular components represents a unique survival strategy for bacterial cells under stressed conditions. A highly ordered, layered structure is often formed in such a process, which may involve one or more than one type of bio-macromolecules. The main advantage of biocrystallization has been attributed to the fact that it is a physical process and thus is independent of energy consumption. Dps is a protein that crystallizes to form a multi-layered structure in starved cells in order to protect DNA against oxidative damage and other detrimental factors. The multi-layered crystal structure of a Dps protein from Bacillus brevis has been revealed for the first time at atomic resolution in the absence of DNA. Inspection of the structure provides the first direct evidence for the existence of a di-nuclear ferroxidase center, which possesses unique features among all the di-iron proteins identified so far. It constitutes the structural basis for the ferroxidase activity of Dps in the crystalline state as well as in solution. This finding proves that the enzymatic process of detoxification of metal ions, which may cause severe oxidative damage to DNA, is the other important aspect of the defense mechanism performed by Dps. In the multi-layered structure, Dps dodecamers are organized in a highly ordered manner. They adopt the classic form of hexagonal packing in each layer of the structure. Such arrangement results in reinforced structural features that would facilitate the attraction and absorption of metal ions from the environment. The highly ordered layered structure may provide an ideal basis for the accommodation of DNA between the layers so that it can be isolated and protected from harmful factors under stress conditions.

Extracellular production of an intact and biologically active human growth hormone by the Bacillus brevis system

The characteristic features of the Bacillus brevis system are very high productivity of heterologous proteins and very low extracellular protease activity. However, degradation of some heterologous proteins, especially mammalian proteins, can be observed and resulted in a lowering of protein productivity. By using a mutant expressing low levels of proteases and the addition of EDTA to the medium, intact human growth hormone (hGH) was successfully produced with the B. brevis system. Signal peptide modification with higher basicity in the amino terminal region and higher hydrophobicity in the middle region brought about a twelve-fold increase in hGH production. The hGH yield was further elevated to 240 mg L−1 by optimization of culture conditions. Thus, biologically active and mature hGH can be efficiently produced directly in the medium with the B. brevis system.

Thermally stable and hydrogen peroxide tolerant manganese peroxidase (MnP) from Lenzites betulinus

A thermally stable and hydrogen peroxide tolerant manganese peroxidase (MnP) was purified from the culture medium of Lenzites betulinus by ion exchange chromatography, gel filtration and isoelectric focusing chromatography. The MnP purified from L. betulinus (L‐MnP) has a molecular mass of 40 kDa and its isoelectric point was determined to be 6.2. The first 19 amino acids at the N‐terminal end of the L‐MnP sequence were found to exhibit 74% identity with those of a Phlebia radiata MnP. L‐MnP was proved to have the highest hydrogen peroxide tolerance among MnPs reported so far. It retained more than 60% of the initial activity after thermal treatment at 60°C for 60 min, and also retained more than 60% of the initial activity after exposure to 10 mM hydrogen peroxide for 5 min at 37°C.

Monitor of antibodies in human saliva using a piezoelectric quartz crystal biosensor

A piezoelectric quartz crystal immunosensor has been developed for the detection of antibodies in diluted human saliva. The system consists of the quartz piezoelectric crystal, oscillator and frequency counter. The antibodies against human immunoglobulin A are immobilized onto the gold electrode surface on the quartz crystal. The immobilization techniques used in this work are the adsorption method and the covalent bond method. The latter gives more stability in repetitive assays.

Classification of polycyclic aromatic hydrocarbons based on mutagenicity in lung tissue through DNA microarray

Polycyclic aromatic hydrocarbons (PAHs) are widespread environmental pollutants produced in the combustion of organic matter. Exposure to PAHs raises the risk of lung cancer and inflammatory and allergic disorders such as asthma. DNA microarray technologies have been applied to research on toxicogenomics in the recent years. To evaluate the mutagenicity of PAHs and constituents of environmental pollutants in lung tissue, including metabolic activation, human alveolar epithelial type II cells (A549) were treated with nonmutagenic PAH pyrene and with the mutagenic PAHs benzo‐[a]‐pyrene, 1‐nitropyrene, or 1,8‐dinitropyrene. Comparison of genome‐wide microarray expression profiles between a nonmutagenic and a mutagenic PAH‐treated group revealed that xenobiotic response genes such as CYP1B1 were commonly upregulated in two groups and that DNA damage induced genes, especially p53‐downstream genes such as p21 (CDKN1A) were upregulated only in the mutagenic PAH‐treated group. Pretreatment with cytochrome P450 inhibitor α‐naphthoflavone or p53 inhibitor pifithrin‐α inhibited the benzo‐[a]‐pyrene‐induced p21 expression. These data suggest that when PAHs enter the cells, lung epithelium induces PAH metabolic activating enzymes, and then the DNA damages‐recognition signal is converged with p53 downstream genes. This metabolic activation and DNA damage is induced in lung epithelium, and the mutagenicity of PAHs can be classified by DNA microarray expression profiles.

Crystal structure of the liganded anti-gibberellin A4 antibody 4-B8(8)/E9 Fab fragment

Gibberellins, a class of plant hormones, consist of more than 120 members. Only a few of them are recognized by a receptor that remains unknown. The haptenic mouse monoclonal antibody, 4-B8(8)/E9, was generated against gibberellin A(4) (GA(4)) to recognize biologically active GA selectivity, and we attempted to confirm the binding properties between the antibody and GA(4). We carried out an X-ray crystallographic analysis of the 4-B8(8)/E9 Fab fragment complexed with GA(4) at a 2.8 A resolution by using the molecular replacement method. The crystal structure of the Fab fragment showed the typical immunoglobulin fold of the beta-barrel structure which is the common motif of all antibodies. A small hapten-combining site was made up of three heavy chain CDR loops. On the other hand, CDRs of the light chain did not interact directly with GA(4). The C/D rings of the GA(4) molecule were in van der Waals contact mainly with the aromatic side chain of Tyr100AH and Phe100BH of CDR-H3. The 3 beta-hydroxyl and 6 beta-carboxyl groups were, respectively, hydrogen-bonded to the main chain of Ala33H and to the Thr53H heavy chain.

THERMOPHILIC FUNGAL PROTEIN DISULFIDE ISOMERASE

Publisher Summary For the study the authors have isolated and characterized a thermostable protein disulfide isomerase (PDI) from a thermophilic fungus, Humicola insolens . The cDNA encoding the fungal PDI has been cloned and expressed in Bacillus brevis. PDIs from vertebrates and yeast are relatively heat labile. Even in the case of an algal enzyme that is most stable of the known PDIs, the stability against heat is not enough for industrial use of the enzyme. Hence, there is continuing interest in finding new, stable PDIs. Refolding activity using scrambled ribonuclease (RNase) as a substrate is discussed. The fungal PDI cDNA is expressed in a heterologous protein production system using B. brevis as a host. In the purification process, anion-exchange chromatography, lectin affinity chromatography, and high-performance liquid chromatography are discussed. The chapter also presents the data for purification of PDI from the fungus, Humicola insolens . To amplify the DNA fragment around the N-terminal consensus region of fungal PDI, two oligonucleotides corresponding to the amino acid sequence are synthesized and used as primers for a reverse transcriptase-mediated polymerase chain reaction (RT-PCR).

Isolation of a protease-deficient mutant of Bacillus brevis and efficient secretion of a fungal protein disulfide isomerase by the mutant

The efficient production of a thermostable protein disulfide isomerase (PDI) was successfully achieved using the newly isolated protease-deficient mutant, Bacillus brevis 31-OK. Extracellular protease (exoprotease) activity was about a quarter of that in the parent, and the mutant was deficient in at least one of the major exoproteases. The cDNA encoding the fungal PDI was inserted downstream of the signal peptide-encoding region in an expression-secretion vector for B. brevis. Efficient production of PDI was feasible using B. brevis 31-OK as a host and modified signal sequences composed of three leucine residues inserted in the hydrophobic region of the MWP (middle wall protein) signal sequence. The maximal secretion of PDI into the culture medium was 1.1 g/l, which is about twice that by the parent strain and fifty times greater than the amount of rat and murine PDIs produced by Escherichia coli. The enzymatic properties such as the specific activity and thermal stability of the recombinant PDI are similar to those of natural PDI derived from Humicola insolens mycelia. B. brevis 31-OK was able to maintain its exoprotease activity at a low level throughout the cultivation and is considered to be useful host for production of a protease-sensitive protein and for increase of protein productivity due to stable accumulation.

Disulfide Bond Formation in Refolding of Thermophilic Fungal Protein Disulfide Isomerase.

Disulfide bond formation in the refolding of thermophilic fungal protein disulfide isomerase (PDI) was investigated. It was revealed that (i) a disulfide bond buried inside the molecule is preferentially formed and contributes to the thermal stability and the isomerizing power of PDI, and (ii) formation of disulfide bonds in active sites located on the molecular surface causes deformation of the optimum conformation resulting in a decrease in the thermal stability.