Roles of the hydroxy group of tyrosine in crystal structures of Sulfurisphaera tokodaiiO6-methylguanine-DNA methyltransferase.

O6-Methylguanine-DNA methyltransferase (MGMT) removes cytotoxic O6-alkyl adducts on the guanine base and protects the cell from genomic damage induced by alkylating agents. Although there are reports of computational studies on the activity of the enzyme with mutations at tyrosine residues, no studies concerning the crystal structure of its mutants have been found. In this study, the function of Tyr91 was investigated in detail by comparing the crystal structures of mutants and their complexes with substrate analogs. In this study, tyrosine, a conserved amino acid near the active-site loop in the C-terminal domain of Sulfurisphaera tokodaii MGMT (StoMGMT), was mutated to phenylalanine to produce a Y91F mutant, and the cysteine which is responsible for receiving the methyl group in the active site was mutated to a serine to produce a C120S mutant. A Y91F/C120S double-mutant StoMGMT was also created. The function of tyrosine is discussed based on the crystal structure of Y91F mutant StoMGMT. The crystal structures of StoMGMT were determined at resolutions of 1.13-2.60 Å. They showed no structural changes except in the mutated part. No electron density for deoxyguanosine or methyl groups was observed in the structure of Y91F mutant crystals immersed in O6-methyl-2'-deoxyguanosine, nor was the group oxidized in wild-type StoMGMT. Therefore, the hydroxy group of Tyr91 may prevent the oxidant from entering the active site. This suggests that tyrosine, which is highly conserved at the N-terminus of the helix-turn-helix motif across species, protects the active site of MGMTs, which are deactivated after repairing only one alkyl adduct. Overall, the results may provide a basis for understanding the molecular mechanisms by which high levels of conserved amino acids play a role in ensuring the integrity of suicide enzymes, in addition to promoting their activity.

Characteristics of established KSG cells derived from the scorpionfish Sebastiscus marmoratus: what happens under the hydrostatic pressure like the deep sea?

Advances in cell biology depend, partly, on the development of new cell lines and culture methods. Our research focused on a fibroblast-like cell line, "KSG," which is derived from scorpionfish fin tissue (Sebastiscus marmoratus). Cells were grown in Leibovitz's L-15 medium with 10% fetal bovine serum following standard procedures. The optimum growth temperatures for these lines ranged from 15°C to 25°C. All cells survived storage for at least 3 yr at -80°C. Subsequently, they were continuously cultured until the 78th generation without evident changes in their morphology. Moreover, we were able to culture KSG cells in the absence of fetal bovine serum in a culture medium containing the fish serum "SeaGrow." Optimum SeaGrow concentrations for these cells ranged from 5% to 20%. The growth rate of KSG cells decreased when the concentration of SeaGrow was reduced to 1%. However, this decrease could be partially reversed by adding 0.5% "Hy-Fish." In addition, the inclusion of Hy-Fish improved cell adhesion. KSG cells that were cultured in serum-free culture media containing 0.5% and 1% Hy-Fish had been added and were able to survive at low densities. Furthermore, we successfully transfected this cell line with a commercial plasmid vector coding a fluorescent protein using the cationic lipid. Finally, the analyses of cell behavior under hydrostatic pressure showed that some pressures (10 MPa) helped the cells to proliferate more.

Evaluation of commercially available reagents as a reference material for intramolecular carbon isotopic measurements of acetic acid.

RATIONALE: Recent advances in analytical techniques for the intramolecular carbon isotopic ratio measurement of some organic compounds have provided important information on carbon cycles in biochemistry, organic geochemistry and food chemistry. These advances have made it necessary to prepare intramolecular isotopic reference materials (RMs) to use for inter-laboratory calibration and/or inter-calibration among different analytical methods. METHODS: We evaluated the feasibility of preparing RMs using commercially available reagents for intramolecular carbon isotopic ratio measurement of acetic acid. The intramolecular carbon isotopic distribution of nine acetic acid and four sodium acetate reagents was determined with high precision using off-line pyrolysis combined with gas chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS). We also evaluated the potential alteration in the isotopic signature of acetic acid reagents by evaporation. RESULTS: The intramolecular carbon isotopic distributions for the acetic acid and sodium acetate reagents were determined with a precision of better than 0.45‰. We found that the isotopic values of these reagents spanned the carbon isotopic range of acetic acid in biological and environmental samples. We also found that the isotope fractionation associated with the evaporation of acetic acid occurs solely on the methyl position, the carboxyl position being unaffected. CONCLUSIONS: These commercially available reagents will be used as RMs in the future for inter-laboratory calibration and/or inter-calibration with another intramolecular isotopic measurement technique, namely quantitative (13) C NMR. In cases where acetic acid is being used as a RM, its storage must be carefully controlled to prevent evaporation.

Intramolecular carbon isotope distribution of acetic acid in vinegar.

Compound-specific carbon isotope analysis of acetic acid is useful for origin discrimination and quality control of vinegar. Intramolecular carbon isotope distributions, which are each carbon isotope ratios of the methyl and carboxyl carbons in the acetic acid molecule, may be required to obtain more detailed information to discriminate such origin. In this study, improved gas chromatography-pyrolysis-gas chromatography-combustion-isotope ratio mass spectrometry (GC-Py-GC-C-IRMS) combined with headspace solid-phase microextraction (HS-SPME) was used to measure the intramolecular carbon isotope distributions of acetic acid in 14 Japanese vinegars. The results demonstrated that the methyl carbons of acetic acid molecules in vinegars produced from plants were mostly isotopically depleted in (13)C relative to the carboxyl carbon. Moreover, isotopic differences (δ(13)C(carboxyl) - δ(13)C(methyl)) had a wide range from -0.3 to 18.2‰, and these values differed among botanical origins, C3, C4, and CAM plants.

[Improvement of the system for collecting blood samples, at the Division of Clinical Laboratory in Yamagata University Hospital, based on the complaints of patients: The way to perform the newly-developed 30-min changeover method for collecting blood samples by medical technologists].

The collection of blood samples is one of the most essential procedures in laboratory examinations for the clinical diagnosis of patients. However, it is not always easy to carry out the procedure smoothly. At the Division of Clinical Laboratory in Yamagata University Hospital, we have tried to employ the best way to collect blood samples without any troubles or complaints. However, there were some complaints made by patients over several years, and one of these was that the waiting time for patients was too long. Therefore, we established a new system: all medical technologists joined the program and one took charge of collecting blood samples for 30 min, and then another technologist took over. The system was important for medical technologists since the duty allocation was impartial, and their routine work was not disturbed. We are proud of this newly-developed 30-min turn in collecting blood samples in our hospital.

Intersite structural rearrangement of the blue copper site induced by substrate binding: spectroscopic studies of a copper-containing nitrite reductase from Alcaligenes xylosoxidans NCIMB 11015.

A copper-containing nitrite reductase from Alcaligenes xylosoxidans NCIMB 11015 has its own unique blue or type 1 copper protein resonance Raman spectrum in the usual Cu-S(Cys) stretching region, nu(Cu-S(Cys)), with a pair of strong peaks at 412 and 420 cm(-1) and a weak peak at 364 cm(-1). The predominantly nu(Cu-S(Cys)) Raman bands at 412, 420, and 364 cm(-1) of the type 1 copper site all shifted to higher frequencies upon binding of nitrite to the type 2 copper site, and the resonance Raman difference spectra progressively intensified with the increments of nitrite ion concentration. Positive support for substrate binding to the type 2 copper is provided by the nu(Cu-S(Cys)) bands in the resonance Raman spectrum of a type 2 copper-depleted enzyme, which is insensitive to the presence of NO2-. The shift to higher frequency of the Raman bands of the type 1 copper center with the addition of nitrite ions suggests a stronger Cu-S(Cys) interaction in the substrate-bound A. xylosoxidans nitrite reductase.

Prehospital management of sarin nerve gas terrorism in urban settings: 10 years of progress after the Tokyo subway sarin attack.

Chemical agents have been used previously in wartime on numerous occasions, from World War I to the Gulf War. In 1994 and 1995, sarin nerve gas was used first in peacetime as a weapon of terrorism in Japan. The Tokyo subway sarin attack was the first large-scale disaster caused by nerve gas. A religious cult released sarin gas into subway commuter trains during morning rush hour. Twelve passengers died and about 5500 people were harmed. Sarin is a highly toxic nerve agent that can be fatal within minutes to hours. It causes the clinical syndrome of cholinergic hyperstimulation by inhibition of the crucial enzyme acetylcholinesterase. Therapy of nerve agent toxicity is divided into three categories, decontamination, respiratory support, and antidotes. All of these therapies may be given simultaneously. This article reviews toxicology and management of this acute chemical emergency. To help minimize the possible catastrophic impact on the public, we make several recommendations based on analysis of the Tokyo subway sarin attack and systematically review the current scientific literature.

Introduction of a pi-pi interaction at the active site of a cupredoxin: characterization of the Met16Phe Pseudoazurin mutant.

The Met16Phe mutant of the type 1 copper protein pseudoazurin (PACu), in which a phenyl ring is introduced close to the imidazole moiety of the His81 ligand, has been characterized. NMR studies indicate that the introduced phenyl ring is parallel to the imidazole group of His81. The mutation has a subtle effect on the position of the two S(Cys)-->Cu(II) ligand-to-metal charge transfer bands in the visible spectrum of PACu(II) and a more significant influence on their intensities resulting in a A(459)/A(598) ratio of 0.31 for Met16Phe as compared to a A(453)/A(594) ratio of 0.43 for wild-type PACu(II) at pH 8. The electron paramagnetic resonance spectrum of the Met16Phe variant is more axial than that of the wild-type protein, and the resonance Raman spectrum of the mutant exhibits subtle differences. A C(gamma)H proton of Met86 exhibits a much smaller hyperfine shift in the paramagnetic (1)H NMR spectrum of Met16Phe PACu(II) as compared to its position in the wild-type protein, which indicates a weaker axial Cu-S(Met86) interaction in the mutant. The Met16Phe mutation results in an approximately 60 mV increase in the reduction potential of PACu. The pK(a) value of the ligand His81 decreases from 4.9 in wild-type PACu(I) to 4.5 in Met16Phe PACu(I) indicating that the pi-pi contact with Phe16 stabilizes the Cu-N(His81) interaction. The Met16Phe variant of PACu has a self-exchange rate constant at pH 7.6 (25 degrees C) of 9.8 x 10(3) M(-)(1) s(-)(1) as compared to the considerably smaller value of 3.7 x 10(3) M(-)(1) s(-)(1) for the wild-type protein under identical conditions. The enhanced electron transfer reactivity of Met16Phe PACu is a consequence of a lower reorganization energy due to additional active site rigidity caused by the pi-pi interaction between His81 and the introduced phenyl ring.