Crystallization and preliminary X-ray diffraction analysis of a recombinant bacterial heme oxygenase (Hmu O) from Corynebacterium diphtheriae.

Hmu O is a 24-kDa soluble bacterial heme degradation enzyme found in the pathogen Corynebacterium diphtheriae, the causative agent of diphtheria. Similar to the mammalian heme oxygenase, it binds hemin stoichiometrically and catalyzes the oxygen-dependent conversion of hemin to biliverdin, carbon monoxide, and free iron. Iron is an essential nutrient for bacteria and especially important for pathogenesis. Here we report the first crystallization and preliminary crystallographic study of the heme-Hmu O complex formed from hemin and a recombinant Hmu O, which was expressed in Escherichia coli from a synthetic gene based on the putative hmu O gene sequence. Crystals of the heme-Hmu O complex were obtained by the sitting drop vapor diffusion method using a precipitant solution containing 18% (w/v) PEG 8000 and 0.2 M calcium acetate in 0.1 M sodium cacodylate (pH 6.5). Using synchrotron radiation, the heme-Hmu O crystal diffracted to 2.8 A resolution. It belongs to the monoclinic space group C2, with unit cell parameters a = 123.18 A, b = 44.51 A, c = 92.10 A, and beta = 123.3 degrees. Assuming one molecule of the heme-Hmu O complex per asymmetric unit, the calculated value of Vm is 2.89 A3/Da.     

Novel cytotoxicity test based on menadione-catalyzed H2O2 productivity for food safety evaluation

Menadione-catalyzed H₂O₂ production by viable cells was proportional to viable cell number, and the assay of this H₂O₂ production was applied to the cytotoxicity test of 17 substances which were used for international validation of fixed-dose procedure as an alternative to the classical LD₅₀ test. The cytotoxicity of substances tested was observed 4 h after the incubation with animal cells, and the viability was determined in 10 min according to menadione-catalyzed H₂O₂ production assay. IC₅₀ of each substance required for 50% inhibition of menadione-catalyzed H₂O₂ production was similar among HepG2, HuH-6KK, HUVE, Vero, Intestine407, NIH/3T3 and Neuro-2a cells. Twelve substances, 3 substances and 2 substances showed the difference of one, two and three orders in the magnitude between LD₅₀ and IC₅₀, respectively. These results show that menadione-catalyzed H₂O₂ production assay is useful for the rapid detection of toxic compounds having the basal cytotoxicity common to various cells, but is unfit for the detection of organ-specific toxic compounds. This revised version was published online in August 2006 with corrections to the Cover Date.     

Advantage of menadione-catalyzed chemiluminescent assay for the determination of viable mammalian cell number

A chemiluminescent assay composed of TCPO [bis(2,4,6-trichlorophenyl)oxalate] and harmless rhodamine B is proposed to be superior in the determination of menadione-catalyzed hydrogen peroxide (H₂O₂) production by viable mammalian cells to that composed of TCPO and harmful pyrene [Anal. Biochem. 207 (1992) 255–260]. In tests, the proposed assay showed that the measurable concentration of H₂O₂ and the viable cell number ranged from 10^?9 to 10^?3 M and from 2 × 10² to 2 × 10⁶ cells/100 μl/well in the presence of 10% bovine serum, respectively. The measuring time was approximately 10 min. On the other hand, the measurable cell numbers by the colorimetric WST-1 and MTT assays requiring several hours ranged only from 10³ to 10⁴ cells/100 μl/well and from 10⁴ to 10⁵ cells/100 μl/well, respectively. The cytotoxicity of sodium dodecyl sulfate was also observed at intervals of 1 min by the proposed assay, but not by the above colorimetric assays.     

Genome-wide association study of idiopathic hypersomnia in a Japanese population

Sleep and Biological Rhythms - Idiopathic hypersomnia (IH) is a rare sleep disorder characterized by excessive daytime sleepiness, great difficulty upon awakening, and prolonged sleep time. In...     

The Senescence-accelerated Mouse (SAM): A Higher Oxidative Stress and Age-dependent Degenerative Diseases Model

The SAM strain of mice is actually a group of related inbred strains consisting of a series of SAMP (accelerated senescence-prone) and SAMR (accelerated senescence-resistant) strains. Compared with the SAMR strains, the SAMP strains show a more accelerated senescence process, a shorter lifespan, and an earlier onset and more rapid progress of age-associated pathological phenotypes similar to human geriatric disorders. The higher oxidative stress status observed in SAMP mice is partly caused by mitochondrial dysfunction, and may be a cause of this senescence acceleration and age-dependent alterations in cell structure and function. Based on our recent observations, we discuss a possible mechanism for mitochondrial dysfunction resulting in the excessive production of reactive oxygen species, and a role for the hyperoxidative stress status in neurodegeneration in SAMP mice. These SAM strains can serve as a useful tool to understand the cellular mechanisms of age-dependent degeneration, and to develop clinical interventions. Special issue article in honor of Dr. Akitane Mori.