Crystal structure of chloroplastic ascorbate peroxidase from tobacco plants and structural insights into its instability

Ascorbate peroxidase (APX) is a heme-containing protein that plays a central role in scavenging H(2)O(2) in higher plants. The structure of stromal APX (sAPX) was determined at 1.6 A to an R-factor of 19.1% and an R-free-factor of 22.3%. The electrostatic potential of the gamma-channel that connects the molecular surface of sAPX to the gamma-edge of heme was more positive than that of cytosolic APX (cAPX) from pea, so sAPX might bind more easily with ascorbate than cAPX. The overall structure of sAPX was similar to those of cAPX from pea and cytochrome c peroxidase (CCP) from yeast, with a substantial difference in a loop structure located in the vicinity of the heme. The side chain of Arg169 in sAPX corresponding to His169 in cAPX and His181 in CCP extended in the opposite direction from the heme, forming two hydrogen bonds with carbonyl groups in the loop structure. The rapid inactivation of sAPX might be due to the characteristic conformation of Arg169 owing to the loop structure of sAPX.     

Investigation of parenchymal cell differentiation in organotypic slice culture of mouse fetal liver under administration of sodium butyrate

The fetal mouse liver tissues in our organotypic slice culture were spread and flattened for at least 3 weeks; small, round cells were distributed in the center and polygonal cells were seen in the periphery. Ultrastructurally, polygonal cells showed abundant rough endoplasmic reticulum and mitochondria. They expressed albumin (ALB) and α-fetoprotein (AFP) for at least 3 weeks, and Cx32-immunoreactivity was also seen in a plaque on the cells. Many proliferating cell nuclear antigen (PCNA)-positive cells were observed at the periphery, and there were scattered CK-19-positive cells. The spreading of the fetal liver tissue in organotypic slice culture was reduced in medium containing sodium butyrate (SB). The expression of ALB was well maintained in polyglonal cells of the SB(+) group 3 weeks after culture and AFP-immunoreactivity was decreased in the SB(+) group. The concentration of ALB in the medium was significantly higher in the SB(+) than in the SB(-) group. CK-19-positive cells in the SB(+) group were increased in number more than those in the SB(-) group. PCNA-positive cells were less numerous in the SB(+) group, and Cx32-positive plaques were increased. SB can help immature hepatocytes to differentiate into the mature type and the cholangiocytic lineage, reducing their proliferation. These findings suggest that parenchymal cells in our organotypic slice culture of the fetal mouse liver can maintain structure and function as in vivo for the long term, and SB is shown to be a differentiation inducer of parenchymal cells in the slice culture. This revised version was published online in July 2006 with corrections to the Cover Date.     

Genotoxic activation of benzophenone and its two metabolites by human cytochrome P450s in SOS/umu assay

The genotoxic potential of benzophenone and its metabolically related compounds, benzhydrol and p-benzoylphenol, was investigated using human cytochrome P450 (P450) enzymes. Benzophenone and its two metabolites (0.1-1mM) showed a suppression of bacterial growth without any P450 system, but no induction of umu gene expression was observed in Salmonella typhimurium TA1535/pSK1002. Human liver microsomes induced the bacterial cytotoxicity of these compounds without any umu gene expression. On the other hand, with the addition of Escherichia coli membranes expressing recombinant human P450 2A6 and NADPH-cytochrome P450 reductase (NPR), benzophenone showed umu gene expression (64 umu units/min/nmol) P450 2A6). Moderate activation of benzophenone by P450 1A1/NPR membranes, 1A2/NPR membranes, or 1B1/NPR membranes was also observed. Activation of benzhydrol and p-benzoylphenol by the P450/NPR system was similar to that of benzophenone. These results suggest that benzophenone and its metabolically related benzhydrol and p-benzoylphenol can be bioactivated by P450 2A6 and P450 family 1 enzymes. Until now, benzophenone has been investigated mainly in terms of estrogenic activity and cytotoxicity, however, the genotoxic activation of benzophenone by human cytochrome P450s should be examined in terms of the risks to humans.     

Allele frequencies of single nucleotide polymorphisms in the second exon of the myoglobin gene among the Japanese

The difference in the allele frequencies of two single nucleotide polymorphisms (SNPs) in the second exon of the myoglobin gene between Japanese and other populations is reported. These SNPs are the substitutions of (A79G) and (T109C), and they were investigated by a single polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) analysis followed by direct sequencing. The substitutions were always linked and two alleles were found in the samples used: the A-T allele with no substitution at positions (79A) and (109T) and the G-C allele with substitutions of (79G) and (109C). The frequencies of these alleles were 0.755 and 0.245, respectively, and they were found to be in Hardy-Weinberg equilibrium. The distribution of alleles in the Japanese population was significantly different from that reported among whites, blacks, and Hispanics (p < 0.0001).     

Purification and characterization of multiple forms of rat liver xanthine oxidoreductase expressed in baculovirus-insect cell system

cDNA of rat liver xanthine oxidoreductase (XOR), a molybdenum-containing iron-sulfur flavoprotein, was expressed in a baculovirus-insect cell system. The expressed XOR consisted of a heterogeneous mixture of native dimeric, demolybdo-dimeric, and monomeric forms, each of which was separated and purified to homogeneity. All the expressed forms contained flavin, of which the semiquinone form was stable during dithionite titration after dithiothreitol treatment, indicating that the flavin domains of all the expressed molecules have the intact conformations interconvertible between NAD(+)-dependent dehydrogenase (XDH) and O(2)-dependent oxidase (XO) types. The absorption spectrum and metal analyses showed that the monomeric form lacks not only molybdopterin but also one of the iron-sulfur centers. The reductive titration of the monomer with dithionite showed that the monomeric form required only three electrons for complete reduction, and the redox potential of the iron-sulfur center in the monomeric form is a lower value than that of FAD. In contrast to native or demolybdo-dimeric XDHs, the monomer showed a very slow reductive process with NADH under anaerobic conditions, although the conformation around FAD is a dehydrogenase form, suggesting the important role of the iron-sulfur center in the reductive process of FAD with the reduced pyridine nucleotide.     

Generation of amyloid beta protein from a presenilin-1 and betaAPP complex

Presenilin-1 (PS1) is a causative gene in early onset familial Alzheimer's disease (FAD). FAD-linked mutant PS1s significantly increased Abeta40 and Abeta42(43) levels (P < 0.001) and decreased the production of an 11.4 kD (beta-stub) and an 8.7 kD (alpha-stub) carboxyl-terminal fragment of amyloid beta precursor protein (betaAPP-CTFs) (P < 0.01). In the 2% CHAPS extracted lysates, the complex containing the amino-terminal fragment of PS1 (PS1-NTF), the carboxyl-terminal fragments of PS1 (PS1-CTF), and betaAPP-CTFs was identified. Incubation of this isolated complex at pH 6.4 showed the direct generation of Abeta40 and gamma-stub from this complex. This reaction was inhibited by a gamma-secretase inhibitor. The degrading rate of a co-precipitated beta-stub was facilitated under the presence of FAD-linked mutant PS1s. This findings suggest that the direct generation of Abeta from the complex may play an important role in the pathogenesis of Alzheimer's disease.     

A long CAG repeat in the mouse Sca1 locus replicates SCA1 features and reveals the impact of protein solubility on selective neurodegeneration

A single nicotine exposure increases dopamine levels in the mesolimbic reward system for hours, but nicotine concentrations experienced by smokers desensitize nAChRs on dopamine neurons in seconds to minutes. Here, we show that persistent modulation of both GABAergic and glutamatergic synaptic transmission by nicotine can contribute to the sustained increase in dopamine neuron excitability. Nicotine enhances GABAergic transmission transiently, which is followed by a persistent depression of these inhibitory inputs due to nAChR desensitization. Simultaneously, nicotine enhances glutamatergic transmission through nAChRs that desensitize less than those on GABA neurons. The net effect is a shift toward excitation of the dopamine reward system. These results suggest that spatial and temporal differences in nicotinic receptor activity on both excitatory and inhibitory neurons in reward areas coordinate to reinforce nicotine self-administration.     

Variability of ground reaction forces during treadmill walking.

The purpose of this study was to investigate whether or not the neuromuscular locomotor system is optimized at a unique speed by examining the variability of the ground reaction force (GRF) pattern during walking in relation to different constant speeds. Ten healthy male subjects were required to walk on a treadmill at 3.0, 4.0, 5.0, 6.0, 7.0, and 8.0 km/h. Three components [vertical (F(z)), anteroposterior (F(y)), and mediolateral (F(x)) force] of the GRF were independently measured for approximately 35 steps consecutively for each leg. To quantify the GRF pattern, five indexes (first and second peaks of F(z), first and second peaks of F(y), and F(x) peak) were defined. Coefficients of variation were calculated for these five indexes to evaluate the GRF variability for each walking speed. It became clear for first and second peaks of F(z) and F(x) peak that index variabilities increased in relation to increments in walking speed, whereas there was a speed (5.5-5.8 km/h) at which variability was minimum for first and second peaks of F(y), which were related to forward propulsion of the body. These results suggest that there is "an optimum speed" for the neuromuscular locomotor system but only for the propulsion control mechanism.