Mutations at Asp112 adjacent to the trinuclear Cu center in CueO as the proton donor in the four-electron reduction of dioxygen

Asp112 adjacent to the trinuclear Cu center of a multicopper oxidase, CueO was mutated for Glu, Ala and Asn. Mutations on Asp112 affected not only spectroscopic and magnetic properties derived from the trinuclear Cu center but also enzyme activities. The uncoordinated Asp112 was found to play multiple roles to promote the binding of dioxygen at the trinuclear Cu center and to accelerate the conversion of dioxygen to water molecules by facilitating the supply of H+ to the reaction intermediates.     

Novel functions of ribosomal protein S6 in growth and differentiation of Dictyostelium cells

We have previously shown that in Dictyostelium cells a 32 kDa protein is rapidly and completely dephosphorylated in response to starvation that is essential for the initiation of differentiation (Akiyama & Maeda 1992). In the present work, this phosphoprotein was identified as a homologue (Dd-RPS6) of ribosomal protein S6 (RPS6) that is an essential member for protein synthesis. As expected, Dd-RPS6 seems to be absolutely required for cell survival, because we failed to obtain antisense-RNA mediated cells as well as Dd-rps6-null cells by homologous recombination in spite of many trials. In many kinds of cell lines, RPS6 is known to be located in the nucleus and cytosol, but Dd-RPS6 is predominantly located in the cell cortex with cytoskeletons, and in the contractile ring of just-dividing cells. In this connection, the overexpression of Dd-RPS6 greatly impairs cytokinesis during axenic shake-cultures in growth medium, resulting in the formation of multinucleate cells. Much severe impairment of cytokinesis was observed when Dd-RPS6-overexpressing cells (Dd-RPS6(OE) cells) were incubated on a living Escherichia coli lawn. The initiation of differentiation triggered by starvation was also delayed in Dd-RPS6(OE) cells. In addition, Dd-RPS6(OE) cells exhibit defective differentiation into prespore cells and spores during late development. Thus, it is likely that the proper expression of Dd-RPS6 may be of importance for the normal progression of late differentiation as well as for the initiation of differentiation.     

A high‐fat diet exacerbates the Alzheimer's disease pathology in the hippocampus of the App NL−F/NL−F knock‐in mouse model

Insulin resistance and diabetes mellitus are major risk factors for Alzheimer''s disease (AD), and studies with transgenic mouse models of AD have provided supportive evidence with some controversies. To overcome potential artifacts derived from transgenes, we used a knock‐in mouse model, App^{NL−F/NL−F} , which accumulates Aβ plaques from 6 months of age and shows mild cognitive impairment at 18 months of age, without the overproduction of APP. In the present study, 6‐month‐old male App^{NL−F/NL−F} and wild‐type mice were fed a regular or high‐fat diet (HFD) for 12 months. HFD treatment caused obesity and impaired glucose tolerance (i.e., T2DM conditions) in both wild‐type and App^{NL−F/NL−F} mice, but only the latter animals exhibited an impaired cognitive function accompanied by marked increases in both Aβ deposition and microgliosis as well as insulin resistance in the hippocampus. Furthermore, HFD‐fed App^{NL−F/NL−F} mice exhibited a significant decrease in volume of the granule cell layer in the dentate gyrus and an increased accumulation of 8‐oxoguanine, an oxidized guanine base, in the nuclei of granule cells. Gene expression profiling by microarrays revealed that the populations of the cell types in hippocampus were not significantly different between the two mouse lines, regardless of the diet. In addition, HFD treatment decreased the expression of the Aβ binding protein transthyretin (TTR) in App^{NL−F/NL−F} mice, suggesting that the depletion of TTR underlies the increased Aβ deposition in the hippocampus of HFD‐fed App^{NL−F/NL−F} mice.     

Environmental dependency of gene knockouts on phenotype microarray analysis in Escherichia coli

Systematic studies have revealed that single gene deletions often display little phenotypic effects under laboratory conditions and that in many cases gene dispensability depends on the experimental conditions. To elucidate the environmental dependency of genes, we analyzed the effects of gene deletions by Phenotype MicroArray? (PM), a system for quantitative screening of thousands of phenotypes in a high-throughput manner. Here, we proposed a new statistical approach to minimize error inherent in measurements of low respiration rates and find which mutants showed significant phenotypic changes in comparison to the wild-type. We show analyzing results from comprehensive PM assays of 298 single-gene knockout mutants in the Keio collection and two additional mutants under 1,920 different conditions. We focused on isozymes of these genes as simple duplications and analyzed correlations between phenotype changes and protein expression levels. Our results revealed divergence of the environmental dependency of the gene among the knockout genes and have also given some insights into possibilities of alternative pathways and availabilities of information on protein synthesis patterns to classify or predict functions of target genes from systematic phenotype screening.     

Fine mapping of <Emphasis Type="Italic">Sta1</Emphasis>, a quantitative trait locus determining stele transversal area,on rice chromosome 9

The stele (root vascular cylinder) in plants plays an important role in the transport of water and nutrients from the root to the shoot. A quantitative trait locus (QTL) on rice chromosome 9 that controls stele transversal area (STA) was previously detected in an F₃ mapping population derived from a cross between the lowland cultivar ‘IR64’, with a small STA, and the upland cultivar ‘Kinandang Patong’, with a large STA. To identify the gene(s) underlying this QTL, we undertook fine mapping of the locus. We screened eight plants from BC₂F₃ lines in which recombination occurred near the QTL. Progeny testing of BC₂F₄ plants was used to determine the genotype classes for the QTL in each BC₂F₃ line. Accordingly, the STA QTL Sta1 (Stele Transversal Area 1) was mapped between the InDel markers ID07_12 and ID07_14. A candidate genomic region for Sta1 was defined more precisely between markers RM566 and RM24334, which delimit a 359-kb interval in the reference cultivar ‘Nipponbare’. A line homozygous for the ‘Kinandang Patong’ allele of Sta1 had an STA approximately 28.4% larger than that of ‘IR64’. However, Sta1 did not influence maximum or total root length, suggesting that this QTL specifically controls STA.     

Cytodiagnostic problems in uterine sarcoma. Analysis according to a novel classification of tumor growth types

OBJECTIVE: To clarify the cytologic diagnostic problems of uterine sarcomas and the differential properties between pure sarcomas and carcinosarcomas. STUDY DESIGN: Four leiomyosarcomas and 21 carcinosarcomas (homologous and heterologous) treated at the Saitama Cancer Center from 1991 to 2000 were analyzed macroscopically and microscopically. RESULTS: Of 4 leiomyosarcomas, 3 were intramuscular, localized type, with a negative diagnosis for sarcoma. Of 21 carcinosarcomas, 7 were exophytic type with little necrosis (B-1), 5 were exophytic type with marked necrosis (B-2), 6 were exophytic type with a small sarcomatous component (B-3), and 3 were endophytic type (B-4). All endometrial smears were positive for sarcoma in B-1, whereas 5 of 14 (36%) were positive in the latter 3 types (B-2, 3 and 4). CONCLUSION: In pure leiomyosarcomas, the sarcomatous portions are usually covered with normal endometrium. In carcinosarcomas, sarcomatous component is relatively limited in some cases and frequently covered with marked necrosis or carcinomatous tissue. These pathologically specific findings should make cytologic diagnosis difficult in uterine sarcomas.     

Analysis of medium-chain acyl-coenzyme A esters in mouse tissues by liquid chromatography-electrospray ionization mass spectrometry

Medium-chain acyl-coenzyme A (CoA) esters are key metabolites in lipid metabolism. Liquid chromatography-electrospray ionization mass spectrometry analysis of medium-chain acyl-CoA esters is described. Eight medium-chain acyl-CoA esters were well separated on a C(8)-MS reversed-phase column using a linear gradient of ammonium acetate buffer (pH 5.3)-acetonitrile. The positive-ion mass spectra of all the saturated and unsaturated medium-chain acyl-CoA esters gave dominant [M+H](+) ions, whereas their negative-ion mass spectra showed abundant [M-H](-) and [M-2H](2-) ions. The positive-ion mode of operation was slightly less sensitive than the negative-ion detection mode. Five medium-chain acyl-CoA esters of C(6:0), C(8:0), C(8:1), C(10:0), and C(10:1) in liver, heart, kidney, and brain from the mouse were identified. The predominant acyl-CoA peaks were C(6:0), C(8:0), and C(10:0). Small amounts of medium-chain acyl-CoAs of C(8:1) and C(10:1) were detected only in heart and kidney. The analytical method is very useful for the analysis of medium-chain acyl-CoA esters in the tissues.     

Syntheses and crystal structures of the first iridium complexes with m- and p-terphenyl (tp). {[Ir2(p-tp)(cod)2](BF4)2 · 2CH2Cl2}3 and [Ir(m-tp)(η-C5Me5)](BF4)2

Novel two iridium terphenyl complexes were prepared and their structures were characterized crystallographically. The reaction of [Ir(cod)₂]BF₄ with p-terphenyl (p-tp) in CH₂Cl₂ was carried out to afford dinuclear Ir(I) complex {[Ir₂(p-tp)(cod)₂](BF₄)₂ · 2CH₂Cl₂}₃ (cod=1,5-cyclooctadiene) (1 · 2CH₂Cl₂), whereas the reaction of the intermediate [Ir(η⁵-C₅Me₅)(Me₂CO)₃]³⁺ in Me₂CO with m-terphenyl (m-tp) was done to provide mononuclear Ir(III) complex [Ir(m-tp)(η⁵-C₅Me₅)](BF₄)₂ (2). In complex 1 · 2CH₂Cl₂, two Ir atoms are η⁶-coordinated to both sides of terminal benzene rings from the upper and lower sides in the p-tp ligand, while one Ir atom is η⁶-coordinated to one side of the terminal benzene ring in the m-tp ligand in complex 2. Each crystal structure describes the first coordination mode found in metal complexes with the m- and p-tp ligands.