MIG-seq is an effective method for high-throughput genotyping in wheat (Triticum spp.)

MIG-seq (Multiplexed inter-simple sequence repeats genotyping by sequencing) has been developed as a low cost genotyping technology, although the number of polymorphisms obtained is assumed to be minimal, resulting in the low application of this technique to analyses of agricultural plants. We applied MIG-seq to 12 plant species that include various crops and investigated the relationship between genome size and the number of bases that can be stably sequenced. The genome size and the number of loci, which can be sequenced by MIG-seq, are positively correlated. This is due to the linkage between genome size and the number of simple sequence repeats (SSRs) through the genome. The applicability of MIG-seq to population structure analysis, linkage mapping, and quantitative trait loci (QTL) analysis in wheat, which has a relatively large genome, was further evaluated. The results of population structure analysis for tetraploid wheat showed the differences among collection sites and subspecies, which agreed with previous findings. Additionally, in wheat biparental mapping populations, over 3,000 SNPs/indels with low deficiency were detected using MIG-seq, and the QTL analysis was able to detect recognized flowering-related genes. These results revealed the effectiveness of MIG-seq for genomic analysis of agricultural plants with large genomes, including wheat.     

Some properties of the apoenzyme of NADPH-adreno-ferredoxin reductase from bovine adrenocortical mitochondria

1. An apo-NADPH-adreno-ferredoxin reductase (EC 1.18.1.2) was obtained from bovine adrenocortical mitochondria and its physicochemical properties were investigated. 2. The effects of various substances such as NADPH, FAD and adreno-ferredoxin on the interaction of the apo-reductase were investigated by various column chromatographies. 3. The apo- and holo-reductases were found to be separated by adreno-ferredoxin affinity chromatography. 4. The removal of FAD from NADPH-adreno-ferredoxin reductase did not affect the net charge of the reductase. 5. The values of s20,w of apo- and holo-reductases were 3.8 x 10(-13) sec and 3.9 x 10(-13) sec, respectively. 6. The apo-reductase was more easily denatured by heat treatment than the holo-reductase. 7. FAD, and adreno-ferredoxin and both could protect the apo-reductase from thermal inactivation.     

Accumulation and aggregation of amyloid beta-protein in late endosomes of Niemann-pick type C cells

There is growing evidence suggesting that cholesterol metabolism is linked to susceptibility to Alzheimer's disease by influencing amyloid beta-protein (Abeta) metabolism. However, the precise cellular linkage sites between cholesterol and Abeta have not yet been clarified. To address this issue, we investigated Niemann-Pick type C (NPC) model cells and NPC mutant cells, which showed aberrant cholesterol trafficking. We observed a remarkable Abeta accumulation in late endosomes of both NPC model cells and mutant cells where cholesterol accumulates and a significant accumulation in the NPC mouse brain. This Abeta accumulation was independent of its constitutive secretion and production through an endocytic pathway. In addition, it is characterized by a marked predominance of Abeta42 and insolubility in SDS, suggesting the presence of aggregated Abeta in late endosomes. Most importantly, Abeta accumulation is coupled with the cholesterol levels in late endosomes. Thus, late endosomes of NPC cells are a novel pool of aggregated Abeta42 as well as cholesterol, suggesting a direct interaction between aggregated Abeta and cholesterol.     

Mechanical unfolding of single filamin A (ABP-280) molecules detected by atomic force microscopy

Filamin A (ABP-280), which is an actin-binding protein of 560 kDa as a dimer, can, together with actin filaments, produce an isotropic cross-linked three-dimensional network (actin/filamin A gel) that plays an important role in mechanical responses of cells in processes such as maintenance of membrane stability and translational locomotion. In this study, we investigated the mechanical properties of single filamin A molecules using atomic force microscopy. In force-extension curves, we observed sawtooth patterns corresponding to the unfolding of individual immunoglobulin (Ig)-fold domains of filamin A. At a pulling speed of 0.37 microm/s, the unfolding interval was sharply distributed around 30 nm, while the unfolding force ranged from 50 to 220 pN. This wide distribution of the unfolding force can be explained by variation in values of activation energy and the width of activation barrier of 24 Ig-fold domains of the filamin A at the unfolding transition. This unfolding can endow filamin A with great extensibility. The refolding of the unfolded chain of filamin A occurred when the force applied to the protein was reduced to near zero, indicating that its unfolding is reversible. Based on these results, we discuss here the physiological implications of the mechanical properties of single filamin A molecules.     

Metabolic activation of carcinogenic 1-nitropyrene by human cytochrome P450 1B1 in Salmonella typhimurium strain expressing an O-acetyltransferase in SOS/umu assay

Metabolic activation of 1-nitropyrene (1-NP) by human cytochrome P450 (P450) family 1 enzymes co-expressed with NADPH-cytochrome P450 reductase (NPR) in Escherichia coli membranes was investigated. 1-NP induced umu gene expression in Salmonella typhimurium TA1535/pSK1002 in the absence of any P450 system, but the activities were influenced by the levels of bacterial O-acetyltransferase (OAT) and nitroreductase. Metabolic activation of 1-NP by human P450 1B1/NPR membranes was observed and was influenced by the levels of OAT levels in tester strains. Metabolic activation of 1-NP (0.3microM) by P450 1B1 was 750 umu units/min/nmol P450 1B1 in an OAT-overexpressing strain NM2009. The metabolic activation of 1-NP (3-30microM) was similar (approximately 300 umu units/min/nmol P450 1B1) using TA1535/pSK1002 or OAT-deficient strain NM2000. P450 1B1 had the highest catalytic activities among P450 family 1 enzymes for the activation of 1-aminopyrene (1-AP) in the OAT-overexpressing strain NM2009, suggesting nitrenium ion formation via N-hydroxylation/O-acetylation. High-performance liquid chromatography (HPLC) analyses revealed the formation of 1-nitropyrene-6-ol and also 1-nitropyrene-3-ol, 1-nitropyrene-8-ol, and trans-4,5-dihydroxy-4,5-diol-1-nitropyrene from 1-NP (10microM), catalyzed by P450 1B1. These results indicate that 1-NP can be activated by human P450 1B1 to a genotoxic agent by nitroreduction/O-acetylation at low substrate concentrations and probably by epoxidation (independent of OAT) at high concentrations.     

One-electron-transfer reactions in biochemical systems V. Difference in the mechanism of quinone reduction by the NADH dehydrogenase and the NAD(P)H dehydrogenase (DT-diaphorase)

The mitochondrial NADH dehydrogenase catalyzes a one-electron reduction of quinones. Semiquinones thus formed have the hyperfine structures of their free anion radicals and are suggested to be detached from the enzyme. In the presence of suitable electron acceptors electron transfer occurs from the semiquinone to the acceptor. The mechanism of quinone reduction by spinach ferredoxin-NADP reductase is the same as that by the NADH dehydrogenase.

On the other hand, the NAD(P)H dehydrogenase (DT-diaphorase) prepared from liver soluble fraction catalyzes a typical two-electron reduction of quinones such as p-benzoquinone and 2-methyl-1,4-naphthoquinone. The mechanisms of one-electron and two-electron reduction of quinones are readily distinguishable by the use of an electron spin resonance spectrometer equipped with a flow apparatus and also by the use of an appropriate set of electron acceptors.

It is concluded that the reduction of quinones and oxygen by flavoproteins falls into three mechanistic categories: one-electron, two-electron and mixed-type reactions.