Analysis of Intraspecies Diversity in Wheat and Barley Genomes Identifies Breakpoints of Ancient Haplotypes and Provides Insight into the Structure of Diploid and Hexaploid Triticeae Gene Pools

A large number of wheat (Triticum aestivum) and barley (Hordeum vulgare) varieties have evolved in agricultural ecosystems since domestication. Because of the large, repetitive genomes of these Triticeae crops, sequence information is limited and molecular differences between modern varieties are poorly understood. To study intraspecies genomic diversity, we compared large genomic sequences at the Lr34 locus of the wheat varieties Chinese Spring, Renan, and Glenlea, and diploid wheat Aegilops tauschii. Additionally, we compared the barley loci Vrs1 and Rym4 of the varieties Morex, Cebada Capa, and Haruna Nijo. Molecular dating showed that the wheat D genome haplotypes diverged only a few thousand years ago, while some barley and Ae. tauschii haplotypes diverged more than 500,000 years ago. This suggests gene flow from wild barley relatives after domestication, whereas this was rare or absent in the D genome of hexaploid wheat. In some segments, the compared haplotypes were very similar to each other, but for two varieties each at the Rym4 and Lr34 loci, sequence conservation showed a breakpoint that separates a highly conserved from a less conserved segment. We interpret this as recombination breakpoints of two ancient haplotypes, indicating that the Triticeae genomes are a heterogeneous and variable mosaic of haplotype fragments. Analysis of insertions and deletions showed that large events caused by transposable element insertions, illegitimate recombination, or unequal crossing over were relatively rare. Most insertions and deletions were small and caused by template slippage in short homopolymers of only a few base pairs in size. Such frequent polymorphisms could be exploited for future molecular marker development.     

A method for terminus proteomics: Selective isolation and labeling of N-terminal peptide from protein through transamination reaction

A novel method for selectively labeling and isolating N-terminal peptide from protein has been developed. An N^α-amino group of protein was converted to a carbonyl group through transamination reaction and the resulting carbonyl group was modified with O-(4-nitrobenzyl)hydroxylamine (NBHA). After proteolytic digestion using Grifola frondosa metalloendopeptidase (LysN), the modified N-terminal peptide remained unbound in the following treatment using amino-reactive p-phenylenediisothiocyanate (DITC) glass, whereas peptides other than the N-terminal peptide were effectively scavenged from the supernatant solution. The modified N-terminal peptide was thus successfully isolated and sequenced by matrix-assisted laser desorption/ionization tandem mass spectrometry (MALDI-MS/MS) analysis.     

Dominant-negative mutant hepatocyte nuclear factor 1α induces diabetes in transgenic-cloned pigs

Pigs have been recognized as an excellent biomedical model for investigating a variety of human health issues. We developed genetically modified pigs that exhibit the apparent symptoms of diabetes. Transgenic cloned pigs carrying a mutant human hepatocyte nuclear factor 1α gene, which is known to cause the type 3 form of maturity-onset diabetes of the young, were produced using a combined technology of intracytoplasmic sperm injection-mediated gene transfer and somatic cell nuclear transfer. Although most of the 22 cloned offspring obtained died before weaning, four pigs that lived for 20–196 days were diagnosed as diabetes mellitus with nonfasting blood glucose levels greater than 200 mg/dl. Oral glucose tolerance test on a cloned pig also revealed a significant increase of blood glucose level after glucose loading. Histochemical analysis of pancreas tissue from the cloned pigs showed small and irregularly formed Langerhans Islets, in which poor insulin secretion was detected.     

A newly designed and constructed 20 kHz magnetic field exposure facility for in vivo study

Exposure to man-made electromagnetic fields has increased over the past century. As a result of exposure to these fields, concerns have been raised regarding the relationship between electromagnetic fields and human health. Interest in the biological and health effects of intermediate frequency (IF) magnetic fields has grown recently because of the increase in public concern. In order to investigate whether IF magnetic fields have biological effects, we have developed a 20 kHz (IF) magnetic field exposure system for in vivo studies. The exposure facility was designed to study the biological effects of IF magnetic field on laboratory animals. The facility consists of a 9 m x 9 m x 5 m high room containing seven separate rooms including a 5.3 m x 4.5 m x 3 m high specific-pathogen free exposure room. The dimensions of the exposure system are 1.6 m x 1.6 m x 1.616 m high located inside this exposure room. The system is designed to provide magnetic fields up to 200 microT at 20 kHz with the uniformity within +/-5% over the space occupied by animals. After constructing the facility, performance tests were carried out. As a result, it was confirmed that our facility met requirements for evaluation of the biological effects of IF magnetic field on small animal experiments. In this paper, the design, construction, and results of evaluation of an animal exposure facility for the in vivo biological effects of an IF magnetic field are described.     

Identification of genetic loci involved in diabetes using a rat model of depression

While diabetic patients often present with comorbid depression, the underlying mechanisms linking diabetes and depression are unknown. The Wistar Kyoto (WKY) rat is a well-known animal model of depression and stress hyperreactivity. In addition, the WKY rat is glucose intolerant and likely harbors diabetes susceptibility alleles. We conducted a quantitative trait loci (QTL) analysis in the segregating F₂ population of a WKY × Fischer 344 (F344) intercross. We previously published QTL analyses for depressive behavior and hypothalamic-pituitary-adrenal (HPA) activity in this cross. In this study we report results from the QTL analysis for multiple metabolic phenotypes, including fasting glucose, post-restraint stress glucose, postprandial glucose and insulin, and body weight. We identified multiple QTLs for each trait and many of the QTLs overlap with those previously identified using inbred models of type 2 diabetes (T2D). Significant correlations were found between metabolic traits and HPA axis measures, as well as forced swim test behavior. Several metabolic loci overlap with loci previously identified for HPA activity and forced swim behavior in this F₂ intercross, suggesting that the genetic mechanisms underlying these traits may be similar. These results indicate that WKY rats harbor diabetes susceptibility alleles and suggest that this strain may be useful for dissecting the underlying genetic mechanisms linking diabetes, HPA activity, and depression.     

Screening system for <Emphasis Type="SmallCaps">d</Emphasis>-Asp-containing proteins using <Emphasis Type="SmallCaps">d</Emphasis>-aspartyl endopeptidase and two-dimensional gel electrophoresis

d-Asp-containing proteins have been implicated in many aging-related diseases. To clarify the role of d-Asp-containing proteins in such diseases, we developed a screening system for these proteins using a d-aspartyl endopeptidase that specifically cleaves the proteins at the C-terminus. The digested proteins were detected by means of two-dimensional gel electrophoresis and identified using nano-liquid chromatography/tandem mass spectrometry. We were able to detect myelin basic protein, a known d-Asp-containing protein, in the brain tissues of mice; this indicates that our system is effective for screening d-Asp-containing proteins.     

Treatment and phosphorus removal from high-concentration organic wastewater by the yeast Hansenula anomala J224 PAWA

A flocculent yeast, Hansenula anomala J224 PAWA, bred in this study, accumulated twice as much phosphorus as the wild type. Over a 30-d period, PAWA removed 70–80% of dissolved total phosphorus from sweet-potato and barley shochu wastewaters (alcoholic distillery wastewaters) while the wild type removed only 30%. Waste sludge was easily separated from effluent wastewater because PAWA cells made large flocks that rapidly settled. Component analysis suggested that PAWA sludge could be used as a protein source for feedstuff and as a phosphorus source for fertilizer. Under anaerobic conditions, denitrification was rapid, resulting in the removal of large amounts of nitrogen from barley shochu wastewater. These results suggest that small shochu manufacturers could benefit from using PAWA to remove phosphorus and organic compounds and then by using a combination of the upflow anaerobic sludge blanket and the downflow hanging sponge method (UASB-DHS method) for nitrification/denitrification.     

MCP-1 expressed by osteoclasts stimulates osteoclastogenesis in an autocrine/paracrine manner

Monocyte chemoattractant protein-1 (MCP-1) is a chemokine that plays a critical role in the recruitment and activation of leukocytes. Here, we describe that multinuclear osteoclast formation was significantly inhibited in cells derived from MCP-1-deficient mice. MCP-1 has been implicated in the regulation of osteoclast cell-cell fusion; however defects of multinuclear osteoclast formation in the cells from mice deficient in DC-STAMP, a seven transmembrane receptor essential for osteoclast cell-cell fusion, was not rescued by recombinant MCP-1. The lack of MCP-1 in osteoclasts resulted in a down-regulation of DC-STAMP, NFATc1, and cathepsin K, all of which were highly expressed in normal osteoclasts, suggesting that osteoclast differentiation was inhibited in MCP-1-deficient cells. MCP-1 alone did not induce osteoclastogenesis, however, the inhibition of osteoclastogenesis in MCP-1-deficient cells was restored by addition of recombinant MCP-1, indicating that osteoclastogenesis was regulated in an autocrine/paracrine manner by MCP-1 under the stimulation of RANKL in osteoclasts.