Putative quantitative trait loci associated with calcium content in soybean seed

Seed calcium content is an important quality attribute of specialty soybean [Glycine max (L.) Merr.] for soyfoods. However, analyzing seed for calcium content is time consuming and labor intensive. Knowing quantitative trait loci (QTL) for seed calcium will facilitate the development of elite cultivars with proper calcium content through marker-assisted selection (MAS). The objective of this study was to identify major QTL associated with calcium content in soybean seed. Calcium content was tested in 178 F(2:3) and 157 F(2:4) lines derived from the cross of SS-516 (low calcium) x Camp (high calcium). The F(2:3) lines were genotyped with 148 simple sequence repeat markers in a previous study on seed hardness, and the genotypic data were used in the QTL analysis of the current study. Four QTL designated as Ca1, Ca2, Ca3, and Ca4 on linkage groups (LGs) A2, I, and M were identified by both single-marker analysis and composite-interval mapping, and the QTL accounted for 10.7%, 16.3%, 14.9%, and 9.7% of calcium content variation, respectively. In addition, multiple-interval mapping analysis revealed a significant dominant-by-dominant interaction effect between Ca1 and Ca3, which accounted for 4.3% calcium content variation. These QTL will facilitate the implementation of MAS for calcium content in soybean-breeding programs.     

Thermodynamic Analyses of Nucleotide Binding to an Isolated Monomeric β Subunit and the α3β3γ Subcomplex of F1-ATPase

Rotation of the γ subunit of the F₁-ATPase plays an essential role in energy transduction by F₁-ATPase. Hydrolysis of an ATP molecule induces a 120° step rotation that consists of an 80° substep and 40° substep. ATP binding together with ADP release causes the first 80° step rotation. Thus, nucleotide binding is very important for rotation and energy transduction by F₁-ATPase. In this study, we introduced a βY341W mutation as an optical probe for nucleotide binding to catalytic sites, and a βE190Q mutation that suppresses the hydrolysis of nucleoside triphosphate (NTP). Using a mutant monomeric βY341W subunit and a mutant α₃β₃γ subcomplex containing the βY341W mutation with or without an additional βE190Q mutation, we examined the binding of various NTPs (i.e., ATP, GTP, and ITP) and nucleoside diphosphates (NDPs, i.e., ADP, GDP, and IDP). The affinity (1/K_d) of the nucleotides for the isolated β subunit and third catalytic site in the subcomplex was in the order ATP/ADP > GTP/GDP > ITP/IDP. We performed van’t Hoff analyses to obtain the thermodynamic parameters of nucleotide binding. For the isolated β subunit, NDPs and NTPs with the same base moiety exhibited similar ΔH⁰ and ΔG⁰ values at 25°C. The binding of nucleotides with different bases to the isolated β subunit resulted in different entropy changes. Interestingly, NDP binding to the α₃β(Y341W)₃γ subcomplex had similar K_d and ΔG⁰ values as binding to the isolated β(Y341W) subunit, but the contributions of the enthalpy term and the entropy term were very different. We discuss these results in terms of the change in the tightness of the subunit packing, which reduces the excluded volume between subunits and increases water entropy.     

Critical role of a methyl group on the γ-lactone ring of annonaceous acetogenins in the potent inhibition of mitochondrial complex I

C34-epi and C34-epi-C35-trifluoro analogues of solamin, a mono-THF annonaceous acetogenin, were synthesized. Their inhibitory activity, along with previously synthesized analogues (C35-fluoro, C35-difluoro, and C35-trifluorosolamins), against bovine mitochondrial NADH–ubiquinone oxidoreductase (complex I) was determined. The present study revealed that the methyl group on the γ-lactone moiety is critical to the potent inhibition of complex I by natural acetogenins.     

The specific Na(+)/Ca(2+) exchange inhibitor SEA0400 prevents nitric oxide-induced cytotoxicity in SH-SY5Y cells

The Na⁺/Ca²⁺ exchanger (NCX) plays a role in the regulation of intracellular Ca²⁺ levels, and nitric oxide (NO) is involved in many pathological conditions including neurodegenerative disorders. We have previously found that sodium nitroprusside (SNP), an NO donor, causes apoptotic-like cell death in cultured glial cells via NCX-mediated pathways and the mechanism for NO-induced cytotoxicity is cell type-dependent. The present study examined using the specific NCX inhibitor 2-[4-[(2,5-difluorophenyl)methoxy]phenoxy]-5-ethoxyaniline (SEA0400) whether NCX is involved in NO-induced injury in cultured neuronal cells. The treatment of neuroblastoma SH-SY5Y cells with SNP resulted in apoptosis and the cytotoxicity was blocked by the mitogen-activated protein (MAP)/extracellular signal-regulated kinase (ERK) kinase inhibitor U0126 and the p38 MAP kinase (MAPK) inhibitor SB203580, but not by the c-Jun N-terminal kinase (JNK) inhibitor SP60012. SNP increased Ca²⁺ influx and intracellular Ca²⁺ levels. In addition, SNP increased ERK and p38 MAPK phosphorylation, and production of reactive oxygen species (ROS) in an extracellular Ca²⁺-dependent manner. These effects of SNP were prevented by SEA0400. SNP-induced cytotoxicity was not affected by inhibitors of the Ca²⁺, Na⁺ and store-operated/capacitative channels. Moreover, SNP-induced increase in intracellular Ca²⁺ levels, ROS production and decrease in cell viability were blocked by a cGMP-dependent protein kinase (PKG) inhibitor. These results suggest that Ca²⁺ influx via the reverse of NCX is involved in the cascade of NO-induced neuronal apoptosis and NO activates the NCX through guanylate cyclase/PKG pathway.     

Purification of Cytochrome P450 and Ferredoxin, Involved in Bisphenol A Degradation, from Sphingomonas sp. Strain AO1

In a previous study (M. Sasaki, J. Maki, K. Oshiman, Y. Matsumura, and T. Tsuchido, Biodegradation 16:449-459, 2005), the cytochrome P450 monooxygenase system was shown to be involved in bisphenol A (BPA) degradation by Sphingomonas sp. strain AO1. In the present investigation, we purified the components of this monooxygenase, cytochrome P450 (P450_bisd), ferredoxin (Fd_bisd), and ferredoxin reductase (Red_bisd). We demonstrated that P450_bisd and Fd_bisd are homodimeric proteins with molecular masses of 102.3 and 19.1 kDa, respectively, by gel filtration chromatography analysis. Spectroscopic analysis of Fd_bisd revealed the presence of a putidaredoxin-type [2Fe-2S] cluster. P450_bisd, in the presence of Fd_bisd, Red_bisd, and NADH, was able to convert BPA. The K_m and k_cat values for BPA degradation were 85 ± 4.7 μM and 3.9 ± 0.04 min⁻¹, respectively. NADPH, spinach ferredoxin, and spinach ferredoxin reductase resulted in weak monooxygenase activity. These results indicated that the electron transport system of P450_bisd might exhibit strict specificity. Two BPA degradation products of the P450_bisd system were detected by high-performance liquid chromatography analysis and were thought to be 1,2-bis(4-hydroxyphenyl)-2-propanol and 2,2-bis(4-hydroxyphenyl)-1-propanol based on mass spectrometry-mass spectrometry analysis. This is the first report demonstrating that the cytochrome P450 monooxygenase system in bacteria is involved in BPA degradation.     

Genome-wide search for genes that modulate inflammatory arthritis caused by Ali18 mutation in mice

Many of inflammatory diseases, including inflammatory arthritis, are multifactorial bases. The Ali18 semidominant mutation induced by N-ethyl-N-nitrosourea in the C3HeB/FeJ (C3H) genome causes spontaneous inflammation of peripheral limbs and elevated immunoglobulin E (IgE) levels in mice. Although the Ali18 locus was mapped to a single locus on chromosome 4, the arthritic phenotype of Ali18/+ mice was completely suppressed in F1 hybrid genetic backgrounds. To determine the chromosomal locations of the modifier loci affecting the severity of arthritis, an autosomal genome scan of 22 affected Ali18/+ F2 mice was conducted using C57BL/6J as a partner strain. Interestingly, regions on chromosomes 1 and 3 in C3H showed significant genetic interactions. Moreover, 174 N2 (backcross to Ali18/Ali18) and 267 F2 animals were used for measurement of arthritis scores and plasma IgE levels, and also for genotyping with 153 genome-wide single nucleotide polymorphism (SNP) markers. In N2 populations, two significant trait loci for arthritis scores on chromosomes 1 and 15 were detected. Although no significant scores were detected in F2 mice besides chromosome 4, a suggestive score was detected on chromosome 3. In addition, a two-dimensional genome scan using F2 identified five suggestive scores of chromosomal combinations, chromosomes 1 × 10, 2 × 6, 3 × 4, 4 × 9, and 6 × 15. No significant trait loci affecting IgE levels were detected in both N2 and F2 populations. Identification of the Ali18 modifier genes by further detailed analyses such as congenic strains and expression profiling may dissect molecular complexity in inflammatory diseases.     

Synthesis of C4-fluorinated solamins and their growth inhibitory activity against human cancer cell lines

C4-Fluorinated analogues of solamin, an antitumor acetogenin, were synthesized and investigated for their antitumor activities against 39 tumor cell lines. C4-Fluorinated solamins showed more potent growth inhibitory activity against cancer cell lines than solamin.     

Xenogenic macrophage immunization reduces atherosclerosis in apolipoprotein E knockout mice

Atherosclerosis is a complex chronic inflammatory disease in which macrophages play a critical role, and the intervention of the inflammatory process in atherogenesis could be a therapeutic strategy. In this study, we investigated the efficacy of xenogenic macrophage immunization on the atherosclerotic lesion formation in a model of murine atherosclerosis. Apolipoprotein E knockout (apoE-KO) mice were repeatedly immunized with formaldehyde-fixed cultured human macrophages (phorbol ester-stimulated THP-1 cells), using human serum albumin as a control protein or HepG2 cells as human control cells, once a week for four consecutive weeks. The vehicle phosphate-buffered saline was injected in the nonimmunized controls. THP-1 immunization induced antibodies that are immunoreactive with mouse macrophages. Although the plasma lipid levels were unchanged by the immunization, the atherosclerotic lesion area in the aortic root was significantly reduced by >50% in 16-wk-old THP-1-immunized apoE-KO mice compared with that in control mice. THP-1 immunization reduced in vivo macrophage infiltration, reduced in vitro macrophage adhesion, and changed cytokine production by macrophages to the antiatherogenic phenotype. Xenogenic macrophage immunization protects against the development of atherosclerosis in apoE-KO mice by modulating macrophage function in which antibodies induced by the immunization are likely to be involved. This method is a novel and potentially useful cell-mediated immune therapeutic technique against atherosclerosis. antibody; THP-1 cells     

Mapping of susceptibility and protective loci for acute GVHD in unrelated HLA-matched bone marrow transplantation donors and recipients using 155 microsatellite markers on chromosome 22

Despite matching donors and recipients for the human leukocyte antigens (HLAs) expressed by the major histocompatibility genomic region of the short arm of chromosome 6, several recipients still develop acute graft-versus-host disease (aGVHD) after bone marrow transplantation (BMT). This is possibly due to non-HLA gene polymorphisms, such as minor histocompatibility antigens (mHas) and genes coding for cytokines. However, a detailed genetic background for aGVHD has not yet been established. To find novel susceptibility and/or protective loci for aGVHD, a whole genome-wide association study of donors and recipients needs to be performed. As the first step to such a study, we retrospectively analyzed polymorphisms of 155 microsatellite markers spread across the long arm of chromosome 22 in 70 pairs of HLA-matched unrelated BMT donors and recipients. We performed individual typing and then compared the markers’ allele frequencies (1) between all the aGVHD (grades III and IV GVHD) and GVHD-free (grade 0 GVHD) groups in donors and recipients and (2) between the aGVHD and aGVHD-free groups in donor/recipient pairs that were matched and mismatched for the microsatellite marker’s allele. Screening of the microsatellite markers revealed five loci with a significant difference between the aGVHD and GVHD-free groups and revealed eight loci on chromosome 22, where the microsatellite allele mismatched markers were associated with aGVHD. This screening analysis suggests that several aGVHD-associated susceptible and protective loci exist on chromosome 22, which may encompass novel gene regions that need to be elucidated for their role in aGVHD.     

Mode of Bactericidal Action of Silver Zeolite and Its Comparison with That of Silver Nitrate

The properties of the bactericidal action of silver zeolite as affected by inorganic salts and ion chelators were similar to those of silver nitrate. The results suggest that the contact of the bacterial cell with silver zeolite, the consequent transfer of silver ion to the cell, and the generation of reactive oxygen species in the cell are involved in the bactericidal activity of silver zeolite.