A High-Density Genetic Map with Array-Based Markers Facilitates Structural and Quantitative Trait Locus Analyses of the Common Wheat Genome

The large genome and allohexaploidy of common wheat have complicated construction of a high-density genetic map. Although improvements in the throughput of next-generation sequencing (NGS) technologies have made it possible to obtain a large amount of genotyping data for an entire mapping population by direct sequencing, including hexaploid wheat, a significant number of missing data points are often apparent due to the low coverage of sequencing. In the present study, a microarray-based polymorphism detection system was developed using NGS data obtained from complexity-reduced genomic DNA of two common wheat cultivars, Chinese Spring (CS) and Mironovskaya 808. After design and selection of polymorphic probes, 13,056 new markers were added to the linkage map of a recombinant inbred mapping population between CS and Mironovskaya 808. On average, 2.49 missing data points per marker were observed in the 201 recombinant inbred lines, with a maximum of 42. Around 40% of the new markers were derived from genic regions and 11% from repetitive regions. The low number of retroelements indicated that the new polymorphic markers were mainly derived from the less repetitive region of the wheat genome. Around 25% of the mapped sequences were useful for alignment with the physical map of barley. Quantitative trait locus (QTL) analyses of 14 agronomically important traits related to flowering, spikes, and seeds demonstrated that the new high-density map showed improved QTL detection, resolution, and accuracy over the original simple sequence repeat map.     

Energy and electron transfer in the photosynthetic reaction center complex of Acidiphilium rubrum containing Zn-bacteriochlorophyll a studied by femtosecond up-conversion spectroscopy

A photosynthetic reaction center (RC) complex was isolated from a purple bacterium, Acidiphilium rubrum. The RC contains bacteriochlorophyll a containing Zn as a central metal (Zn-BChl a) and bacteriopheophytin a (BPhe a) but no Mg-BChl a. The absorption peaks of the Zn-BChl a dimer (P_Zn), the accessory Zn-BChl a (B_Zn), and BPhe a (H) at 4 K in the RC showed peaks at 875, 792, and 753 nm, respectively. These peaks were shorter than the corresponding peaks in Rhodobacter sphaeroides RC that has Mg-BChl a. The kinetics of fluorescence from P_Zn^*, measured by fluorescence up-conversion, showed the rise and the major decay with time constants of 0.16 and 3.3 ps, respectively. The former represents the energy transfer from B_Zn^* to P_Zn, and the latter, the electron transfer from P_Zn to H. The angle between the transition dipoles of B_Zn and P_Zn was estimated to be 36° based on the fluorescence anisotropy. The time constants and the angle are almost equal to those in the Rb. sphaeroides RC. The high efficiency of A. rubrum RC seems to be enabled by the chemical property of Zn-BChl a and by the L168HE modification of the RC protein that modifies P_Zn.     

Bioenergetics and Gene Silencing Approaches for Unraveling Nucleotide Recognition by the Human EIF2C2/Ago2 PAZ Domain

Gene silencing and RNA interference are major cellular processes that control gene expression via the cleavage of target mRNA. Eukaryotic translation initiation factor 2C2 (EIF2C2, Argonaute protein 2, Ago2) is considered to be the major player of RNAi as it is the core component of RISC complexes. While a considerable amount of research has focused on RNA interference and its associated mechanisms, the nature and mechanisms of nucleotide recognition by the PAZ domain of EIF2C2/Ago2 have not yet been characterized. Here, we demonstrate that the EIF2C2/Ago2 PAZ domain has an inherent lack of binding to adenine nucleotides, a feature that highlights the poor binding of 3′-adenylated RNAs with the PAZ domain as well as the selective high trimming of the 3′-ends of miRNA containing adenine nucleotides. We further show that the PAZ domain selectively binds all ribonucleotides (except adenosine), whereas it poorly recognizes deoxyribonucleotides. In this context, the modification of dTMP to its ribonucleotide analogue gave a drastic improvement of binding enthalpy and, hence, binding affinity. Additionally, higher in vivo gene silencing efficacy was correlated with the stronger PAZ domain binders. These findings provide new insights into the nature of the interactions of the EIF2C2/Ago2 PAZ domain.     

Ajuga Δ24-Sterol Reductase Catalyzes the Direct Reductive Conversion of 24-Methylenecholesterol to Campesterol

Dimunito/Dwarf1 (DWF1) is an oxidoreductase enzyme that is responsible for the conversion of C₂₈- and C₂₉-Δ²⁴⁽²⁸⁾-olefinic sterols to 24-methyl- and 24-ethylcholesterols. Generally, the reaction proceeds in two steps via the Δ²⁴⁽²⁵⁾ intermediate. In this study, we characterized the ArDWF1 gene from an expression sequence tag library of Ajuga reptans var. atropurpurea hairy roots. The gene was functionally expressed in the yeast T21 strain. The in vivo and in vitro study of the transformed yeast indicated that ArDWF1 catalyzes the conversion of 24-methylenecholesterol to campesterol. A labeling study followed by GC-MS analysis suggested that the reaction proceeded with retention of the C-25 hydrogen. The 25-H retention was established by the incubation of the enzyme with (23,23,25-²H₃,28-¹³C)-24-methylenecholesterol, followed by ¹³C NMR analysis of the resulting campesterol. Thus, it has been concluded that ArDWF1 directly reduces 24-methylenecholesterol to produce campesterol without passing through a Δ²⁴⁽²⁵⁾ intermediate. This is the first characterization of such a unique DWF1 enzyme. For comparison purposes, Oryza sativa DWF1 (OsDWF1) was similarly expressed in yeast. An in vivo assay of OsDWF1 supported the generally accepted two-step mechanism because the C-25 hydrogen of 24-methylenecholesterol was eliminated during its conversion to 24-methylcholesterol. As expected, the 24-methylcholesterol produced by OsDWF1 was a mixture of campesterol and dihydrobrassicasterol. Furthermore, the 24-methylcholesterol contained in the Ajuga hairy roots was determined to be solely campesterol through its analysis using chiral GC-MS. Therefore, ArDWF1 has another unique property in that only campesterol is formed by the direct reduction catalyzed by the enzyme.     

Novel Methodology for Rapid Detection of KRAS Mutation Using PNA-LNA Mediated Loop-Mediated Isothermal Amplification

Detecting point mutation of human cancer cells quickly and accurately is gaining in importance for pathological diagnosis and choice of therapeutic approach. In the present study, we present novel methodology, peptide nucleic acid—locked nucleic acid mediated loop-mediated isothermal amplification (PNA-LNA mediated LAMP), for rapid detection of KRAS mutation using advantages of both artificial DNA and LAMP. PNA-LNA mediated LAMP reactions occurred under isothermal temperature conditions of with 4 primary primers set for the target regions on the KRAS gene, clamping PNA probe that was complimentary to the wild type sequence and LNA primers complementary to the mutated sequences. PNA-LNA mediated LAMP was applied for cDNA from 4 kinds of pancreatic carcinoma cell lines with or without KRAS point mutation. The amplified DNA products were verified by naked-eye as well as a real-time PCR equipment. By PNA-LNA mediated LAMP, amplification of wild type KRAS DNA was blocked by clamping PNA probe, whereas, mutant type KRAS DNA was significantly amplified within 50 min. Mutant alleles could be detected in samples which diluted until 0.1% of mutant-to-wild type ratio. On the other hand, mutant alleles could be reproducibly with a mutant-to-wild type ratio of 30% by direct sequencing and of 1% by PNA-clamping PCR. The limit of detection (LOD) of PNA-LNA mediated LAMP was much lower than the other conventional methods. Competition of LNA clamping primers complementary to two different subtypes (G12D and G12V) of mutant KRAS gene indicated different amplification time depend on subtypes of mutant cDNA. PNA-LNA mediated LAMP is a simple, rapid, specific and sensitive methodology for the detection of KRAS mutation.     

Tanshinone II-A inhibits low density lipoprotein oxidation in vitro

Tanshinone II-A (TSII-A) is a major component of Salvia miltorrhiza Bunge which has long been used for preventing and ameliorating anginal pain in China. However the effect of TSII-A on low density lipoprotein (LDL) oxidation has not been studied. The present study was performed to investigate the effects of TSII-A on LDL oxidation using four oxidizing systems, including copper-, peroxyl radical- and peroxynitriteinitiated and macrophage-mediated LDL oxidation. LDL oxidation was measured in terms of formation of thiobarbituric acid-reactive substances (TBARS), relative electrophoretic mobility (REM) on agarose gel and lag time. In all four systems, TSII-A has apparent antioxidative effects against LDL oxidation, as evidenced by its dose-dependent inhibition of TBARS formation, prolongation of lag time and suppression of increased REM.

Regarding the mechanism underlying its antioxidative effect, TSII-A neither scavenged superoxide nor peroxynitrite. It also did not chelate copper. But it has mild peroxyl radical scavenging activity. The direct binding to LDL particles and conformational change of LDL structure by TSII-A were suggested, because it increased negative charge of LDL which was shown by increased REM on agarose gel. In conclusion, TSII-A is an effective antioxidant against LDL oxidation in vitro. The underlying mechanism appears to be related to its peroxyl radical scavenging and LDL binding activity.     

Complex of GM1- and GD1a-Like Lipo-Oligosaccharide Mimics GM1b,Inducing Anti-GM1b Antibodies

Objective

Molecular mimicry between Campylobacter jejuni lipo-oligosaccharides (LOSs) and human gangliosides GM1 and GD1a induces the production of anti-GM1 and anti-GD1a antibodies, and the development of Guillain-Barré syndrome. Complexes of two different gangliosides form new molecular shapes capable of enhancing recognition by anti-ganglioside antibodies. To test the hypothesis that the complex of GM1-like and GD1a-like LOSs of C. jejuni induces the development of anti-GM1b antibodies in Guillain-Barré syndrome patients.

Methods

Mass spectrometry analysis determined the LOS outer core structures, with which mice were immunized. IgG antibodies to single gangliosides and complex of gangliosides were tested in sera from Guillain-Barré syndrome patients from whom C. jejuni LOS had been isolated.

Results

Two isolates from GBS patients who had anti-GM1b antibodies, but neither anti-GM1 nor -GD1a antibodies, expressed both GM1-like and GD1a-like LOSs, but not GM1b-like LOS. Anti-GM1b antibodies were induced in one of the mice immunized with the C. jejuni bearing GM1-like and GD1a-like LOS. Sera from 20 patients had antibodies to the complex of GM1 and GD1a, all of which carried anti-GM1b reactivity. Five of these sera harbored neither anti-GM1 nor anti-GD1a antibodies. IgG antibodies to the complex were absorbed by GM1b, but by neither GM1 nor GD1a.

Conclusions

GM1-like and GD1a-like LOSs form a GM1b epitope, inducing the development of anti-GM1b antibodies in patients with Guillain-Barré syndrome subsequent to C. jejuni enteritis. Here, we present a new paradigm that the complex of two different structures forms a new molecular mimicry, inducing the production of autoantibodies.     

Regional Gray Matter Density Associated with Cognitive Reflectivity–Impulsivity: Evidence from Voxel-Based Morphometry

When faced with a problem or choice, humans can use two different strategies: “cognitive reflectivity,” which involves slow responses and fewer mistakes, or “cognitive impulsivity,” which comprises of quick responses and more mistakes. Different individuals use these two strategies differently. To our knowledge, no study has directly investigated the brain regions involved in reflectivity–impulsivity; therefore, this study focused on associations between these cognitive strategies and the gray matter structure of several brain regions. In order to accomplish this, we enrolled 776 healthy, right-handed individuals (432 men and 344 women; 20.7 ± 1.8 years) and used voxel-based morphometry with administration of a cognitive reflectivity–impulsivity questionnaire. We found that high cognitive reflectivity was associated with greater regional gray matter density in the ventral medial prefrontal cortex. Our finding suggests that this area plays an important role in defining an individual’s trait associated with reflectivity and impulsivity.