含铬重组液激活部分缺失金属原子簇的钼铁蛋白的研究

棕色固氮菌（Ａｚｏｔｏｂａｃｔｅｒ ｖｉｎｅｌａｎｄｉｉ）固氮酶钼铁蛋白经邻菲口罗啉和Ｏ２ 处理后,变为部分缺失ＦｅＭｏｃｏ 和Ｐ－ｃｌｕｓｔｅｒ的失活蛋白。与由Ｋ２ＣｒＯ４、高柠檬酸铁、Ｎａ２Ｓ和二硫苏糖醇组成的重组液保温后,处理蛋白对乙炔和质子还原的活性都得以显著恢复;然而,它的吸收光谱和圆二色谱虽有明显恢复,但仍与还原钼铁蛋白有所不同。这表明,激活蛋白中也许存在功能与钼铁蛋白相似,而结构则有所差异的含铬（ＣｒＦｅ）蛋白     

An Update on the Functional Molecular Immunology (FIMM) Database

Data on the major histocompatibility complex, T-cell epitopes, B-cell epitopes, antigens and diseases are heterogeneous and scattered among different databases and the literature. Since it has become increasingly difficult to obtain an integrated view of functional immune response components, we have developed and updated over several years the Functional molecular IMMunology (FIMM) database (http:// research.i2r.a-star.edu.sg/fimm/). FIMM contains integrated expert-curated data on protein antigens, and on human immunological receptors that recognise and bind them in healthy or disease states. Interfaces with multiple, intuitive query options and query reports provide immunologists with prioritised information that aids data interpretation, vaccine target discovery and immune disease research.     

The plant glycosyltransferase clone collection for functional genomics

The glycosyltransferases (GTs) are an important and functionally diverse family of enzymes involved in glycan and glycoside biosynthesis. Plants have evolved large families of GTs which undertake the array of glycosylation reactions that occur during plant development and growth. Based on the Carbohydrate‐Active enZymes (CAZy) database, the genome of the reference plant Arabidopsis thaliana codes for over 450 GTs, while the rice genome (Oryza sativa) contains over 600 members. Collectively, GTs from these reference plants can be classified into over 40 distinct GT families. Although these enzymes are involved in many important plant specific processes such as cell‐wall and secondary metabolite biosynthesis, few have been functionally characterized. We have sought to develop a plant GTs clone resource that will enable functional genomic approaches to be undertaken by the plant research community. In total, 403 (88%) of CAZy defined Arabidopsis GTs have been cloned, while 96 (15%) of the GTs coded by rice have been cloned. The collection resulted in the update of a number of Arabidopsis GT gene models. The clones represent full‐length coding sequences without termination codons and are Gateway^® compatible. To demonstrate the utility of this JBEI GT Collection, a set of efficient particle bombardment plasmids (pBullet) was also constructed with markers for the endomembrane. The utility of the pBullet collection was demonstrated by localizing all members of the Arabidopsis GT14 family to the Golgi apparatus or the endoplasmic reticulum (ER). Updates to these resources are available at the JBEI GT Collection website http://www.addgene.org/ .     

A Homogeneous,High-Throughput Assay for Phosphatidylinositol 5-Phosphate 4-Kinase with a Novel,Rapid Substrate Preparation

Phosphoinositide kinases regulate diverse cellular functions and are important targets for therapeutic development for diseases, such as diabetes and cancer. Preparation of the lipid substrate is crucial for the development of a robust and miniaturizable lipid kinase assay. Enzymatic assays for phosphoinositide kinases often use lipid substrates prepared from lyophilized lipid preparations by sonication, which result in variability in the liposome size from preparation to preparation. Herein, we report a homogeneous 1536-well luciferase-coupled bioluminescence assay for PI5P4Kα. The substrate preparation is novel and allows the rapid production of a DMSO-containing substrate solution without the need for lengthy liposome preparation protocols, thus enabling the scale-up of this traditionally difficult type of assay. The Z’-factor value was greater than 0.7 for the PI5P4Kα assay, indicating its suitability for high-throughput screening applications. Tyrphostin AG-82 had been identified as an inhibitor of PI5P4Kα by assessing the degree of phospho transfer of γ-³²P-ATP to PI5P; its inhibitory activity against PI5P4Kα was confirmed in the present miniaturized assay. From a pilot screen of a library of bioactive compounds, another tyrphostin, I-OMe tyrphostin AG-538 (I-OMe-AG-538), was identified as an ATP-competitive inhibitor of PI5P4Kα with an IC₅₀ of 1 µM, affirming the suitability of the assay for inhibitor discovery campaigns. This homogeneous assay may apply to other lipid kinases and should help in the identification of leads for this class of enzymes by enabling high-throughput screening efforts.     

An integrative approach to the identification of Arabidopsis and rice genes involved in xylan and secondary wall development

Xylans constitute the major non-cellulosic component of plant biomass. Xylan biosynthesis is particularly pronounced in cells with secondary walls, implying that the synthesis network consists of a set of highly expressed genes in such cells. To improve the understanding of xylan biosynthesis, we performed a comparative analysis of co-expression networks between Arabidopsis and rice as reference species with different wall types. Many co-expressed genes were represented by orthologs in both species, which implies common biological features, while some gene families were only found in one of the species, and therefore likely to be related to differences in their cell walls. To predict the subcellular location of the identified proteins, we developed a new method, PFANTOM (plant protein family information-based predictor for endomembrane), which was shown to perform better for proteins in the endomembrane system than other available prediction methods. Based on the combined approach of co-expression and predicted cellular localization, we propose a model for Arabidopsis and rice xylan synthesis in the Golgi apparatus and signaling from plasma membrane to nucleus for secondary cell wall differentiation. As an experimental validation of the model, we show that an Arabidopsis mutant in the PGSIP1 gene encoding one of the Golgi localized candidate proteins has a highly decreased content of glucuronic acid in secondary cell walls and substantially reduced xylan glucuronosyltransferase activity.     

A novel light‐dependent selection marker system in plants

Photosensitizers are common in nature and play diverse roles as defense compounds and pathogenicity determinants and as important molecules in many biological processes. Toxoflavin, a photosensitizer produced by Burkholderia glumae, has been implicated as an essential virulence factor causing bacterial rice grain rot. Toxoflavin produces superoxide and H₂O₂ during redox cycles under oxygen and light, and these reactive oxygen species cause phytotoxic effects. To utilize toxoflavin as a selection agent in plant transformation, we identified a gene, tflA, which encodes a toxoflavin‐degrading enzyme in the Paenibacillus polymyxa JH2 strain. TflA was estimated as 24.56 kDa in size based on the amino acid sequence and is similar to a ring‐cleavage extradiol dioxygenase in the Exiguobacterium sp. 255‐15; however, unlike other extradiol dioxygenases, Mn²⁺and dithiothreitol were required for toxoflavin degradation by TflA. Here, our results suggested toxoflavin is a photosensitizer and its degradation by TflA serves as a light‐dependent selection marker system in diverse plant species. We examined the efficiencies of two different plant selection systems, toxoflavin/tflA and hygromycin/hygromycin phosphotransferase (hpt) in both rice and Arabidopsis. The toxoflavin/tflA selection was more remarkable than hygromycin/hpt selection in the high‐density screening of transgenic Arabidopsis seeds. Based on these results, we propose the toxoflavin/tflA selection system, which is based on the degradation of the photosensitizer, provides a new robust nonantibiotic selection marker system for diverse plants.     

The genetic effect of copy number variations on the risk of type 2 diabetes in a Korean population

Background

Unlike Caucasian populations, genetic factors contributing to the risk of type 2 diabetes mellitus (T2DM) are not well studied in Asian populations. In light of this, and the fact that copy number variation (CNV) is emerging as a new way to understand human genomic variation, the objective of this study was to identify type 2 diabetes–associated CNV in a Korean cohort.

Methodology/Principal Findings

Using the Illumina HumanHap300 BeadChip (317,503 markers), genome-wide genotyping was performed to obtain signal and allelic intensities from 275 patients with type 2 diabetes mellitus (T2DM) and 496 nondiabetic subjects (Total n = 771). To increase the sensitivity of CNV identification, we incorporated multiple factors using PennCNV, a program that is based on the hidden Markov model (HMM). To assess the genetic effect of CNV on T2DM, a multivariate logistic regression model controlling for age and gender was used. We identified a total of 7,478 CNVs (average of 9.7 CNVs per individual) and 2,554 CNV regions (CNVRs; 164 common CNVRs for frequency>1%) in this study. Although we failed to demonstrate robust associations between CNVs and the risk of T2DM, our results revealed a putative association between several CNVRs including chr15:45994758–45999227 (P = 8.6E-04, P^corr = 0.01) and the risk of T2DM. The identified CNVs in this study were validated using overlapping analysis with the Database of Genomic Variants (DGV; 71.7% overlap), and quantitative PCR (qPCR). The identified variations, which encompassed functional genes, were significantly enriched in the cellular part, in the membrane-bound organelle, in the development process, in cell communication, in signal transduction, and in biological regulation.

Conclusion/Significance

We expect that the methods and findings in this study will contribute in particular to genome studies of Asian populations.     

Mutations affecting early distribution of primordial germ cells in Medaka (Oryzias latipes) embryo

The development of germ cells has been intensively studied in Medaka (Oryzias latipes). We have undertaken a large-scale screen to identify mutations affecting the development of primordial germ cells (PGCs) in Medaka. Embryos derived from mutagenized founder fish were screened for an abnormal distribution or number of PGCs at embryonic stage 27 by RNA in situ hybridization for the Medaka vasa homologue (olvas). At this stage, PGCs coalesce into two bilateral vasa-expressing foci in the ventrolateral regions of the trunk after their migration and group organization. Nineteen mutations were identified from a screen corresponding to 450 mutagenized haploid genomes. Eleven of the mutations caused altered PGC distribution. Most of these alterations were associated with morphological abnormalities and could be grouped into four phenotypic classes: Class 1, PGCs dispersed into bilateral lines; Class 2, PGCs dispersed in a region more medial than that in Class 1; Class 3, PGCs scattered laterally and over the yolk sac area; and Class 4, PGCs clustered in a single median focus. Eight mutations caused a decrease in the number of PGCs. This decrease was observed in the offspring of heterozygous mothers, indicating the contribution of a maternal factor in determining PGC abundance. Taken together, these mutations should prove useful in identifying molecular mechanisms underlying the early PGC development and migration.