小型猪葡萄糖转运子4外显子4a的基因多态性分析

目的分析我国特有的3个小型猪品系巴马小型猪、中国农大小型猪、五指山小型猪葡萄糖转运子4基因外显子4a的单核苷酸多态性（SNPs）分布特点,为我国小型猪在糖尿病和代谢性疾病研究中提供基础资料。方法以3个品系小型猪基因组DNA为模板,应用特异性引物进行PCR扩增,扩增产物纯化测序,进行BLAST比对分析。结果在小型猪GLUT-4基因外显子4a上有2个SNP位点：SNP1：GCT→GCC（Ala133 Ala）,3个品系均发生了变化,均为纯合突变,其突变率为（22/22,100%）。SNP2：GGC→GGT（Gly146 Gly）,巴马小型猪突变率为（6/6,100%）,均为纯合突变;五指山小型猪突变率为（6/6,100%）,均为纯合突变;中国农大小型猪突变率为（10/10,100%）,其中包括6例纯合突变（6/10,60%）和4例杂合突变（4/10,40%）。结论SNPs1,在所有测定的小型猪品系中检出,这可能是所测小型猪的共有特征。SNPs2可能是小型猪品系之间的差异。     

小鼠胚胎干细胞p16^INK4a基因外显子1a打靶研究

在活体水平上,小鼠p16^INK4a基因是否具有抑制肿瘤发生和发展的功能是一个悬而未决的问题。利用筛选基因组文库得到的小鼠p16^INK4a基因组DNA片段,构建了针对小鼠p16^INK4a基因外显子1a的基因打靶载体,其短臂为1.5kbEco81 I/AccⅡ片段,长壁为5.9kb Xba I/Xho I片段。打靶载体经线性化和纯化后通过电穿孔转导小鼠R1 ES细胞,获得37个G418和Gancyclovir双药抗性克隆。用Southern杂交法对双药抗性克隆进行鉴定,获得一个敲除了p16^INK4a基因外显子1a的阳性ES细胞克隆。     

The Arabidopsis gene ATST4a is?not a typical brassinosteroids catabolic gene

Brassinosteroid (BR) homeostasis is maintained in part by this hormone’s catabolism. The presence of multiple BR-catabolic pathways in Arabidopsis demonstrates the importance of this process in growth and development. Previous biochemical analyses suggest that ATST4a has BR catalytic activity. We have used both overexpression and loss-of-function genetic approaches to further explore the role of ATST4a in Arabidopsis. Up to 1000-fold overexpression of the ATST4a gene did not result in any characteristic BR-deficient phenotypes. In addition, the T-DNA insertion null mutant atst4a1–1 did not display enhanced seedling hypocotyl growth in the presence or absence of the active BR brassinolide when grown in white light. This lack of hallmark characteristics for BR-inacitivion genes suggests that ATST4a encodes an atypical BR catabolic enzyme.     

Is N-acetyl-D-glucosamine a rigid 4C1 chair?

Understanding microsecond-timescale dynamics is crucial to establish three-dimensional (3D) structure-activity relationships in sugars but has been intractable to experiments and simulations. As a consequence, whether arguably the most important chemical scaffold in glycobiology, N-acetyl-d-glucosamine (GlcNAc), deviates from a rigid (4)C(1) chair is unknown. Here, conformer populations and exchange kinetics were quantified from the longest aqueous carbohydrate simulations to date (0.2 ms total) of GlcNAc, four derivatives from heparan sulfate and their methylglycosides. Unmodified GlcNAc took 3-5 μs to reach a conformational equilibrium, which comprised a metastable (4)C(1) chair that underwent (4)C(1) ? (1)C(4) transitions at a predicted forward rate of 0.8 μs(-1) with an average (1)C(4)-chair lifetime of 3 ns. These predictions agree with high-resolution crystallography and nuclear magnetic resonance but not with the hypothesis that GlcNAc is a rigid (4)C(1) chair, concluded from previous experimental analyses and non-aqueous modeling. The methylglycoside was calculated to have a slower forward rate (0.3 μs(-1)) and a more stable (4)C(1) conformer (0.2 kcal mol(-1)), suggesting that pivotal 3D intermediates (particularly (2)S(O), (1)S(5) and B(2,5)) increased in energy, and water was implicated as a major cause. Sulfonation (N-, 3-O and 6-O) significantly augmented this effect by blocking pseudorotation, but did not alter the rotational preferences of hydroyxl or hydroxymethyl groups. We therefore propose that GlcNAc undergoes puckering exchange that is dependent on polymerization and sulfo substituents. Our analyses, and 3D model of the equilibrium GlcNAc conformer in water, can be used as dictionary data and present new opportunities to rationally modify puckering and carbohydrate bioactivity, with diverse applications from improving crop yields to disease amelioration.     

Deletion of a 236 kb region around S 4-RNase in a stylar-part mutant S 4 sm -haplotype of Japanese pear

Japanese pear (Pyrus pyrifolia Nakai) has a gametophytic self-incompatibility (GSI) mechanism controlled by a single S-locus with multiple S-haplotypes, each of which contains separate genes that determine the allelic identity of pistil and pollen. The pistil S gene is the S-ribonuclease (S-RNase) gene, whereas good candidates for the pollen S gene are the F-box protein genes. A self-compatible (SC) cultivar, ‘Osa-Nijisseiki’, which is a bud mutant of ‘Nijisseiki’ (S ₂ S ₄), has a stylar-part mutant -haplotype, which lacks the S ₄-RNase gene but retains the pollen S gene. To delineate the deletion breakpoint in the -haplotype, we constructed a bacterial artificial chromosome (BAC) library from an S ₄-homozygote, and assembled a BAC contig of 570 kb around the S ₄-RNase. Genomic PCR of DNA from S ₄- and -homozygotes and the DNA sequence of the BAC contig allowed the identification of a deletion of 236 kb spanning from 48 kb upstream to 188 kb downstream of S ₄-RNase. The -haplotype lacks 34 predicted open reading frames (ORFs) including the S ₄-RNase and a pollen-specific F-box protein gene (termed as S ₄ F-box0). Genomic PCR with a primer pair designed from the deletion junctions yielded a product specific for the -haplotype. The product could be useful as a maker for early selection of SC cultivars harboring the -haplotype. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

CXCR4, a therapeutic target in rare immunodeficiencies?

Currently, more than 200?primary immunodeficiency diseases have been discovered. In most cases, genetic defects affect the expression or the function of proteins involved in immune development and homeostasis. Some orphan immuno-hematological disorders are characterized by an abnormal leukocyte trafficking, a notion predictive of an anomaly of the chemokine/chemokine receptor system. In this review, we focus on recent advances in the characterization of dysfunctions of the CXCL12 (SDF-1)/CXCR4 signaling axis in two rare human immunodeficiencies, one associated with a loss of CXCR4 function, the Idiopathic CD4(+) T-cell Lymphocytopenia, and the other with a gain of CXCR4 function, the WHIM syndrome.     

p16INK4a基因的功能及其调控

p16INK4a蛋白能抑制CDK4和CDK6的活性,使pRb处于非磷酸化或低磷酸化状态而能与转录因子E2Fs结合,从而抑制DNA 的合成,阻止细胞由G1期进入S期.p16INK4a的表达受Ets1和Ets2的正调控,受Bmi-1的负调控.p16INK4a基因缺失、突变、甲基化、RNA剪接加工错误可导致细胞周期失控和癌变.应用p16INK4a对某些肿瘤进行基因治疗的研究正在进行中.     

过表达NtMYB4a基因增强烟草抗旱能力

为探究转录因子NtMYB4a参与调控烟草(Nicotiana tabacum)抵抗干旱胁迫的能力,以NtMYB4a过表达株系(OE)、敲除株系(KO)和野生型(WT)为材料,通过盆栽实验模拟自然干旱胁迫,对比分析三个株系在干旱胁迫0、2、4、6、8 d和复水2 d后的光合特征、根系活力、叶绿素、脯氨酸(Pro)和丙二醛(MDA)含量以及抗氧化酶活性等指标。结果表明,OE株系的净光合速率(Pn)、根系活力、叶绿素含量、Pro含量以及SOD、POD和CAT活性均显著高于WT株系,而蒸腾速率(Tr)、气孔导度(Gs)、MDA含量则显著低于WT株系;KO株系多数时期Pn、根系活力、叶绿素含量、Pro含量及SOD、POD和CAT活性显著低于WT株系,Tr、Gs、MDA含量则显著高于WT株系。因此,超表达NtMYB4a可能通过提高烟草的光合能力、根系活力和抗氧化酶活性,减少细胞膜损伤来提高烟苗抗旱性。     

Desulfoferrodoxin structure determined by MAD phasing and refinement to 1.9-Å resolution reveals a unique combination of a tetrahedral FeS4 centre with a square pyramidal FeSN4 centre

The structure of desulfoferrodoxin (DFX), a protein containing two mononuclear non-heme iron centres, has been solved by the MAD method using phases determined at 2.8?Å resolution. The iron atoms in the native protein were used as the anomalous scatterers. The model was built from an electron density map obtained after density modification and refined against data collected at 1.9?Å. Desulfoferrodoxin is a homodimer which can be described in terms of two domains, each with two crystallographically equivalent non-heme mononuclear iron centres. Domain I is similar to desulforedoxin with distorted rubredoxin-type centres, and domain II has iron centres with square pyramidal coordination to four nitrogens from histidines as the equatorial ligands and one sulfur from a cysteine as the axial ligand. Domain I in DFX shows a remarkable structural fit with the DX homodimer. Furthermore, three β-sheets extending from one monomer to another in DFX, two in domain I and one in domain II, strongly support the assumption of DFX as a functional dimer. A calcium ion, indispensable in the crystallisation process, was assumed at the dimer interface and appears to contribute to dimer stabilisation. The C-terminal domain in the monomer has a topology fold similar to that of fibronectin III.     

trans-4-hydroxy-β-prolinebetaine,a new betaine fromFurcellaria lumbricalis

From the red marine alga Furcellaria lumbricalis (Huds.) Lamour, a novel betaine has been isolated and characterised from infra-red and nuclear magnetic resonance spectroscopic and mass spectrometric data astrans-4-hydroxy-β-prolinebetaine.     

Probing α-310 Transitions in a Voltage-Sensing S4 Helix

The S4 helix of voltage sensor domains (VSDs) transfers its gating charges across the membrane electrical field in response to changes of the membrane potential. Recent studies suggest that this process may occur via the helical conversion of the entire S4 between α and 3₁₀ conformations. Here, using LRET and FRET, we tested this hypothesis by measuring dynamic changes in the transmembrane length of S4 from engineered VSDs expressed in Xenopus oocytes. Our results suggest that the native S4 from the Ciona intestinalis voltage-sensitive phosphatase (Ci-VSP) does not exhibit extended and long-lived 3₁₀ conformations and remains mostly α-helical. Although the S4 of NavAb displays a fully extended 3₁₀ conformation in x-ray structures, its transplantation in the Ci-VSP VSD scaffold yielded similar results as the native Ci-VSP S4. Taken together, our study does not support the presence of long-lived extended α-to-3₁₀ helical conversions of the S4 in Ci-VSP associated with voltage activation.     

Agouti-related protein: more than a melanocortin-4 receptor antagonist?

It is well established that agouti-related protein (AGRP) can act as a competitive antagonist to proopiomelanocortin (POMC)-derived peptides at the melanocortin-4 receptor (MC4R), and that this homeostatic mechanism is important as a means of coordinating appetite with perceived metabolic requirement. However, there are clearly additional facets to the physiological role of AGRP, given that it is active in MC4R knockout mice and it has strikingly long-lasting effects on food intake, compared with MC4R agonists. In this review we focus on: (i) evidence that AGRP is more sensitive to perturbations in energy balance than POMC and is therefore the primary basis of melanocortinergic regulation. (ii) Evidence that the bioactive peptide AGRP83-132, acts by alternate mechanism(s) to elicit its long-term effects on food intake. (iii) Evidence that AGRP is post-translationally cleaved to generate AGRP83-132 and one or more N terminal peptides, which may have an important physiological role(s) that are independent of the melanocortin system. A clear understanding of how proAGRP processing is regulated, and the role of resultant peptides, may define additional therapeutic targets in the treatment of obesity.     

Muscle injury-induced thymosin β4 acts as a chemoattractant for myoblasts

Thymosin β4 (Tβ4) is a major intracellular G-actin-sequestering peptide. There is increasing evidence to support important extracellular functions of Tβ4 related to angiogenesis, wound healing and cardiovascular regeneration. We investigated the expression of 'Tβ4' and 'thymosin β10', a closely related peptide, during skeletal muscle regeneration in mice and chemotactic responses of myoblasts to these peptides. The mRNA levels of 'Tβ4' and 'thymosin β10' were up-regulated in the early stage of regenerating muscle fibres and inflammatory haematopoietic cells in the injured skeletal muscles of mice. We found that both Tβ4 and its sulphoxized form significantly accelerated wound closure and increased the chemotaxis of C2C12 myoblastic cells. Furthermore, we showed that primary myoblasts and myocytes derived from muscle satellite cells of adult mice were chemoattracted to sulphoxized form of Tβ4. These data indicate that muscle injury enhances the local production of Tβ4, thereby promoting the migration of myoblasts to facilitate skeletal muscle regeneration.