Response to RAG-mediated VDJ cleavage by NBS1 and gamma-H2AX

Genetic disorders affecting cellular responses to DNA damage are characterized by high rates of translocations involving antigen receptor loci and increased susceptibility to lymphoid malignancies. We report that the Nijmegen breakage syndrome protein (NBS1) and histone gamma-H2AX, which associate with irradiation-induced DNA double-strand breaks (DSBs), are also found at sites of VDJ (variable, diversity, joining) recombination-induced DSBs. In developing thymocytes, NBS1 and gamma-H2AX form nuclear foci that colocalize with the T cell receptor alpha locus in response to recombination activating gene (RAG) protein-mediated VDJ cleavage. Our results suggest that surveillance of T cell receptor recombination intermediates by NBS1 and gamma-H2AX may be important for preventing oncogenic translocations.     

Function and molecular mechanism of acetylation in autophagy regulation

Protein acetylation emerged as a key regulatory mechanism for many cellular processes. We used genetic analysis of Saccharomyces cerevisiae to identify Esa1 as a histone acetyltransferase required for autophagy. We further identified the autophagy signaling component Atg3 as a substrate for Esa1. Specifically, acetylation of K19 and K48 of Atg3 regulated autophagy by controlling Atg3 and Atg8 interaction and lipidation of Atg8. Starvation induced transient K19-K48 acetylation through spatial and temporal regulation of the localization of acetylase Esa1 and the deacetylase Rpd3 on pre-autophagosomal structures (PASs) and their interaction with Atg3. Attenuation of K19-K48 acetylation was associated with attenuation of autophagy. Increased K19-K48 acetylation after deletion of the deacetylase Rpd3 caused increased autophagy. Thus, protein acetylation contributes to control of autophagy.     

Rtt109 acetylates histone H3 lysine 56 and functions in DNA replication

Acetylation of histone H3 lysine 56 (H3-K56) occurs in S phase, and cells lacking H3-K56 acetylation are sensitive to DNA-damaging agents. However, the histone acetyltransferase (HAT) that catalyzes global H3-K56 acetylation has not been found. Here we show that regulation of Ty1 transposition gene product 109 (Rtt109) is an H3-K56 HAT. Cells lacking Rtt109 or expressing rtt109 mutants with alterations at a conserved aspartate residue lose H3-K56 acetylation and exhibit increased sensitivity toward genotoxic agents, as well as elevated levels of spontaneous chromosome breaks. Thus, Rtt109, which shares no sequence homology with any other known HATs, is a unique HAT that acetylates H3-K56.     

同步化处理后小鼠成纤维细胞表观遗传水平的相对定量分析

供体细胞的同步化处理可能改变其表观遗传特性,进而影响胚胎的克隆效率。研究同步化处理对小鼠胎儿成纤维细胞(mouse embryonicf ibroblasts,MEFs)组蛋白H3K9甲基化、乙酰化及组蛋白H3K4单甲基化、三甲基化表达的影响。分离培养MEFs,增殖稳定的第3代MEFs分别用5mL/L血清饥饿处理4d或15mL/LDM-SO处理2d使细胞处于增殖抑制期,通过免疫组化染色和Image-J图像处理软件,相对定量比较不同处理情况下组蛋白H3K9甲基化、乙酰化和组蛋白H3K4单甲基化、三甲基化变化情况。Ki-67染色检测结果表明,两种同步化处理可使细胞处于G0期或G1期。DMSO处理使MEFs组蛋白H3K9乙酰化表达水平升高,而5mL/L血清饥饿处理则使其表达水平下降;此外,两种同步化处理均导致组蛋白H3K9甲基化和H3K4单甲基化表达下降,但不影响组蛋白H3K4三甲基化的表达水平。研究结论表明:同步化处理可改变MEFs组蛋白乙酰化和甲基化表达水平,进而有可能影响胚胎克隆效率。     

Wistar大鼠GTH细胞的cAMP对FSHβ mRNA表达的影响

[目的]从分子水平阐述大鼠FSHβmRNA表达的GnRH受体后信号转导机制。[方法]采用细胞体外培养技术结合实时荧光定量PCR技术,检测大鼠GTH细胞中cAMP改变对FSHβmRNA表达的影响。[结果]cAMP含量对大鼠GTH细胞表达的FSHβmRNA的Ct值的影响不显著。FSK和SQ22536能有效改变GTH细胞的cAMP含量,却不能改变PKC活性。FSHβ的表达是受GnRH调控的。[结论]cAMP不是FSHβmRNA表达的GnRH受体后的信号转导途径。     

Sir2-dependent activation of acetyl-CoA synthetase by deacetylation of active lysine

Acetyl-coenzyme A (CoA) synthetase (Acs) is an enzyme central to metabolism in prokaryotes and eukaryotes. Acs synthesizes acetyl CoA from acetate, adenosine triphosphate, and CoA through an acetyl-adenosine monophosphate (AMP) intermediate. Immunoblotting and mass spectrometry analysis showed that Salmonella enterica Acs enzyme activity is posttranslationally regulated by acetylation of lysine-609. Acetylation blocks synthesis of the adenylate intermediate but does not affect the thioester-forming activity of the enzyme. Activation of the acetylated enzyme requires the nicotinamide adenine dinucleotide-dependent protein deacetylase activity of the CobB Sir2 protein from S. enterica. We propose that acetylation modulates the activity of all the AMP-forming family of enzymes, including nonribosomal peptide synthetases, luciferase, and aryl- and acyl-CoA synthetases. These findings extend our knowledge of the roles of Sir2 proteins in gene silencing, chromosome stability, and cell aging and imply that lysine acetylation is a common regulatory mechanism in eukaryotes and prokaryotes.     

蛋白质磷酸化对肌动球蛋白解离及其乙酰化水平的影响

[目的]研究肌球蛋白重链和肌动蛋白磷酸化对其乙酰化水平、肌动球蛋白解离及ATP酶活性的影响,为通过调控磷酸化水平改善肉品嫩度提供理论依据.[方法]以羊背最长肌为材料制备肌肉匀浆液,采用碱性磷酸酶抑制剂(抑制去磷酸化)和蛋白激酶抑制剂(抑制磷酸化)调控其磷酸化水平,在4℃分别孵育0、0.5、4、12、24、48和72 h...     

Reduction of naturally occurring motoneuron death in vivo by a target-derived neurotrophic factor

Treatment of chick embryos in ovo with crude and partially purified extracts from embryonic hindlimbs (days 8 to 9) during the normal cell death period (days 5 to 10) rescues a significant number of motoneurons from degeneration. The survival activity of partially purified extract was dose-dependent and developmentally regulated. The survival of sensory, sympathetic, parasympathetic, and a population of cholinergic sympathetic preganglionic neurons was unaffected by treatment with hindlimb extract. The massive motoneuron death that occurs after early target (hindlimb) removal was partially ameliorated by daily treatment with the hindlimb extract. These results indicate that a target-derived neurotrophic factor is involved in the regulation of motoneuron survival in vivo.     

基于不同预处理的竹纤维磺化改性及其产物热塑性能研究

以西南地区特色大型竹材巨龙竹为原料,分别经碱、水热、H₂O₂预处理后,在相对温和条件下进行磺化改性,探究不同预处理对竹纤维磺化改性可及度影响,深入分析磺化改性对竹纤维热塑性的影响调控机制。结果表明：3种预处理均可以提高竹纤维磺化反应可及性,其中基于H₂O₂预处理的竹纤维再经过磺化改性,即先氧化再磺化,磺化效果最好,磺酸基含量0.49 mmol/g。经磺化改性后竹纤维塑性明显改善,改性竹纤维热塑性随着磺酸基含量的增加而提升。3种改性方式中,经H₂O₂预处理再进行磺化改性得到的竹纤维塑性最显著,其玻璃化转变温度为104.71 ℃。     

草莓果实采后NAD激酶活性与NAD（H）、NADP（H）含量及活性氧代谢的关系

【目的】研究草莓采后成熟衰老过程中NADK活性与NAD（H）、NADP（H）及活性氧代谢和膜氧化产物变化的关系，以探讨NADK在非跃变型果实成熟衰老过程中的作用，为调控果实的成熟衰老提供理论依据。【方法】将从果园采回的草莓果实贮藏于不同的温度下并进行每天取样，研究草莓果实在低温（4℃）、常温（20℃）贮藏期间成熟衰老过程中NAD激酶（NADK）活性及其底物NAD（H）、产物NADP（H）以及超氧阴离子O2- •过氧化氢（H2O2）、膜氧化产物丙二醛（MDA）含量的变化并分析NADK 与上述指标的关系。【结果】草莓果实在低温（4℃）贮藏时，其NADK活性比常温（20℃）贮藏的高，NAD（H）含量则相应比常温贮藏的低，NADP（H）含量则高于常温下的；同时，在常温贮藏期间果实O2- •生成速率和H2O2含量、膜氧化产物MDA含量均比低温贮藏的高，暗示NADK可能通过影响NAD（H）、NADP（H）的含量及比例来调控O2- •生成速率和H2O2含量，从而调控果实的成熟衰老。【结论】非跃变型果实草莓采后成熟衰老过程中，保持较高的NADK活性有利于延缓果实的成熟衰老，降低NADK活性可导致NAD和NAD（H）含量的积累，进而加速电子传递，产生大量的活性氧O2- •和H2O2，从而促进膜过氧化作用和积累较多的MDA，最终导致果实衰老变质。     

Regulated genes in transgenic plants

Transgenic plants are an effective system for the study of regulated gene expression. Developmental control of expression can be monitored by assaying different tissues or by assaying a plant at different developmental stages. Analysis of the petunia 5-enolpyruvylshikimate-3-phosphate synthase gene, which is highly expressed in flowers, allowed identification of an upstream region that confers tissue-specific and developmentally regulated expression. The cell specificity of expression in floral tissues has been defined by histochemical localization. This expression is contrasted to that of the 35S promoter of cauliflower mosaic virus, a nominally constitutive promoter that shows a definite specificity of expression in floral tissues. Moreover, this expression differs in transgenic hosts of different species.     

玉米大斑病菌毒素的结构鉴定和钝化反应

玉米大斑病菌（Ｅｘｓｅｒｏｈｉｌｕｍ ｔｕｒｃｉｃｕｍ）在改良Ｆｒｉｅｓ培养液中培养可以产生ＨＴ－毒素。ＴＬＣ分析表明：１号小种毒素至少有２种毒性成分,２号小种毒素至少有３种毒性成分。通过ＧＣ－ＭＳ、ＨＲＭＳ、ＮＭＲ分析表明组分Ｉ的分子式为Ｃ６Ｈ６Ｏ３,分子量１２６．０３２８,其化学结构为５－羟甲基－２－呋喃甲醛。组分Ⅱ分子量为１２４,初步推测为２,５－二甲醛呋喃。毒素钝化试验表明,代森锰锌、Ｎａ