Site‐specific T–DNA integration in Arabidopsis thaliana mediated by the combined action of CRE recombinase and ϕC31 integrase

Random T–DNA integration into the plant host genome can be problematic for a variety of reasons, including potentially variable transgene expression as a result of different integration positions and multiple T–DNA copies, the risk of mutating the host genome and the difficulty of stacking well‐defined traits. Therefore, recombination systems have been proposed to integrate the T–DNA at a pre‐selected site in the host genome. Here, we demonstrate the capacity of the ?C31 integrase (INT) for efficient targeted T–DNA integration. Moreover, we show that the iterative site‐specific integration system (ISSI), which combines the activities of the CRE recombinase and INT, enables the targeting of genes to a pre‐selected site with the concomitant removal of the resident selectable marker. To begin, plants expressing both the CRE and INT recombinase and containing the target attP site were constructed. These plants were supertransformed with a T–DNA vector harboring the loxP site, the attB sites, a selectable marker and an expression cassette encoding a reporter protein. Three out of the 35 transformants obtained (9%) showed transgenerational site‐specific integration (SSI) of this T–DNA and removal of the resident selectable marker, as demonstrated by PCR, Southern blot and segregation analysis. In conclusion, our results show the applicability of the ISSI system for precise and targeted Agrobacterium‐mediated integration, allowing the serial integration of transgenic DNA sequences in plants.     

Asymmetric Dimethylarginine,an Endogenous NOS Inhibitor,Is Actively Metabolized in Rat Erythrocytes

N ^G,N ^G-Dimethyl-L-arginine (asymmetric dimethylarginine: ADMA) is an endogenous competitive inhibitor of nitric oxide synthase (NOS). Plasma ADMA concentrations have been reported to increase in connection with diseases associated with an impaired endothelial L-arginine/NO pathway. In this study, we investigated the metabolism of ADMA in circulating blood cell populations to elucidate the regulatory mechanism of elevation of plasma ADMA, a novel risk factor for cardiovascular disease. We found by RT-PCR and Western blot analyses that protein arginine methyltransferase (PRMT)1 and dimethylarginine dimethylaminohydrolase (DDAH)-1, responsible for the biosynthesis and degradation of ADMA respectively, are expressed in erythrocytes (ECs), leukocytes, and platelets. We also identified a major ADMA-containing protein in ECs as catalase, confirmed by GST-pull down assay to bind to PRMT1 in vitro. This is the first report that the ADMA-metabolizing system, including the arginine methylation of proteins and the breakdown of free ADMA, occurs in circulating blood cell-populations, and that catalase in ECs might be a potential protein targeted by PRMT1.     

arcA基因提高大肠杆菌对有机溶剂的耐受性

【目的】将来源于恶臭假单胞菌(Pseudomonas putida JUCT1)的基因arc A(编码精氨酸脱亚胺酶)整合到Escherichia coli JM109(DE3)基因组中,以提高该菌对有机溶剂的耐受性。【方法】以P.putida JUCT1的基因组为模板扩增基因arc A,并与p ET-20b(+)连接后导入E.coli JM109(DE3)中,验证该基因提高E.coli JM109(DE3)对有机溶剂的耐受性。利用Red同源重组的方法将arc A整合到E.coli JM109(DE3)基因组中。【结果】E.coli JM109(DE3)/p ET-20b(+)-arc A在添加了2.0%(体积比)环己烷、0.1%(体积比)甲苯、4.0%(体积比)萘烷和0.1%(体积比)丁醇的培养基中培养8 h后,其OD660由初始的0.2分别上升到0.8、0.9、1.8和1.3。将arc A成功整合到E.coli JM109(DE3)基因组中,获得了具有较好遗传稳定性的溶剂耐受E.coli JM109(DE3)宿主菌株。【结论】外源基因arc A能提高大肠杆菌菌株的有机溶剂耐受性,为工业化应用中耐溶剂微生物菌株的构建提供了实验依据和理论基础。     

AM真菌摩西管柄囊霉对干旱胁迫下枳抗氧化酶基因表达的影响

测定分析了接种丛枝菌根（AM）真菌摩西管柄囊霉Funneliformis mosseae对正常供水与干旱处理的盆栽枳Poncirus trifoliata实生苗生长、活性氧代谢及抗氧化酶基因表达量的影响。结果表明,7周干旱处理显著降低了根系菌根侵染率。接种摩西管柄囊霉显著促进了干旱处理的枳植株生长,增加了根系体积和叶片相对含水量,显著降低了叶片脯氨酸含量,同时也上调了干旱处理的枳叶片精氨酸脱羧酶基因（PtADC1和PtADC2）和超氧化物歧化酶基因（PtFe-SOD和PtMn-SOD）、过氧化物酶基因（PtPOD）和过氧化氢酶基因（PtCAT1）的表达,因而维持了一个相对更低的活性氧水平（如过氧化氢）,有利于增强植株的抗旱性。     

Cyanobacterial bioactive metabolites—A review of their chemistry and biology

Cyanobacterial blooms occur when algal densities exceed baseline population concentrations. Cyanobacteria can produce a large number of secondary metabolites. Odorous metabolites affect the smell and flavor of aquatic animals, whereas bioactive metabolites cause a range of lethal and sub-lethal effects in plants, invertebrates, and vertebrates, including humans. Herein, the bioactivity, chemistry, origin, and biosynthesis of these cyanobacterial secondary metabolites were reviewed. With recent revision of cyanobacterial taxonomy by Anagnostidis and Komárek as part of the Süβwasserflora von Mitteleuropa volumes 19(1–3), names of many cyanobacteria that produce bioactive compounds have changed, thereby confusing readers. The original and new nomenclature are included in this review to clarify the origins of cyanobacterial bioactive compounds.Due to structural similarity, the 157 known bioactive classes produced by cyanobacteria have been condensed to 55 classes. This review will provide a basis for more formal procedures to adopt a logical naming system. This review is needed for efficient management of water resources to understand, identify, and manage cyanobacterial harmful algal bloom impacts.     

脑血管内皮细胞Prmt5在脑血管发育过程中的表达及其下游靶分子

目的:研究内皮细胞中蛋白精氨酸甲基转移酶5(PRMT5)在脑血管发育及血脑屏障建成的关键时期的表达变化及其潜在下游靶分子。方法:原位观察PRMT5在小鼠不同发育时期脑血管内皮上的分布;流式分选获得原代脑血管内皮,利用real-time PCR分析Prmt5的表达;体外培养小鼠内皮细胞敲降Prmt5后,利用Western印迹、real-time PCR、ChIP等方法检测其对经典下游分子的影响。结果:PRMT5在胚胎期各时间点的脑血管内皮细胞质均有表达,小鼠出生后主要表达在脑血管内皮细胞核,在胚胎期18.5 d时表达量显著升高;小鼠脑血管内皮细胞体外细胞系中基因敲降Prmt5后,其经典对称甲基化组蛋白产物H4R3me2s及H3R2me2s均明显下降,Bmp4表达显著上调;免疫共沉淀实验提示Bmp4启动子区域组蛋白具有H3R2me2s修饰,Prmt5基因敲降后,该组蛋白修饰显著减少。结论:脑血管发育过程中PRMT5在脑血管内皮细胞中表达的位置和水平均发生变化,脑血管内皮细胞中PRMT5可以调节H4R3和H3R2对称二甲基化水平,Bmp4启动子区域组蛋白具有H3R2me2s修饰,且PRMT5可以抑制Bmp4表达。     

Role for Btg1 and Btg2 in growth arrest of WEHI-231 cells through arginine methylation following membrane immunoglobulin engagement

Engagement of membrane Ig (mIg) on WEHI-231 murine B lymphoma cells, a cell line model representative of primary immature B cells, results in growth arrest and subsequent apoptosis. Of the several dozen genes upregulated greater than two-fold by anti-IgM treatment through DNA microarray analysis, we focused on B cell translocation gene 1 (Btg1) and Btg2, member of Btg/Tob family of proteins. WEHI-231 cells were infected with the Btg1/EGFP or Btg2/EGFP retroviral vectors, and those expressing either Btg1 or Btg2 accumulated in G1 phase at significantly higher proportions than that seen for cells expressing control vector. Btg1 or Btg2 bound to protein arginine methyltransferase (PRMT) 1 via the box C region, an interaction required for anti-IgM-induced growth inhibition. The arginine methyltransferase inhibitor AdOx partially abrogated growth inhibition induced by Btg1, Btg2, or anti-IgM. The Btg1- or Btg2-induced growth inhibition was also abrogated in PRMT1-deficient cells via introduction of small interference RNA. In addition, we observed anti-IgM-induced arginine methylation of two proteins, a 28-kDa and a 36-kDa protein. Methylation, detected by a monoclonal antibody specific for asymmetric, but not symmetric methyl residues, was observed as early as 1 h-2 h after stimulation and was sustained for up to 24 h. The anti-IgM-induced p36 arginine methylation was abrogated in the PRMT1-deficient cells, suggesting that PRMT1 induces p36 methylation. Together, these results suggest that anti-IgM-induced growth inhibition is mediated via upregulation of Btg1 and Btg2, resulting in the activation of arginine methyltransferase activity and culminating in growth inhibition of WEHI-231 cells.     

Growth hormone and ghrelin secretion in severely obese women before and after bariatric surgery

Objective: The objective was to evaluate ghrelin and growth hormone (GH) interactions and responses to a growth hormone‐releasing hormone (GHRH)/arginine test in severe obesity before and after surgically‐induced weight loss. Research Methods and Procedures: Our study population included 11 severely obese women 39 ± 12 years of age, with a mean BMI of 48.6 ± 2.4 kg/m², re‐studied in a phase of stabilized body weight, with a BMI of 33.4 ± 1.2 kg/m², 18 months after having successfully undergone biliopancreatic diversion (BPD). A GHRH/arginine test was performed before and 18 months after BPD to evaluate ghrelin and GH interactions. Active ghrelin, measured by radioimmunoassay (RIA), and GH, measured by chemiluminescence assay, were assayed before and after the GHRH/arginine test. Results: Fasting serum GH levels and GH area under the curve (AUC) significantly increased from 0.2 ± 0.05 ng/mL to 1 ± 0.3 ng/mL (p < 0.05) and from 514.76 ± 98.7 ng/mL for 120 minutes to 1957.3 ± 665.1 ng/mL for 120 minutes after bariatric surgery (p < 0.05), respectively. Although no significant change in fasting ghrelin levels was observed (573 ± 77.9 before BPD vs. 574.1 ± 32.7 after BPD), ghrelin AUC significantly increased from ?3253.9 ± 2180.9 pg/mL for 120 minutes to 1142.3 ± 916.4 pg/mL for 120 minutes after BPD (p < 0.05). Fasting serum insulin‐like growth factor (IGF)‐1 concentration did not change significantly (133.6 ± 9.9 ng/mL before vs. 153.3 ± 25.2 ng/mL after BPD). Discussion: Our study demonstrates that the mechanisms involved in ghrelin and GH secretion after the secretagogue stimulus (GHRH/arginine) are consistent with patterns observed in other populations.     

厚壳贻贝鸟氨酸-尿素循环中的关键代谢物及基因分析

鸟氨酸-尿素循环(OUC)是生物新陈代谢过程中的重要循环过程,但在贝类中尚缺乏相关研究。为此,以厚壳贻贝为研究对象,分别采用氨基酸分析仪和荧光定量PCR研究了其外套膜和后闭壳肌组织中的鸟氨酸-尿素循环途径的主要代谢物和关键基因的含量及其表达量;进一步测试了在精氨酸注射条件下,各主要代谢物和关键基因的含量及表达量变化,以及¹³C标记尿素注射贻贝后,其贝壳中δ¹³C比值(¹³C/¹²C)变化。结果表明,厚壳贻贝外套膜和后闭壳肌均含有较高浓度的尿素;精氨酸注射导致其两种组织中尿素浓度显著上升(P<0.01),以及瓜氨酸浓度显著下降(P<0.01),但鸟氨酸浓度维持相对稳定的水平。精氨酸注射显著上调了两种组织中的脲酶基因的表达量(P<0.01),但其他基因表达量的变化在两种组织中存在差异,显示出鸟氨酸-尿素循环途径在其两种组织中具有复杂而不同的调控过程。¹³C标记尿素注射贻贝显著上调了贝壳中δ¹³C的比值(P <0.01),表明尿素分子可能参与了贻贝贝壳的生物矿化过程。上述研究为深入了解贻贝鸟氨酸-尿素途径与生物矿化之间的关联,以及探讨贻贝对海水酸化耐受性的内在分子机制奠定了基础。