Isolation and Structure of Gibberellin A18 from Immature Seeds of Lupinus luteus

A new gibberellin, tentatively called Lupinus gibberellin I, was isolated from young yellow lupin seeds. It has been shown to have structure (X), and now named gibberellin A₁₈.     

A New Flavanone with Antifungal Activity Isolated from Hops

6-Isopentenylnaringenin 1, which has previously been synthesized by other workers, was isolated together with xanthohumol 2 and isoxanthohumol 3 from hard resins of hops ( Humulus lupulus L.). The structures of 1 and sophoraflavanone B were examined; that of the latter previously reported as 6-isopentenylnaringenin, has been revised to 8-isopentenylnaringenin. 1, 2 and 3 were found to have antifungal activities.     

Plant regeneration from hypocotyl segments of Lupinus luteus cv. L. Aurea

Complete plants of Lupinus luteus L. cv. Aurea that were regenerated from hypocotyl segments, bloomed, produced seeds and were efficiently nodulated by Bradyrhizobium sp. strains. The highest rates of shoot formation were obtained on A medium plus 1.3% agar with 10.0 M 2-isopentenyladenine (2iP) and 0.11 M naphthaleneacetic acid (NAA); the best rooting was achieved on a medium with 0.5 M NAA plus 0.05 M 2iP. Afterward, plantlets were transferred to either perlite or peat-containing pots and irrigated with a N-free nutrient solution until maturity. Direct rooting of hypocotyls could also be obtained on A medium with 1% agar.     

Characterization of linear-oblong pyrenoids with cp-DNA along their sides in Nitzschia sigmoidea (Bacillariophyceae)

In the past 10 years Nitzschia sigmoidea (Nitzsch) W. Sm. has begun to occur in Japanese rivers in various areas. It is a common diatom in Europe but was previously absent in Japan. Each chloroplast of N. sigmoidea contains many unusual linear‐oblong structures. The internal structure of the chloroplast in this species was observed using epifluorescence and electron microscopy with immunolocalization techniques. The linear‐oblong structures in the chloroplasts could hardly be observed by conventional light microscopy of living cells, but were obvious in cells stained with propionocarmine. Transmission electron microscopy showed that the cross sections of this structure were lanceolate to fusiform with penetration by a single thylakoid. In cells stained with DAPI, chloroplast DNA was detected along both sides of the linear‐oblong structures, and DNA fibrils were detected by electron microscopy. Immunofluorescence microscopy of sectioned cells and also immunoelectron microscopy revealed specific localization of Rubisco between these DNA‐containing areas, which divided at the same time as the chloroplast. Our observations confirmed that the linear‐oblong structures are pyrenoids. The diversity of localization patterns of chloroplast DNA in diatoms is discussed.     

A PSTAIRE CDK-like protein localizes in nuclei and cytoplasm of Physarum polycephalum and functions in the mitosis

CDKs play key roles in controlling cell cycle progression in all eukaryotes. In plants, multiple CDKs are present,among which the best characterized CDKs are PSTAIRE CDKs. In this study, we carried out Western blot,immunoelectron microscopy and antibody treatment with an anti-PSTAIRE monoclonal antibody to explore the subcellular localization and functions of PSTAIRE CDKs in Physarum polycephalum. The results of Western blot and immunoelectron microscopy showed that in P. polycephalum, a PSTAIRE CDK-like protein was 34 kD in molecular weight and located in both nuclei and cytoplasm. In nuclei, the protein was mainly associated with chromosomes and nucleoli. The expression of the PSTAIRE CDK-like protein in both the plasmodia and nuclei showed little fluctuation through the whole cell cycle. When treated with an anti-PSTAIRE monoclonal antibody at early S phase, the cells were arrested in S phase, and the mitotic onset of P. polycephalum was blocked for about 1 h when treated at early G2 phase.Our data indicated that the PSTAIRE CDK-like protein has a direct bearing on the mitosis.     

多头绒泡菌SC35类蛋白的免疫电镜研究

经抗SC35单克隆抗体标记后,在电子显微镜下观察到多头绒泡菌S、G2、前期、中期和后末期细胞核中存在大量金颗粒,说明多头绒泡菌细胞核含有SC35类蛋白。在G2期和前期时,SC35类蛋白主要分布在细胞核的核仁区域和非核仁区域的染色质间区域;中期和后-末期时,SC35类蛋白主要分布在细胞核内染色体间区域;说明染色质(体)间区域和核仁区域是富含SC35类蛋白的区域。对核仁的进一步观察指出,在核仁中金颗粒主要分布在DFC,FC中的金颗粒很少,说明在核仁中SC35类蛋白主要存在于DFC组分中。     

The C5 domain of Col6A3 is cleaved off from the Col6 fibrils immediately after secretion.

In articular cartilage, type VI collagen is concentrated in the pericellular matrix compartment. During protein synthesis and processing at least the alpha3(VI) chain undergoes significant posttranslational modification and cleavage. In this study, we investigated the processing of type VI collagen in articular cartilage. Immunostaining with a specific polyclonal antiserum against the C5 domain of alpha3(VI) showed strong cellular staining seen in nearly all chondrocytes of articular cartilage. Confocal laser-scanning microscopy and immunoelectron microscopy allowed localization of this staining mainly to the cytoplasm and the immediate pericellular matrix. Double-labeling experiments showed a narrow overlap of the C5 domain and the pericellular mature type VI collagen. Our results suggest that at least in human adult articular cartilage the C5 domain of alpha3(VI) collagen is synthesized and initially incorporated into the newly formed type VI collagen fibrils, but immediately after secretion is cut off and is not present in the mature pericellular type VI matrix of articular cartilage.     

一株新的药用植物内生菌Burkholderia sp. H-6

植物内生菌(Plant endophyte)是微生物中的一个重要类群,能参与植物次级代谢产物的合成与转化或独立合成次级代谢产物,其物种丰富,数量庞大,已经成为新医(农)药活性物质的潜在资源.研究显示,目前从植物内生菌中分离出的生物活性物质,大约51%属于未报道过的新化合物.因此,植物内生菌相关领域的研究工作,愈来愈受到国内外同行的关注.     

嗜铬粒蛋白N端片段基因在枯草杆菌中的表达及表达产物的活性分析

嗜铬粒蛋白(CGA)是存在于分泌细胞的由439个氨基酸组成的可溶性蛋白。近年的研究发现CGA的N端具有抗血管收缩、抗细菌和抗真菌的功能。为了寻找高效低毒的抗真菌片段,利用PCR技术扩增了编码人嗜铬粒蛋白N端1-76位氨基酸（CGA1-76）的DNA片段,并将之克隆进本实验构建的枯草杆菌诱导型表达载体pSBPTQ,获得含CGA1-76基因的重组质粒pSVTQ,转化蛋白酶三缺陷的枯草杆菌DB403。经蔗糖诱导后,CGA1-76片段在枯草杆菌DB403（pSVTQ）中获得表达,产物分泌到细胞外,分泌量约为5mg/L,占总分泌蛋白的133% 。测试了表达产物对几种丝状真菌和酵母的抑制作用,发现在4μmol/L的浓度下,枯草杆菌表达的重组CGA1-76对镰刀菌、链格孢霉及白假丝酵母有明显的抑制作用。     

杜仲抗真菌蛋白晶体生长的原子力显微成像研究——快速生长与晶面生长速率

杜仲抗真菌蛋白(Eucommiaantifungalprotein,EAFP)的单晶体具有在几小时内就可长大的快速生长特性.用原子力显微成像(atomicforcemicroscope,AFM)技术,原位实时观测了EAFP单斜晶体生长过程中的｛10 0｝表面形貌动态变化,并分别在不同的过饱和度下测量了其生长速率.结果表明,EAFP晶体生长的速率与蛋白质溶液的过饱和度相关,在过饱和度高时(σ =1 78)晶面生长极快;在中等过饱和度(σ =1 5)下,其晶面台阶的生长速率沿b,c方向分别为 12nm/s和 2 4 2nm/s,比溶菌酶生长速率(6～ 7nm/s)快很多;在蛋白质浓度很低的情况下,其生长速率仍与其他蛋白质相当.EAFP晶体快速生长可能与该分子尺寸较小,内部结构紧凑,分子骨架呈刚性和分子表面性质等其固有特性密切相关.沉淀剂浓度对EAFP晶体生长也有影响.过饱和度很低时,提高沉淀剂浓度会干扰晶体生长.     

萝卜抗真菌蛋白1(Rs-AFP1)在大肠杆菌中的融合表达及其对大丽轮枝菌抑制活性的研究(英文)

利用聚合酶链式反应 (PCR)获得了萝卜 (RaphanussativusL .)抗真菌蛋白 1(Rs_AFP1)基因编码区核苷酸序列。将整个阅读框架片段和去除了N_端信号肽序列的片段分别装入原核表达载体pET_32b( )中 ,在大肠杆菌中表达 ,发现带有信号肽的Rs_AFP1不能在大肠杆菌中表达 ,而当这一序列去除后 ,表达出约 2 7kD的Rs_AFP1的融合蛋白。用凝血酶处理融合蛋白以去除N_端His.tag的部分序列 ,然后用处理后的融合蛋白进行了抑制真菌生长的实验。结果表明 ,在加入 0 .3g/L的Rs_AFP1的融合蛋白的培养液中 ,大丽轮枝菌 (VerticilliumdahliaeKleb .)的生长受到抑制 ,分别比加入对照细菌蛋白和PBS下降 5 7.5 %和 6 9.8% ;孢子的萌发也受到抑制。显然 ,细菌表达的融合蛋白对大丽轮枝菌的生长有抑制作用。     

枯草芽孢杆菌TF26抗菌蛋白抑菌活性的初步研究

目的:研究枯草芽孢杆菌TF26抗菌蛋白的抑菌活性和生物稳定性,为菌株及抗菌蛋白的应用提供理论依据.方法:采用硫酸铵盐析方法提取抗菌蛋白,采用菌丝生长速率法检测其对13种植物病原真菌的抑菌活性,采用抑菌圈方法对其生物稳定性进行分析.结果:抗菌蛋白粗提物能够抑制13种植物病原真菌的生长,平皿抑制率为74.3％ ～91.3％,对葵花菌核病菌、番茄和黄瓜枯萎病菌、黄瓜菌核病菌和立枯病菌、水稻恶苗病菌和大豆根腐病菌抑制作用较强.抗菌蛋白在100℃以下,pH＜ 10范围内抑菌活性稳定,对紫外线照射不敏感,室温(20℃)和4℃储存150d抑菌活性稳定.结论:抗菌蛋白具有较强的热、酸碱、紫外和储存稳定性以及广谱的抑菌活性.     

A global view of protein expression in human cells,tissues, and organs

Defining the protein profiles of tissues and organs is critical to understanding the unique characteristics of the various cell types in the human body. In this study, we report on an anatomically comprehensive analysis of 4842 protein profiles in 48 human tissues and 45 human cell lines. A detailed analysis of over 2 million manually annotated, high‐resolution, immunohistochemistry‐based images showed a high fraction (>65%) of expressed proteins in most cells and tissues, with very few proteins (<2%) detected in any single cell type. Similarly, confocal microscopy in three human cell lines detected expression of more than 70% of the analyzed proteins. Despite this ubiquitous expression, hierarchical clustering analysis, based on global protein expression patterns, shows that the analyzed cells can be still subdivided into groups according to the current concepts of histology and cellular differentiation. This study suggests that tissue specificity is achieved by precise regulation of protein levels in space and time, and that different tissues in the body acquire their unique characteristics by controlling not which proteins are expressed but how much of each is produced.     

A New Monoterpene from the Rhizomes of Alpinia galanga and Its Anti-Vpr Activity

A new menthane-type monoterpene, alpigalanol ( 1 ), together with four known terpenes ( 2 – 5 ) were isolated from the ethyl acetate soluble fraction of the 70 % ethanol extract of the Alpinia galanga rhizomes. The structure of 1 was determined by spectroscopic analyses, including 1D- and 2D-NMR. The extract of the A. galanga rhizomes and all isolated compounds ( 1 – 5 ) possessed Vpr inhibitory activities against the TREx-HeLa-Vpr cells at a concentration of 1.25 μM without showing any cytotoxicity.     

一个能与水稻未成熟种子核蛋白特异结合的31bp的DNA片段

水稻蜡质基因5'上游序列中有5个能与水稻胚乳核蛋白结合的片段,其中HinfId和RsaIe片段有部分重叠,而重叠部分含AACGT顺序。按重叠区上下游顺序合成了其中含有3次重复的AACGT顺序的31bp寡核苷酸,并以此为探针进行凝胶滞后实验,表明它不仅能与未成熟水稻种子的胚乳核蛋白特异结合,而且也与HinfId和RsaIe片段有强烈的竞争作用。因此31bp顺序中含有与水稻胚乳核蛋白专一结合位点,从而有可能应用此31bp的寡核苷酸作探针来分离编码蜡质基因的调控蛋白基因。     

萝卜抗真菌蛋白1（Rs-AFP1)在大肠杆菌中的融合表达 及其对大丽轮枝菌抑制活性的研究

The Raphanus sativus L. antifungal protein 1 (Rs-AFP1) gene was isolated by polymerase chain reaction (PCR). The complete open reading frame and the fragment encoding the putative mature protein were inserted into the prokaryotic expression vector pET-32b(+), respectively. Subsequent expression showed that the Rs-AFP1 was produced in E. coli as a 27 kD fusion protein only when the N-terminal signal peptide was removed. After treatment with thrombin to remove part of the N-terminal His.tag sequence, the bacterially expressed Rs-AFP1 was used for fungal growth inhibition assay which was conducted on Verticillium dahliae Kleb., a soil-born fungus causing the cotton wilt disease. Results showed that, in the liquid medium, the Rs-AFP1 fusion protein at a concentration of 0.3 g/L clearly inhibited the growth of V. dahliae and the germination of spores. Thus the bacterially expressed fusion protein had the antifungal activity against V. dahliae.     

麻疯树核糖体失活蛋白Curcin2的原核可溶性表达及其抗真菌活性研究

Curcin2是麻疯树幼苗在真菌侵染、干旱及高低温胁迫下诱导产生的一种核糖体失活蛋白.采用分子克隆的方法,从麻疯树基因组中扩增到Curcin2成熟肽编码基因,分别连接到质粒载体pGEX-6p-1、pMAL-c5E上,转化大肠杆菌最终获得重组菌PGC(pGEX-6p-1-curcin2)和PMC(pMAL-c5E-curcin2).在不同温度、IPTG浓度、时间诱导下,Curcin2在重组菌PGC中均以包涵体形式表达,在重组菌PMC中主要以可溶性融合蛋白形式表达,且蛋白质的表达量与诱导条件相关.重组菌PMC表达可溶性curcin2的优化条件为:温度28℃,IPTG 0.3mmol/L,时间8h,此条件下目的蛋白占总蛋白表达量的30.6％,1L培养物中可获得19.74mg电泳纯的重组蛋白.重组蛋白可被MBP TrapTM HP柱亲和纯化并与curcin抗体发生抗原抗体反应.体外抗真菌活性实验表明,纯化后的curcin2融合蛋白有抑制真菌生长作用,且对小麦赤霉、油菜菌核的抑制作用强于curcin.此蛋白的获得为其相关功能的研究奠定了基础.     

Molecular characterization of a nuclear topoisomerase II from Nicotiana tabacum that functionally complements a temperature-sensitive topoisomerase II yeast mutant

We have successfully expressed enzymatically active plant topoisomerase II in Escherichia coli for the first time, which has enabled its biochemical characterization. Using a PCR-based strategy, we obtained a full-length cDNA and the corresponding genomic clone of tobacco topoisomerase II. The genomic clone has 18 exons interrupted by 17 introns. Most of the 5 and 3 splice junctions follow the typical canonical consensus dinucleotide sequence GU-AG present in other plant introns. The position of introns and phasing with respect to primary amino acid sequence in tobacco TopII and Arabidopsis TopII are highly conserved, suggesting that the two genes are evolved from the common ancestral type II topoisomerase gene. The cDNA encodes a polypeptide of 1482 amino acids. The primary amino acid sequence shows a striking sequence similarity, preserving all the structural domains that are conserved among eukaryotic type II topoisomerases in an identical spatial order. We have expressed the full-length polypeptide in E. coli and purified the recombinant protein to homogeneity. The full-length polypeptide relaxed supercoiled DNA and decatenated the catenated DNA in a Mg²⁺- and ATP-dependent manner, and this activity was inhibited by 4-(9-acridinylamino)-3-methoxymethanesulfonanilide (m-AMSA). The immunofluorescence and confocal microscopic studies, with antibodies developed against the N-terminal region of tobacco recombinant topoisomerase II, established the nuclear localization of topoisomerase II in tobacco BY2 cells. The regulated expression of tobacco topoisomerase II gene under the GAL1 promoter functionally complemented a temperature-sensitive TopII ^ts yeast mutant.     

An MTA phosphorylase gene discovered in the metagenomic library derived from Antarctic top soil during screening for lipolytic active clones confers strong pink fluorescence in the presence of rhodamine B

Eubacteria encode proteins that are required for nucleoid organization and for regulation of DNA-dependent processes. Of these histone-like proteins (Hlps), Escherichia coli HU has been shown to associate with the nucleoid and to regulate processes such as DNA repair and recombination. In contrast, the divergent HU homologs encoded by mycobacteria have been variously identified as involved in the physiology of dormancy, in the response to cold shock, or as laminin-binding proteins associated with the cell envelope. We show here, contrary to previous reports that the HU-related Hlp from Mycobacterium smegmatis associates with the nucleoid in vivo . Using indirect fluorescent antibody microscopy we show that cold shock causes Hlp to accumulate in the cytoplasm of M. smegmatis . No evidence of surface-associated Hlp was found in M. smegmatis cells treated for cell wall permeabilization. Quantitative Western blots indicate that exponentially growing cells contain c . 120 molecules per cell, with upregulation of Hlp after cold shock estimated to be c . 10-fold. That Hlp associates with the nucleoid in vivo suggests functions in DNA metabolism, perhaps in adaptation to environmental stress.