Transmembrane kinase 1-mediated auxin signal regulates membrane-associated clathrin in Arabidopsis roots

Clathrin-mediated endocytosis (CME) is the major endocytic pathway in eukaryotic cells that directly regulates abundance of plasma membrane proteins. Clathrin triskelia are composed of clathrin heavy chains (CHCs) and light chains (CLCs), and the phytohormone auxin differentially regulates membrane-associated CLCs and CHCs, modulating the endocytosis and therefore the distribution of auxin efflux transporter PIN-FORMED2 (PIN2). However, the molecular mechanisms by which auxin regulates clathrin are still poorly understood. Transmembrane kinase (TMKs) family proteins are considered to contribute to auxin signaling and plant development; it remains unclear whether they are involved in PIN transport by CME. We assessed TMKs involvement in the regulation of clathrin by auxin, using genetic, pharmacological, and cytological approaches including live-cell imaging and immunofluorescence. In tmk1 mutant seedlings, auxin failed to rapidly regulate abundance of both CHC and CLC and to inhibit PIN2 endocytosis, leading to an impaired asymmetric distribution of PIN2 and therefore auxin. Furthermore, TMK3 and TMK4 were shown not to be involved in regulation of clathrin by auxin. In summary, TMK1 is essential for auxin-regulated clathrin recruitment and CME. TMK1 therefore plays a critical role in the establishment of an asymmetric distribution of PIN2 and an auxin gradient during root gravitropism.     

Systematics of Mukdenia and Oresitrophe (Saxifragaceae): Insights from genome skimming data

Oresitrophe and Mukdenia (Saxifragaceae) are epilithic sister genera used in traditional Chinese medicine. The taxonomy of Mukdenia, especially of M. acanthifolia, has been controversial. To address this, we produced plastid and mitochondrial data using genome skimming for Mukdenia acanthifolia and Mukdenia rossii, including three individuals of each species. We assembled complete plastomes, mitochondrial CDS and nuclear ribosomal ETS/ITS sequences using these data. Comparative analysis shows that the plastomes of Mukdenia and Oresitrophe are relatively conservative in terms of genome size, structure, gene content, RNA editing sites and codon usage. Five plastid regions that represent hotspots of change (trnH-psbA, psbC-trnS, trnM-atpE, petA-psbJ and ccsA-ndhD) are identified within Mukdenia, and six regions (trnH-psbA, petN-psbM, trnM-atpE, rps16-trnQ, ycf1 and ndhF) contain a higher number of species-specific parsimony-informative sites that may serve as potential DNA barcodes for species identification. To infer phylogenetic relationships between Mukdenia and Oresitrophe, we combined our data with published data based on three different datasets. The monophyly of each species (Oresitrophe rupifraga, M. acanthifolia and M. rossii) and the inferred topology ((M. rossii, M. acanthifolia), O. rupifraga) are well supported in trees reconstructed using the complete plastome sequences, but M. acanthifolia and M. rossii did not form a separate clade in the trees based on ETS + ITS data, while the mitochondrial CDS trees are not well-resolved. We found low recovery of genes in the Angiosperms353 target enrichment panel from our unenriched genome skimming data. Hybridization or incomplete lineage sorting may be the cause of discordance between trees reconstructed from organellar and nuclear data. Considering its morphological distinctiveness and our molecular phylogenetic results, we strongly recommend that M. acanthifolia be treated as a distinct species.     

Probing the structure and mobility of Pseudomonas aeruginosa azurin by circular dichroism and dynamic fluorescence anisotropy.

The UV dynamic fluorescence and CD of several Pseudomonas aeruginosa azurins bearing single amino acid mutation have been studied. Two classes of mutants were examined. In the first class, two hydrophobic residues in the core of the protein, Ile 7 and Phe 110, nearest to the azurin single tryptophan Trp 48, were substituted by a serine (mutants 17S and F110S). In the second class, two residues in the outer sphere of the copper ligand field were changed, obtaining the following mutants: M44K, H35F, H35L, and H35Q. All these proteins showed two fluorescence lifetimes in the copper-containing form, but only one in the copper-free form. The lifetime of the latter derivatives was different from either those of the metal-bound samples, definitely ruling out the presence of apo-like species in the holo protein. Copper-free 17S and F110S showed a more complex fluorescence decay profile requiring a distribution of lifetimes rather than a single lifetime. Holo F110S was also better fitted, in the limit of confidence, with two distributions rather than a pair of lifetimes. Time-resolved anisotropy of these two mutants as well as of wild-type (wt) protein showed two components (rotational times for wt < or = 200 ps and 7 ns, respectively). These components were not affected significantly by copper removal in the case of wt protein. Instead, the short rotational component of the mutants dropped dramatically to values near zero, indicating a much greater mobility of the tryptophanyl residue in the mutant apo azurins. These data were supported by CD measurements showing a small effect of the copper presence in the region below 250 nm, i.e., in the secondary structure, but almost a collapse of the aromatic asymmetry at 270-295 nm related to a relaxation of the structural constraint around the tryptophan. Altogether these data show that copper does not play a structural role in wt azurin, whereas it is crucial in the stabilization of 17S and F110S mutants. Furthermore, although the metal site geometry is rigidly kept in wt apo-azurin, it regains the native form only in the presence of the metal in the "core" mutants. This finding is important for the theory of entatic states in metalloproteins (Williams RJP, 1995, Eur J Biochem 234:363-381).     

细叶马先蒿根系发育形态学研究

细叶马先蒿为玄参科多年生草本植物。年生产周期明显缩短。根系营养生长至花期为粗壮主根与纤细侧根并存,果期侧根几乎全部枯萎脱落,所存留根系皆呈乳白色。由胚根形成的初生主根根毛密集,初生木质部二原型。侧生分生组织只有形成层而无木栓形成层。根表皮细胞经解离后略呈不规则方形片状,横切面为平周长梭形,进行垂周分裂增加梭形根表皮细胞长度,以适应根的增粗生长。根表皮脱落时,外皮层以同样生长方式代替脱落的表皮。在年     

丝氨酸蛋白酶抑制剂的研究及应用

丝氨酸蛋白酶抑制剂（serpin）是一类结构、序列同源的蛋白酶抑制剂,它是体内许多蛋白水解级联反应的调节因子,其遗传性结构或分泌异常将导致许多疾病．因此对于其结构及作用机理的研究将为临床应用提供依据．     

固定化酵母细胞生产1,6-二磷酸果糖研究

本文研究了固定化酵母细胞制备果糖1,6二磷酸(FDP)的方法及其生产。用卡拉胶包埋方法固定化酿酒酵母(Sacchromyces cerevisae),对含葡萄糖1.0M,磷酸盐0.8M的糖磷液,pH6.5,在37℃下进行磷酸化反应。反复分批转化20天以上,可达到平均产FDPH_427.58mg/ml,最高为59.94mg/ml。用100ml固定化细胞生物反应器连续运转309h,稀释速率D=0.097h~(-1),平均产FDPH_4 21.51mg/ml。20L反应器连续运转,生产能力达到1.7g/h.L。用层析方法制备FDPNa_3结晶粉,提取收率为72.08％,制备质量达到或超过了国内外同类产品的质量要求。     

通过染色体外同源重组建立乳腺中表达外源基因的转基因小鼠

为研究染色体外重组方法在创建转基因动物中的应用,选取牛asl酪蛋白基因的5′及3′侧翼区和氯霉素乙酰化酶编码区,构建了两个具有3 kb相互重叠的融合基因.将这两个DNA片段末端脱磷酸后,以摩尔比1:1的比例混合,通过显微注射导入小鼠受精原核,最后获得了11个品系的转基因小鼠.对其中10个品系的小鼠的整合分析表明,所注射的两个DNA片段均发生了染色体外同源重组,而且除了 1个品系的小鼠丢失了大约1kb的序列外,其余品系小鼠的重组产物与预想的结构符合.在当代和后代的转基因小鼠乳汁中均可测到氯霉素乙酰化酶的活性.这表明融合基因在转基因小鼠乳腺中得到表达和分泌,也说明显微共注射两个相互重叠的基因片段是建立转基因动物的一个可行途径.     

龙血树真菌群及其对血竭形成的影响

从柬埔寨龙血树（Ｄｒａｃａｅｎａｃｏｃｈｉｎｃｈｉｎｅｎｓｉｓ）茎杆中分离到３０３株真菌,其中镰刀菌属（Ｆｕｓａｒｉｕｍ）菌株占总分离频率的５２％,其次是短梗霉（Ａｕｒｅｏｂａｓｉｄｉｕｍ）和枝孢霉（Ｃｌａｄｏｓｐｏｒｉｕｍ）。通过活体接种对血竭产生的影响试验表明,对血竭形成起重要作用的真菌主要是禾谷镰刀菌龙血树变种（Ｆ．ｇｒａｍｉｎｕｍｖａｒ．ｄｒａｃａｅｎａ）等４株红色镰刀菌,可使血竭形成量提高６６％－１２０％。     

l-Fucose residues on cellulose-based dialysis membranes: Quantification of membrane-associatedl-fucose and analysis of specific lectin binding

Contact of mononuclear human leukocytes with cellulose dialysis membranes may result in complement-independent cell activation, i.e. enhanced synthesis of cytokines, prostaglandins and an increase in 2-microglobulin synthesis. Cellular contact activation is specifically inhibited by the monosaccharidel-fucose suggesting that dialysis membrane associatedl-fucose residues are involved in leukocyte activation. In this study we have detected and quantitatedl-fucose on commercially-available cellulose dialysis membranes using two approaches. A sensitive enzymatic fluorescence assay detectedl-fucose after acid hydrolysis of flat sheet membranes. Values ranged from 79.3±3.6 to 90.2±5.0 pmol cm^–2 for Hemophan® or Cuprophan® respectively. Enzymatic cleavage of terminal -l-fucopyranoses with -l-fucosidase yielded 7.7±3.3 pmoll-fucose per cm² for Cuprophan. Enzymatic hydrolysis of the synthetic polymer membranes AN-69 and PC-PE did not yield detectable amounts ofl-fucose. In a second approach, binding of the fucose specific lectins ofLotus tetragonolobus andUlex europaeus (UEAI) demonstrated the presence of biologically accessiblel-fucose on the surface of cellulose membranes. Specific binding was observed with Cuprophan®, and up to 2.6±0.3 pmoll-fucose per cm² was calculated to be present from Langmuir-type adsorption isotherms. The data presented are in line with the hypothesis that surface-associatedl-fucose residues on cellulose dialysis membranes participate in leukocyte contact activation.