Determination of the main hydrolysis product of O-ethylS-2-diisopropylaminoethyl methylphosphonothiolate, ethyl methylphosphonic acid, in human serum

For the unequivocal proof of the use of a nerve agent O-ethyl S-2-diisopropylaminoethyl methylphosphonothiolate (VX), a rapid, accurate and sensitive method which allows us to identify its main hydrolysis product ethyl methylphosphonic acid (EMPA) in human serum was explored by GC-MS. GC-MS analysis was performed after solvent extraction with acetonitrile in acidic conditions from the serum sample, which was previously deproteinized by micro-ultrafiltration, and subsequent tert.-butyldimethylsilyl derivatization with N-methyl-N-(tert.-butyldimethylsilyl)trifluoroacetamide (MTBSTFA) with 1% tert.-butyldimethylsilyl chloride (t-BDMSC). Linear calibration curves were obtained in the concentration range from 50 to 500 ng/ml for EMPA in the full-scan EI mode and from 5 to 50 ng/ml for EMPA in the SIM EI mode. The relative standard deviation obtained at a sample concentration of 50 ng/ml was 8.4% in the full-scan mode and 7.3% in the SIM mode. Upon applying the full-scan EI and CI mode, 40 ng/ml and 80 ng/ml were the detection limits. Using the SIM-EI mode, in which the ion at m/z 153 was chosen, the limit was 3 ng/ml.     

A comparison of the genotoxicity of ethylnitrosourea and ethyl methanesulfonate in lacZ transgenic mice (Muta™Mouse)

We compared the induction of gene mutations and chromosomal aberrations by ethylating agents in lacZ transgenic mice (Muta™Mouse). Chromosomal aberrations were detected by the peripheral blood micronucleus assay. Gene mutations were detected in the lacZ transgene. A small amount of blood was sampled from a tail vessel during the expression time for fixation of gene mutations in vivo; this enabled us to detect and compare clastogenicity and gene mutations in the identical mouse. Single intraperitoneal injections of ENU (50–200 mg/kg) and EMS (100–400 mg/kg) strongly induced micronucleated reticulocytes (MN) detectable in peripheral blood 48 h after treatment. The maximum MN frequencies induced were 6.6% and 3.3% for ENU (100 mg/kg) and EMS (400 mg/kg), respectively (the control value was 0.3%). lacZ mutant frequency (MF) was analyzed in bone marrow and liver 7 days after treatment. Spontaneous MFs were 2.0–4.6x10⁻⁶. MF in bone marrow was increased by ENU to 3.4x10⁻⁵ at 200 mg/kg and induced by EMS to 1.8x10⁻⁵ at 400 mg/kg. In liver, however, both chemicals at their highest doses induced only slight increases in MF. The induction of both micronuclei and lacZ mutations in bone marrow by both ENU and EMS correlated better with O⁶-ethylguanine adducts than with N7-ethylguanine adducts. The mutants (19 for ENU and 12 for EMS) were subjected to DNA sequence analysis. Among EMS-induced mutations, 75% were GC to AT transitions, which were probably caused by O⁶-ethylguanine. Among ENU-induced mutations, in contrast, 40% occurred as AT base pair substitutions (6 AT to TA transversions and 2 AT to GC transitions) (no such mutations were induced by EMS). These results, together with the known reactivity of ENU to thymine suggest that thymine adducts play a significant role in the ENU mutagenesis.     

Mutational specificity of ethyl methanesulfonate in excision-repair-proficient and -deficient strains of Drosophila melanogaster

Summary The vermilion gene was used as a target to determine the mutational specificity of ethyl methanesulfonate (EMS) in germ cells of Drosophila melanogaster. To study the impact of DNA repair on the type of mutations induced, both excision-repair-proficient (exr ⁺) and excision-repair-deficient (exr ^–) strains were used for the isolation of mutant flies. In all, 28 mutants from the exr ⁺ strain and 24 from the exr ^– strain, were characterized by sequence analysis. In two mutants obtained from the exr ⁺ strain, small deletions were observed. All other mutations were caused by single base-pair changes. In two mutants double base-pair substitutions had occurred. Of the mutations induced in the exr ⁺ strain, 22 (76%) were GCAT transitions, 3 (10%) ATTA transversions, 2 (6%) GCTA transversions and 2 (6%) were deletions. As in other systems, the mutation spectrum of EMS in Drosophila is dominated by GCAT transitions. Of the mutations in an exr ^– background, 12 (48%) were GCAT transitions, 7 (28%) ATTA transversions, 5 (20%) GCTA transversions and 1 (4%) was a ATGC transition. The significant increase in the contribution of transversion mutations obtained in the absence of an active maternal excision-repair mechanism, clearly indicates efficient repair of N-alkyl adducts (7-ethyl guanine and 3-ethyl adenine) by the excision-repair system in Drosophila germ cells.     

The role of different anions in ionic liquids on Pseudomonas cepacia lipase catalyzed transesterification and hydrolysis

Lipase Pseudomonas cepacia (PS) catalyzed transesterification of ethyl 3-phenylpropanoate with eleven alcohols was investigated in three ionic liquids [ILs], [Bmim]BF₄, [Bmim]PF₆, and [Bmim]Tf₂N, consisting of an identical cation and different anions. The yields were higher in hydrophobic ILs [Bmim]Tf₂N (55–96%) and [Bmim]PF₆ (22–95%), than in hydrophilic [Bmim]BF₄ (0–19%). The incubation of lipase PS in hydrophobic ILs for a period of 20–300 days at room temperature resulted in an increased yield of 62–98% in [Bmim]Tf₂N and 45–98% in [Bmim]PF₆, respectively. The lipase PS-hydrophobic IL mixture was recycled five times without any decrease in the yield of the products. In another set of experiments, the hydrolytic activity of the enzyme was determined after incubation in each of the three ILs and in hexane for 20 days at room temperature. It was found to be 1.8- and 1.6-fold higher in [Bmim]Tf₂N and [Bmim]PF₆, respectively, remained unchanged in [Bmim]BF₄ and was 1.6 times lower in hexane as compared to the non-incubated enzyme.     

A novel carbonyl reductase from Pichia stipitis for the production of ethyl (S)-4-chloro-3-hydroxybutanoate

An NADPH-dependent carbonyl reductase (PsCR) gene from Pichia stipitis was cloned. It contains an open reading frame of 849 bp encoding 283 amino acids whose sequence had less than 60% identity to known reductases that produce ethyl (S)-4-chloro-3-hydroxybutanoates (S-CHBE). When expressed in Escherichia coli, the recombinant PsCR exhibited an activity of 27 U/mg using ethyl 4-chloro-3-oxobutanoate (COBE) as a substrate. Reduction of COBE to (S)-CHBE by transformants in an aqueous mono-phase system for 18 h, gave a molar yield of 94% and an optical purity of the (S)-isomer of more than 99% enantiomeric excess.     

麻竹miR172a靶基因DlAP2的克隆及其表达

为了解开花麻竹(Dendrocalamus latiflorus)的Dl AP2基因功能,采用RT-PCR和RACE技术克隆了mi R172a靶基因AP2同源序列c DNA全长,命名为Dl AP2。结果表明,Dl AP2基因c DNA全长为1729 bp,包含5′端非编码区81 bp、开放阅读框1464 bp、3′端非编码区160 bp和24个碱基的Poly A尾巴,在编码框靠近3′端130 bp处有1个高度匹配mi R172a的结合位点(CTGCAGCATCATCAGGATTCT)。Dl AP2编码487个氨基酸的蛋白,具有两个AP2结构域,属于AP2/ERF家族AP2亚家族的AP2组,与来自其它单子叶植物的AP2蛋白均有较高同源性。RLM-5′RACE分析表明,mi R172a主要在靶序列的第11~12个碱基之间剪切靶基因Dl AP2的mi RNA。q RT-PCR结果表明,麻竹花芽中Dl AP2基因的表达规律与mi R172a表达变化正好相反,证明mi R172a对Dl AP2基因的表达具有调控作用。     

Efficient in vitro synthesis of cis-polyisoprenes using a thermostable cis-prenyltransferase from a hyperthermophilic archaeon Thermococcus kodakaraensis

The Tk-idsB encoding cis-prenyltransferase which catalyzes consecutive cis-condensation of isopentenyl diphosphate to allylic diphosphate was isolated from a hyperthermophilic archaeon Thermococcus kodakaraensis, and enzymatic characteristics of the recombinant Tk-IdsB were examined. Tk-IdsB was not fully denatured even at 90 °C and preferably utilizes both C₁₀ and C₁₅ allylic diphosphates to yield mainly the C₆₀–C₆₅ products. Based on structural models, single alanine-substitution mutants at Glu68, Lys109, or Leu113 were constructed, showing that all the three produced longer chains (C₆₅–C₇₀) than the wild-type and the substitution at 109 (K109A) was the most effective. Tk-IdsB was applied to an organic-aqueous dual-phase system and more than 90% of the products were recovered from the organic phase when 1-butanol or 1-pentanol was overlaid. When 1-octanol was overlaid, 70% of the products were obtained from the upper organic phase. The product distributions were changed depending on the hydrophobicity of organic solvents used. Tk-IdsB was then immobilized onto silica beads to make Tk-IdsB more tolerant, showing that half-life of enzyme at 80 °C was prolonged by immobilization. When the immobilized Tk-IdsB was applied in the organic-aqueous dual-phase system, immobilized Tk-IdsB catalyzed consecutive condensation more efficiently than the unimmobilized one.     

The nifHDK genes are contiguous with a nifA-like regulatory gene in Rhodobacter capsulatus

Summary A 15.2 kb DNA fragment was isolated from Rhodobacter capsulatus (ex. Rhodopseudomonas capsulata), which was able to complement mutations both in a nifA-like regulatory gene and in the nifH gene. Physical mapping of this fragment revealed that the nifA-like gene was adjacent to, and downstream from, the nifHDK operon. Hybridization experiments were carried out using a cloned Klebsiella pneumoniae DNA fragment containing nifA and the flanking portions of nifB and nifL. This fragment failed to hybridize with a 2.15 kb HindIII fragment of R. capsulatus DNA containing the nifA-like gene, but hybridized instead with a 2.6 kb EcoRI fragment adjacent to the nifA-like gene. The homologous region was found to be located within the K. pneumoniae nifB gene. The adjacent 2.6 kb and 2.15 kb fragments also hybridized with each other, indicating the presence of repeated sequences in this region.     

一株嗜麦芽窄食单胞菌裂解性噬菌体的分离及生物学特性

【背景】嗜麦芽窄食单胞菌是一种广泛存在于医院和自然环境中的条件致病菌,其分离率与耐药率逐年增加。噬菌体是一类能特异性感染并杀灭细菌的病毒。【目的】分离一株新型嗜麦芽窄食单胞菌噬菌体,为临床嗜麦芽窄食单胞菌感染及防控提供补充手段。【方法】以临床分离的嗜麦芽窄食单胞菌为宿主菌,用点板法从医院污水中分离鉴定噬菌体;用双层平板法测定噬菌体效价及一步生长曲线等生物学特性;用透射电镜观察噬菌体形态;提取噬菌体基因组DNA进行全基因组测序,拼接噬菌体基因组并进行注释。【结果】分离到一株嗜麦芽窄食单胞菌裂解性噬菌体,命名为v B＿Sma S＿P11。该噬菌体感染宿主菌的潜伏期小于5 min,快速增殖60 min后达到平稳期,暴发量为100 PFU/cell。透射电镜观察该噬菌体为长尾噬菌体,具有典型的二十面体头和不可收缩的尾部。基因组测序结果表明,该噬菌体基因组全长44 600 bp,GC含量为63.7%,无抗生素耐受基因、毒力基因和t RNA,与NCBI数据库中所有已知嗜麦芽窄食单胞菌噬菌体相比同源性很低。基因组注释显示该噬菌体含有66个开放阅读框(open reading frame,ORF),其...     

Construction of a Tn5 derivative encoding bioluminescence and its introduction in Pseudomonas, Agrobacterium and Rhizobium

Summary A simple method based upon the use of a Tn5 derivative, Tn5-Lux, has been devised for the introduction and stable expression of the character of bioluminescence in a variety of gram-negative bacteria. In Tn5-Lux, the luxAB genes of Vibrio harveyi encoding luciferase are inserted on a SalI-BglII fragment between the kanamycin resistance (Km^r) gene and the right insertion sequence. The transposon derivative was placed on a transposition suicide vehicle by in situ recombination with the Tn5 suicide vector pGS9, to yield pDB30. Mating between Escherichia coli WA803 (pDB30) and a strain from our laboratory, Pseudomonas sp. RB100C, gave a Km^r transfer frequency of 10^-6 per recipient, a value 10 times lower than that obtained with the original suicide vehicle pGS9. Tn5-Lux was also introduced by insertion mutagenesis in other strains of gram-negative soil bacteria. The bioluminescence marker was expressed in the presence of n-decanal, and was monitored as chemiluminescence in a liquid scintillation counter. The recorded light intensities were fairly comparable among the strains, and ranged between 0.2 to 1.8x10⁶ cpm for a cell density of 10³ colony forming units/ml. Nodules initiated by bioluminescent strains of Rhizobium leguminosarum on two different hosts were compared for intensity of the bioluminescence they produced.     

Competition in microcosm between a clonal plant species (Bolboschoenusmaritimus) and a rare quillwort (Isoetessetacea) from Mediterranean temporary pools of southern France

Bolboschoenus maritimus, a clonal species, is locally invasive in Mediterranean temporary pools where it threatens endangered rare plant species such as Isoetes setacea. The combination of management modifications (grazing) and of the progressive accumulation of fine sediments in the pools contributed to the establishment of competitive perennial plants such as B. maritimus. The competitive advantage of B. maritimus on I. setacea has been studied in controlled conditions. The goal of this experiment was to assess the role of environmental conditions in the output of the competition between Bolboschoenus and Isoetes, notably hydrology and soil richness. For this purpose, Isoetes was cultivated alone (three individuals/pot) and with Bolboschoenus (three individuals of both species). The experiment was run with five replicates on six types of sediment (gradient of richness in sand/silt/clay) combined with three hydrological treatments (flooded, wet. and dry). The competitive advantage of Bolboschoenus was measured as the ratio of the production of Isoetes in mixture versus monoculture. The results showed that Isoetes was always outcompeted by Bolboschoenus. However, the competitive advantage of Bolboschoenus on Isoetes, was more related to hydrology than to soil richness. The competitive advantage of Bolboschoenus was very high in wet and flooded conditions and very low in dry conditions. This situation may lead to the extinction, medium-term, of the populations of I. setacea. The introduction of ovine grazing or of cut back practices in temporary pools could reduce the B. maritimus biomass and help toward the conservation of I. setacea populations. Guest editors: B. Oertli, R. Cereghino, A. Hull & R. Miracle Pond Conservation: From Science to Practice. 3rd Conference of the European Pond Conservation Network, Valencia, Spain, 14–16 May 2008     

Cesariella, a new genus of Laboulbeniales

Cesariella graeca gen. sp. nov. is described to accommodate a new species of the Laboulbeniales (Fungi, Ascomycota) parasitic on the endogean ground beetles Reicheadella aetolica and R. bischoffi (Coleoptera, Carabidae) from Greece. Cesariella is distinguished from the allied genus Laboulbenia by the presence of two cells borne on the inner side of cell III, and by the presence of a conspicuous remnant of the spore apex protruding laterally near the base of the appendage.     

Identification of 4-O-methyl-D-xylose as a constituent of the cell wall of Chlorella vulgaris

From the cell wall of a strain of Chlorella vulgaris a sugar was isolated after acid hydrolysis and was identified as 4-O-methyl-D-xylose by the following criteria: (i) mass spectroscopy of its alditol acetate revealed characteristic primary fragments with m/e 117 and m/e 261, and, when one deuterium atom was substituted at C-1, with m/e 262 instead of m/e 261; (ii) after demethylation with BCl₃, xylose was identified as its parent sugar by chromatographic methods; (iii) L-iditol: NAD 5-oxidoreductase (sorbitol dehydrogenase) catalyzed the oxidation of its alditol, but not of 4-O-methyl-L-xylitol. 4-O-Methyl-D-xylose amounted to approx. 10% of the cell walls' dry weight or 1.6% of the cells' dry weight.     

Selective grazing of Temora longicornis in different stages of a Phaeocystis globosa bloom—a mesocosm study

Selective grazing of a calanoid copepod Temora longicornis was measured during different stages of a Phaeocystis globosa bloom, in order to reveal (1) if T. longicornis feeds on single cells and/or colonies of P. globosa in the presence of alternative food sources, (2) if copepod food selection changes during the initiation, maintenance, collapse and decay of a P. globosa bloom and (3) if P. globosa dominated food assemblage provides a good diet for copepod egg production. Our results show low but constant feeding on small colonies of P. globosa, irrespective of the type or concentration of alternative food sources. In contrast, feeding on single cells was never significant, and the total contribution of P. globosa to carbon ingestion of T. longicornis was minor. T. longicornis fed most actively on the decaying colonies, whereas during the peak of the bloom copepods selected against P. globosa. Mostly, T. longicornis fed unselectively on different food particles: before the bloom, the major part of the diet consisted of diatoms, whereas during and after the bloom copepod diet was dominated by dinoflagellates and ciliates. Egg production was highest during the decay of the bloom, coinciding with highest proportional ingestion of heterotrophic organisms, but was not seriously reduced even during the peak of the bloom. We conclude that P. globosa blooms should not threaten survival of copepod populations, but the population recruitment may depend on the type (and concentration) of the dominant heterotrophs present during the blooms. Due to relatively unselective grazing, the impact of T. longicornis to the initiation of a Phaeocystis bloom is considered small, although grazing on decaying colonies may contribute to the faster termination of a bloom.     

银杏bHLH91转录因子基因的克隆及表达分析

bHLH转录因子在植物的生长发育、胁迫应答和次生代谢中具有重要的调控作用。该研究通过PCR技术从银杏(Ginkgo biloba)叶中分离得到了一个bHLH基因的cDNA序列,并将其命名为GbbHLH91。序列分析结果显示扩增的GbbHLH91基因cDNA序列长度为1 425 bp,开放阅读框是1 065 bp,编码354个氨基酸,分子量为40.1 kDa,等电点为8.20。系统进化分析结果显示,从用于进化树构建的bHLH蛋白质聚类情况来看,银杏GbbHLH91蛋白与裸子植物油松(Pinus tabuliformis)bHLH蛋白亲缘关系最近,且与被子植物无油樟(Amborella trichopoda)bHLH蛋白相似性达到60%,表明该基因在进化过程中相对比较保守。实时荧光定量PCR分析发现银杏bHLH91基因在银杏的各个组织中均有表达,其中在银杏叶中表达量最高,在根和茎中基因的表达量次之,在银杏雌花和果中表达量较少,在雄花中的表达水平最低;GbbHLH91基因在不同发育时期的银杏叶片中,表达量也存在一定的差异,其中在4月中旬该基因的表达水平达到最高,而后随着叶片的生长发育,该基因的表达水平呈现下降趋势。该研究结果为进一步验证GbbHLH91基因的功能奠定了前期基础。     

Lysis of Aphanizomenon flos-aquae (Cyanobacterium) by a bacterium Bacillus cereus

A phytoplankton-lytic (PL) bacterium, Bacillus cereus, capable of lysing the bloom-forming cyanobacterium Aphanizomenon flos-aquae was isolated from Lake Dianchi of Yunnan province, China. This bacterium showed lytic activities against a wide range of cyanobacteria/algae, including A. flos-aquae, Microcystis viridis, Microcystis wesenbergi, Microcystis aeruginosa, Chlorella ellipsoidea, Oscillatoria tenuis, Nostoc punctiforme, Anabaena flos-aquae, Spirulina maxima, and Selenastrum capricornutum. Chlorophyll a contents, phycocyanin contents, and photosynthetic activities of the A. flos-aquae decreased evidently in an infected culture for a period. Bacterium B. cereus attacked rapidly A. flos-aquae cells by cell-to-cell contact mechanism. It was shown that the lysis of A. flos-aquae began with the breach of the cyanobacterial cell wall, and the cyanobacterial cell appeared abnormal in the presence of the PL bacterium. Moreover, transmission electron microscope examinations revealed that a close contact between the bacterium and the cyanobacterium was necessary for lysis. Some slime extrusions produced from B. cereus assisted the bacterial cells to be in close association with and lyse the cyanobacterial cells. These findings suggested that this bacterium could play an important role in controlling the Aphanizomenon blooms in freshwaters.     

Geocoris punctipes as a predator of Bemisia tabaci: a laboratory evaluation

Geocoris punctipes (Say), a predaceous lygaeid not previously docmented as a whitefly predator, was tested in the laboratory as a natural enemy of the sweet potato whitefly, Bemisia tabaci (Genn.). Its stalking behavior with whiteflies as prey was similar to that observed with aphids. A previously unobserved behavior was noted that involves the predators using salivary secretions to fasten the wings of prey to various surfaces, allowing labial probing and feeding. Prey consumption as a function of prey number appeared to follow the pattern of the Holling type II functional response. Handling time per prey item ranged from about 180 to 240 seconds. No changes were observed in handling time devoted to earlier versus later catches. Nutritional quality of whiteflies was measured using crude protein, lipids and carbohydrates as criteria. Performance, in terms of predator behavior, total daily handling time, functional response, energy budget and nutritional quality all support the hypothesis that G. punctipes is a promising candidate for biological control of sweet potato whiteflies.     

Characterization of a genomic clone of Erwinia carotovora subsp. carotovora TG3 encoding a pectolytic enzyme of apparent molecular weight 78 kDa

Summary A clone containing the gene encoding a pectolytic enzyme of Erwinia carotovora subsp. carotovora was selected as the one that showed maceration on a solid medium containing sodium polypectate. The gene was located on a 3.2-kb DNA fragment flanked by a BglII site and a Hin-dIII site. Via mini-Mudlac mutagenesis, a possible promoter site was located within the gene between the BglII site and the EcoRI site. The mRNA transcribed from the promoter was directed from the BglII site toward the EcoRI site, determined from the orientation of the inserted mini-Mudlac. The probable gene product was identified as a 78 kDa protein. The enzyme activity of the Escherichia coli clone was detected mainly in the periplasmic space. Potato tuber slices were not macerated by the E. coli clone and synthesis of the enzyme in E. coli was not regulated by the enzyme substrate, sodium polypectate.