Acquired resistance to echinostomes in four Biomphalaria glabrata strains

Four strains of Biomphalaria glabrata showed a distinctive pattern of acquired resistance to each of 3 echinostome species. Juvenile albino B. glabrata from our laboratory NIH stock developed a strong resistance to Echinostoma lindoense but only a weak one to E. paraensei and a moderate one to E. liei. Juvenile B. glabrata 10-R2 strain developed a strong acquired resistance to E. lindoense but a weak one to E. paraensei and E. liei. Juvenile B. glabrata M-RLc strain developed a strong acquired resistance to E. lindoense and a moderate one to E. paraensei and E. liei. Juvenile B. glabrata 641 strain developed a moderate acquired resistance to E. lindoense, a weak one to E. liei and no measurable resistance to E. paraensei.     

Spondias purpurea Exudate polysaccharide as affinity matrix for the isolation of a galactose-binding-lectin

Spondias purpurea L., popularly known as ciriguela, is native and widespread tree from Mexico through Northern Peru and Brazil, particularly in semi-arid zones. This tree exudes a water soluble polysaccharide, constituted of a (1→3) linked galactan backbone substituted at C6 with d-galactose, d-xylose, l-arabinose, l-rhamnose and glucuronic acid units. Brazilian polysaccharide differs from Venezuelan on the amount of acid and arabinose as well as the presence of fucose and glucose as minor sugar. The d-galactose substitution (1→6) confers to the polysaccharide the peculiar capacity of binding -d-galactose specific lectins after cross-linking with epichlorohydrin. The gel obtained was able to specifically retain d-galactose-binding-lectins, among with those from Artocarpus incisa, Artocarpus integrifolia, Erythrina velutina and Ricinus communis. On the other hand, no glucose-binding-lectins were retained.     

Ester production in E. coli and C. acetobutylicum

Genetic engineering has improved the product yield of a variety of compounds by overexpressing, inactivating, or introducing new genes in microbial systems. The production of flavor-enhancing ester compounds is an emerging area of heterologous gene expression for desired product yield in Escherichia coli. Isoamyl acetate, butyl acetate, ethyl acetate, and butyl butyrate are reported here to be produced by expressing Saccharomyces cerevisiae genes ATF1 or ATF2 and the strawberry gene SAAT in E. coli when the appropriate substrates are provided. Increasing the concentration of alcohol added to the reaction generally resulted in increased ester production. ATF1 expression was found to produce more isoamyl acetate and butyl acetate than ATF2 expression or SAAT expression in the strains and culture conditions examined. Additionally, SAAT expression resulted in greater isoamyl acetate and butyl acetate production than ATF2 expression. Butyl butyrate is produced by cell-free extracts of E. coli harboring SAAT but not ATF1 or ATF2.     

Ferredoxin reductase enhances heterologously expressed cytochrome CYP105D1 in Escherichia coli and Streptomyces lividans

The ability of two different ferredoxin reductases from Streptomyces coelicolor, to enhance the amount of active recombinant Streptomyces griseus soyC (CYP105D1) was investigated in both Escherichia coli and Streptomyces lividans. In E. coli a two-plasmid system and a single operon construct were used for expression of the CYP105D1 and the ferredoxin reductase(s) under the control of T7 promoters. Expression levels of CYP105D1 were found to range between 85 and 280 nmol l⁻¹ cell culture after prolonged growth. In S. lividans the CYP105D1 and its ferredoxin were cloned downstream of the Pact1 promoter in the E. coli/Streptomyces shuttle vector pBW160. The recombinant E. coli and S. lividans cells converted 7-ethoxycoumarin into 7-hydroxycoumarin efficiently. Expression of a ferredoxin reductase as an operon with CYP105D1 and its ferredoxin enhances the o-dealkylation of 7-ethoxycoumarin. Ferredoxin NADPH reductase was found to enhance the level of the active form of CYP105D1 monooxygenase when no substrate was present.     

Putaminoxin, a phytotoxic nonenolide from Phoma putaminum

Phoma putaminum, the causal agent of leaf necrosis of Erigeron annuus, a common weed of field and pasture, produced toxic metabolites when grown in liquid culture. The main phytotoxin, named putaminoxin, was isolated and characterized using spectroscopic and chemical methods as (5S)5-hydroxy-9-propyl-6-nonen-9-olide, a new 10-macrolide. When assayed on leaves of host and non-host plants, putaminoxin showed a wide range of toxicity, with leaves of E. annuus being most sensitive.     

Optimization of bio-indigo production by recombinant E. coli harboring fmo gene

A bacterial flavin-containing monooxygenase (FMO) gene was cloned from Methylophaga aminisulfidivorans MP^T, and a plasmid pBlue 2.0 was constructed to express the bacterial fmo gene in E. coli. To increase the production of bio-indigo, upstream sequence size of fmo gene was optimized and response surface methodology was used. The pBlue 1.7 plasmid (1686 bp) was prepared by the deletion of upstream sequence of pBlue 2.0. The recombinant E. coli harboring the pBlue 1.7 plasmid produced 662 mg l⁻¹ of bio-indigo in tryptophan medium after 24 h of cultivation in flask. The production of bio-indigo was optimized using a response surface methodology with a 2ⁿ central composite design. The optimal combination of media constituents for the maximum production of bio-indigo was determined as tryptophan 2.4 g l⁻¹, yeast extract 4.5 g l⁻¹ and sodium chloride 11.4 g l⁻¹. In addition, the optimum culture temperature and pH were 30 °C and pH 7.0, respectively. Under the optimized conditions mentioned above, the recombinant E. coli harboring pBlue 1.7 plasmid produced 920 mg of bio-indigo per liter in optimum tryptophan medium after 24 h of cultivation in fermentor. The combination of truncated insert sizes and culture optimization resulted in a 575% increase in the production of bio-indigo.     

Anti-microtubule activity of the traditional Chinese medicine herb Northern Ban Lan (Isatis tinctoria) leads to glucobrassicin

Traditional Chinese medicine (TCM) belongs to the most elaborate and extensive systems of plant-based healing. The herb Northern Ban Lan (Isatis tinctoria) is famous for its antiviral and anti-inflammatory activity. Although numerous components isolated from I. tinctoria have been characterized so far, their modes of action have remained unclear. Here, we show that extracts from I. tinctoria exert anti-microtubular activity. Using time-lapse microscopy in living tobacco BY-2 (Nicotiana tabacum L. cv Bright Yellow 2) cells expressing green fluorescent protein-tubulin, we use activity-guided fractionation to screen out the biologically active compounds of I. tinctoria. Among 54 fractions obtained from either leaves or roots of I. tinctoria by methanol (MeOH/H₂O 8:2), or ethyl acetate extraction, one specific methanolic root fraction was selected, because it efficiently and rapidly eliminated microtubules. By combination of further purification with ultra-high-performance liquid chromatography and high-resolution tandem mass spectrometry most of the bioactivity could be assigned to the glucosinolate compound glucobrassicin. Glucobrassicin can also affect microtubules and induce apoptosis in HeLa cells. In the light of these findings, the antiviral activity of Northern Ban Lan is discussed in the context of microtubules being hijacked by many viral pathogens for cell-to-cell spread.     

Enhancement of the uvrA gene dosage reduces pyrimidine dimer excision in UV-irradiated Escherichia coli

E. coli possesses an efficient repair mechanism able to remove pyrimidine dimers from UV-irradiated DNA, which is catalyzed by UvrABC endonuclease. In E. coli B/r Hcr⁺ cells transformed with a multicopy plasmid harboring a gene coding for UvrA, the excision capacity was greatly reduced. The course of thymine dimer excision was investigated using the enzymatic as well as the radiochromatographic method and the results are discussed in term of nonspecific interaction between the excess of UvrA protein and undamaged DNA duplex.     

Potent induction of the adaptive response by a weak mutagen, methyl iodide, in Escherichia coli

Methyl iodide (MeI), a weakly mutagenic and highly chemoselective chemicals, was tested for its abilities to induced the adaptive and SOS responses in E. coli CSH26/pMCP1000 (alkA′-lacZ′) and CSH26/psK1002 (umuC′-lacZ′). MeI induced the adaptive response effectively but gave a very weak SOS response. Its potent ability in inducing the adaptive response was also demonstrated by adaptation to both the mutagenic and killing effects of N-methyl-N-nitrosourea (MNU) in E. coli WP2 cells. Simultaneous treatment with MeI in a non-growth medium slightly increased the mutagenicity of MNU, probably as a result of depletion of the repair enzyme, O⁶-methylguanine-DNA methyltransferase, which is constitutively present in the cells. As MeI itself proved to be only weakly mutagenic, a small part of the adaptive response which we have observed may involve indirect methylation of the repair enzyme by methyl transfer from MeI-induced O⁶-methylguanine residues in DNA. But the extent of the induced adaptive response seems to be much higher than would be expected from the observed weak mutagenicity of MeI. It is therefore suggested that the mechanism of induction of the adaptive response may involve direct methylation of the O⁶-methylguanine-DNA methyltransferase itself.     

Isolation of de-exined pollen and cytological studies of the pollen intines of Pinus bungeana Zucc. Ex Endl. and Picea wilsonii Mast

To study the cytological and biochemical characteristics of intine, pollen deprived of exine, or de-exined pollen, was isolated from the gymnosperms Pinus bungeana and Picea wilsonii. The factors influencing the isolation rate were examined. Cellulose, callose, pectin, and arabinogalactan proteins (AGPs) were localized in this material using fluorescent probes, and components of the isolated intine were further analyzed by Fourier transform infrared (FTIR) microspectroscopy. The isolation protocol was repeatable and reliable. Cellulose was found to be evenly distributed on the surface of the intine, as indicated by strong calcofluor White ST (CW) fluorescence, and aniline blue staining revealed that callose was present on the intine of P. bungeana but not on that of P. wilsonii. Immunolabeling revealed that acidic pectin epitopes recognized by the monoclonal antibody JIM5 were present on the pollen intine, as well as esterified pectin recognized by the monoclonal antibody JIM7, and AGPs recognized by the LM2 antibody. Two lectin binding sites, the concanavalin agglutinin (Con A) and soybean agglutinin (SBA) binding sites, were present on the intine surface, but no wheat germ agglutinin (WGA) binding sites were detectable. These results were confirmed by FTIR analysis.     

Homologization of storage proteins from Aquilegia vulgaris and Digitalis purpurea

The separation of the main seed proteins from Aquilegia vulgaris and Digitalis purpurea by means of Sephadex G-200 gel filtration and DEAE-Sephadex ion exchange absorption resulted in remarkably similar patterns. Using gel electrophoresis and serological techniques this phenomenon has been attributed to the predominance of two storage proteins present in each taxon: (a) one main protein in Aquilegia (= nigellin) and in Digitalis (= tubiflorin), each, being similar, but probably not homologous. (b) A secondary protein (= aquilegilin) was identified in both taxa. The distribution of only a few storage proteins in Magnoliophytina (angiosperms) with different serological reactivity (= different primary structure) has been presumed, based upon the data obtained.     

Population dynamics of the harmful diatom Eucampia zodiacus Ehrenberg causing bleachings of Porphyra thalli in aquaculture in Harima-Nada, the Seto Inland Sea, Japan

The diatom Eucampia zodiacus Ehrenberg is one of the harmful diatom species which indirectly cause bleachings of Nori (Porphyra thalli) in aquaculture through competitive utilizing of nutrients (especially nitrogen) and resultant nutrient depletion in water columns during the bloom events. The seasonal changes in environmental factors, cell density and cell size of E. zodiacus were investigated for 4 years (April 2002–December 2005) to understand the population ecology of this diatom in Harima-Nada, the eastern part of the Seto Inland Sea, Japan. Vegetative cells of E. zodiacus were usually detected year-round. Total cell densities of E. zodiacus annually peaked from mid-February to early April, and high cell densities were observed in the whole water columns during the bloom-period. Nutrient concentrations decreased with the increase of cell density of E. zodiacus, and low nutrients concentrations continued throughout the E. zodiacus bloom-period. The average cell size (length of apical axis) of E. zodiacus populations ranged from 10.8 μm to 81.2 μm, and the restoration of cell size occurred once in autumn every year just after reaching the minimum cell size. In addition, its great seasonal regularity was confirmed by the decrease and restoration of its cell size through 4-year study period. Temperature and nutrients were suitable in autumn for the growth of E. zodiacus, its blooms never occur in that season. These results strongly suggest that E. zodiacus did not have a resting stage, and it spends autumn for size restoration and starts to bloom thereafter in Harima-Nada in winter and spring, causing fishery damage to Nori aquaculture by resulting nutrient deprivation.     

Bt水稻秸秆还田对赤子爱胜蚓生长发育和生殖的影响

Bt蛋白能通过转Bt基因作物的秸秆还田进入土壤,进而可能会对土壤动物如蚯蚓的生长发育和生殖造成影响.为评估Bt水稻对赤子爱胜蚓的影响,本文模拟秸秆还田,在土壤中添加2.5%、5%、7.5%和10% Bt水稻(b2B138)及其同源水稻(安丰A)秸秆,分别在饲养赤子爱胜蚓7、15、30、45、60、75和90 d后观测蚯蚓的存活率、相对生长率和生殖情况,以及秸秆土壤混合物和蚯蚓体内的Cry1Ab蛋白含量.结果表明:较高还田量(7.5%和10%)Bt水稻秸秆处理对赤子爱胜蚓存活率有抑制作用;Bt水稻秸秆还田对赤子爱胜蚓的相对生长率没有不利影响;还田量为5%、7.5%和10%时,Bt水稻秸秆还田能促进蚯蚓的生殖.酶联免疫吸附测定法(ELISA)结果表明: 在Bt水稻土壤混合物中,蚯蚓体内均能检测到Cry1Ab蛋白,且前者随着时间延长而显著减少.因此,还田量为2.5%和5%时,Bt水稻秸秆还田释放的Cry1Ab蛋白对赤子爱胜蚓的生长发育和生殖没有不利影响.     

Cloning, sequencing, and expression of the meso-diaminopimelate dehydrogenase gene from Bacillus sphaericus

The gene encoding the meso-diaminopimelate dehydrogenase of Bacillus sphaericus was cloned into E. coli cells and its complete DNA sequence was determined. The meso-diaminopimelate dehydrogenase gene consisted of 978 nucleotides and encoded 326 amino acid residues corresponding to the subunit of the dimeric enzyme. The amino acid sequence deduced from the nucleotide sequence of the enzyme gene of B. sphaericus showed 50% identity with those of the enzymes from Corynebacterium glutamicum and Brevibacterium flavum. The enzyme gene from B. sphaericus was highly expressed in E. coli cells. We purified the enzyme to homogeneity from a transformant with 76% recovery. The N-terminal amino acid of both the enzyme from B. sphaericus and the transformant were serine, indicating that the N-terminal methionine is removed by post-translational modification in B. sphaericus and E. coli cells.     

Chemical composition of essential oils of seven species of Eugenia from Monteverde, Costa Rica

The leaf essential oils of seven species of Eugenia from Monteverde, Costa Rica (Eugenia austin-smithii, Eugenia cartagensis, Eugenia haberi, Eugenia monteverdensis, Eugenia zuchowskiae, Eugenia sp. A aff. haberi, and Eugenia sp. B aff. oerstediana) have been obtained by hydrodistillation and analyzed by GC–MS. The seven species were compared to determine the similarities and differences among their volatile chemical compositions. The major component in each of the seven species was as follows: E. austin-smithii and E. cartagensis was trans-2-hexenal, E. haberi and E. zuchowskiae was -pinene, E. monteverdensis was linalool, Eugenia sp. A was zingiberene, and Eugenia sp. B was 1,8-cineole. The following six components were present in all seven species: -copaene, β-caryophyllene, -humulene, δ-cadinene, trans-nerolidol, and torreyol. The complex array and differing abundances of these compounds among the Eugenia species studied suggest that they may provide useful characters in understanding the phylogenetic relationships among closely related species.     

Production of excreted human epidermal growth factor (hEGF) by an efficient recombinant Escherichia coli system

Recombinant Escherichia coli JM101 strains harbouring plasmids pWKW2 or lacUV5par8EGF, both encoding human epidermal growth factor (hEGF), were used in fermentations to optimize levels of excreted hEGF. Medium composition, inducer level, growth stage at induction and culture conditions, were optimized with respect to volumetric production of the recombinant protein. MMBL medium, with glucose at 5 g/l and tryptone as nitrogen source, was chosen. Isopropyl-β- -thiogalactopyranoside(IPTG) concentrations of 0.1 mM for E. coli JM101[pWKW2] and 0.2 mM for E. coli K-12 JM101[lacUV5par8EGF], were found to give the best hEGF production levels. The volumetric yields of hEGF were maximal when the cultures were induced in the mid-logarithmic phase. Growth temperature had a significant effect on hEGF yield. A simple continuous fed-batch process for cultivation of E. coli JM101[pWKW2] was developed. The maximum concentration of excreted hEGF attained in continuous fed-batch cultivation was 325 mg/l, as compared to 175 mg/l, in batch cultivation. The hEGF produced from the continuous fed-batch cultivation was substantiated by SDS-PAGE and immunoblotting.     

氮磷添加对巨桉幼苗生物量分配和C:N:P化学计量特征的影响

巨桉(Eucalyptus grandis)是一种优良的速生用材树种, 了解氮(N)和磷(P)对巨桉生长、养分限制、化学计量特征的影响对于科学合理施肥具有重要意义。该实验以巨桉无性系组培苗为研究对象, 通过在酸性紫色土中设置不同施N或施P梯度, 研究巨桉幼苗各器官(根、茎、叶)生物量及碳(C)、N、P的分配和化学计量特征以及巨桉生长的养分限制状况。结果表明: 施N处理对巨桉根茎叶及总生物量的影响极显著, 增加了地上部分的生物量比例而显著降低了根系的生物量比例; 施P对巨桉幼苗总生物量影响不显著, 但显著提高了根的生物量分配比例, 对茎和叶的生物量分配没有显著影响。施N或施P显著改变了巨桉幼苗的N、P含量和化学计量比, 同时也显著影响了土壤与植物N:P的关系。施N可以促使酸性紫色土条件下巨桉对N的吸收而抑制对P的吸收, 施P则促进巨桉幼苗对P的吸收。施N对巨桉幼苗根茎叶的C、N、P分配特征有极显著影响, 而施P对巨桉幼苗根茎叶的C、N、P分配没有显著影响。施N极显著降低了巨桉幼苗N的利用率, 显著提高了P的利用率, 而施P处理极显著降低了巨桉幼苗P的利用率。从巨桉生物量沿施肥梯度和N:P的变化规律可以判断, 当叶片N:P < 15时, 巨桉的生长主要受到N的限制作用。施N可以显著地提高根茎叶的N:P比值, 缓解巨桉缺N的现象, 施P则进一步加剧了N元素的缺乏。     

Studies on resistance in snails: Specific resistance induced by irradiated miracidia of Echinostoma lindoense in Biomphalaria glabrata snails

In juvenile Biomphalaria glabrata snails exposed to irradiated Echinostoma lindoense miracidia, the sporocysts migrated to the heart at the same speed as did nonirradiated sporocysts in control snails. However, in each snail so exposed to irradiated miracidia, amebocyte clumps in the snail's heart destroyed the sporocysts within 2–9 days post-exposure. This process induced a strong, highly specific resistance to homologous reinfection in these previously susceptible snails. The snails remained susceptible to Schistosoma mansoni and Paryphostomum segregatum (Echinostomatidae), but were partially resistant to Echinostoma paraensei and E. liei, two echinostome species closely related to E. lindoense.