QTLs for nutritional contents of rice seedlings (Oryza sativa L.) in solution cultures and its implication to tolerance to iron-toxicity

Iron toxicity is one of the major constraints for lowland rice production in highly weathered soils, which are widely distributed in tropics and subtropics and often lack macronutrients such as potassium (K) and phosphorus (P). To analyze the genetic factors for excess iron accumulation under K or P deficiency, a set of seedlings in F₃ and F₈ generations from an Oryza sativa cross between a japonica cultivar ‚Gimbozu’ and an indica cultivar ‚Kasalath’ were raised and exposed to nutritional stresses in a short period under nutritional solutions. In the F₈ lines, contents of K, P, Fe, and Mg in dried shoots were measured. Quantitative trait loci (QTL) for the iron accumulation and related mineral contents in each plant were analyzed with composite interval mapping. QTLs for the Fe, P and Mg content in shoots were compared in the maps of F₃ and F₈. The QTLs for the Fe content in shoots varied in three types of nutritional conditions, but consistently indicated two overlapping regions on chromosome 3 and 4. The obtained QTLs were crosschecked with those reported before. Some of these QTLs were indicative of iron excluding the power of the root, which was expressed under reduced P content in solution.     

Isolation, characterization and molecular cloning of new temporins from the skin of the North African ranid Pelophylax saharica

Temporins are small antimicrobial peptides isolated from North American and Eurasian ranid frogs that are particularly active against Gram-positive bacteria. To date, no temporins have been characterized from North African frog species. We isolated three novel members of the temporin family, named temporin-1Sa (FLSGIVGMLGKLF(amide)), -1Sb (FLPIVTNLLSGLL(amide)), and -1Sc (FLSHIAGFLSNLF(amide)), from the skin of the Sahara frog Pelophylax (Rana) saharica originating from Tunisia. These temporins were identified by a combined mass spectrometry/molecular cloning approach. Temporin-1Sa was found to be highly active against Gram-positive and Gram-negative bacteria, yeasts and fungi (MIC=2-30muM). To our knowledge, this is the first 13-residue member of the temporin family with a net charge of +2 that shows such broad-spectrum activity with particularly high potency on the clinically relevant Gram-negative strains, Escherichia coli (MIC=10muM) and Pseudomonas aeruginosa (MIC=31muM). Moreover, temporin-1Sa displays significant antiparasitic activity (IC(50) approximately 20muM) against the promastigote and amastigote stages of Leishmania infantum.     

In-vitro flower formation in leaf explants of tomato: effect of NaCl

Leaf explants of 24 cultivars and 2 F1 hybrids of the common tomato (Lycopersicon esculentum Mill.) and ofL. pimpinellifolium Brezh. were cultured on Murashige-Skoog medium containing different concentrations of NaCl. The cultures of 11 genotypes formed flower buds when cultured on medium containing 0.5% NaCl. Flower formation occurred either by direct differentiation from the leaf cultures or by transition of the apices of regenerated shoots from the vegetative state to floral buds. No flower formation occurred on medium without NaCl or media with 1.0% NaCl or more. There existed great differences in the capacity of in-vitro flower formation in the tomato leaf explants among the genotypes tested. The genotypes whose explants did form flowers were all of determinate growth habit.     

Photosynthetic performance of transplanted ecotypes ofEcklonia cava(Laminariales, Phaeophyta)

Young sporophytes of short-stipe ecotype ofEcklonia cavafrom a warmer locality (Tei, Kochi Pref., southern Japan) and those of long-stipe ecotype from a cooler locality (Nabeta, Shizuoka Pref., central Japan) were transplanted in 1995 to artificial reefs immersed at the habitat of long-stipe ecotype in Nabeta Bay, Shizuoka Pref., central Japan. The characteristics of photosynthesis and respiration of bladelets of the transplanted sporophytes of the two ecotypes were compared in winter and summer 1997; the results were assessed per unit area, per unit chlorophyllacontent and per unit dry weight. In photosynthesis-light curves at 10–29 °C, light saturation occurred at 200–400 mol photon m^–2s^–1in sporophytes from both Tei and Nabeta. The maximum photosynthetic rate (P _max) at 10–29 °C and the light-saturation index (I _k) at 25–29 °C in sporophytes from both localities were generally higher in winter than in summer.P _maxat 25–29 °C (per unit area and chlorophylla) were higher in sporophytes from Tei than those from Nabeta in both seasons. The optimum temperature for photosynthesis was 25 °C in winter and 27 °C in summer at high light intensities of 100–400 mol photon m^–2s^–1. However, at lower light intensities of 12.5–50 mol photon m^–2s^–1, it was 20 °C in winter and 25–27 °C in summer for sporophytes from both locations. Dark respiration increased with temperature rise in the range of 10–29 °C in sporophytes from both locations in summer and winter. The sporophytes transplanted from Tei (warmer area) showed higher photosynthetic activities than those from Nabeta (cooler area) at warmer temperatures even under the same environmental conditions. This indicates that these physiological ecotypes have arisen from genetic differentiation.     

An impedimetric method for rapid screening of cosmetic preservatives

An efficient impedance method was developed for rapid evaluation of cosmetic preservatives. The method used decimal reduction time or D-value to assess preservative efficacies. The D-value, which was calculated from the plot of Log CFU ml^–1 versus time by linear regression analysis, could be obtained within 48 h. Thus, the time required for the challenge test was reduced from 4–8 weeks with the standard procedures (eg US Pharmacopeia), to 2 days with the current method. A calibration curve (r=-0.95) was established by plotting the Log CFU ml^–1 versus capacitance detection time (DT) of 108 samples. With the calibration, CFU can be estimated directly from the impedance test without plating. Two commercial biocides and several other chemicals were evaluated in a shampoo by the impedance procedure againstPseudomonas aeruginosa. The D-values obtained from the impedance test were not significantly different from those produced by the conventional plate count method. The technique was found to be particularly useful when screening a large number of compounds to find novel preservatives and synergistic preservative combinations.     

Antibody-supported screening of alcohol dehydrogenases

Polyclonal antibodies raised against purified (R)-specific alcohol dehydrogenase of Lactobacillus kefir were used in Western blot analyses to search for structurally or immunologically related proteins. No immunochemical reactions were found with commercially available alcohol dehydrogenases (from yeast, horse liver and Thermoanaerobium brockii), but screening among the genus Lactobacillus revealed that each strain of a subgroup of Betabacterium gave positive results whereas strains of the other subgroups of Lactobacillus were found to be inactive. However, enzymatic assays with these antibody-positive strains showed, that besides L. kefir itself, only the strains of L. brevis possess alcohol dehydrogenase activity with acetophenone and NADPH as substrates.     

纳米抗体筛选技术研究进展

纳米抗体(nanobody, Nb)是在骆驼科血清中发现的一种新型抗体,具有体积小、特异性强、稳定性高、易于表达和能识别隐藏的抗原表位等优势,在各个领域具有广泛的应用价值。本文介绍了纳米抗体筛选与优化过程,包括纳米抗体文库构建、体外展示和亲和力成熟3个重要技术阶段的分类与特点。其中,简要描述了天然、免疫及半合成/合成文库的制备方法与重要参数,并系统介绍了应用噬菌体、酵母、细菌、核糖体/mRNA和真核细胞等表面展示系统,以及酵母双杂交、高通量测序和质谱鉴定方法,共8种不同体外展示技术进行快速筛选的方法及其优缺点,汇总用于提升纳米抗体功能可靠性的体外及计算机辅助亲和力成熟技术平台,为综合运用各种技术手段快速获得稳定、可靠、特异的纳米抗体类药物或诊断制剂提供了参考。     

Gas chromatographic-mass spectrometric determination of 20(S)-protopanaxadiol and 20(S)-protopanaxatriol for study on human urinary excretion of ginsenosides after ingestion of ginseng preparations

An improved gas chromatographic-mass spectrometric method (GC-MS) with a fast solid-phase extraction on a newly introduced C₁₈ microcolumn, was applied to study the urinary excretion of 20(S)-protopanaxadiol and 20(S)-protopanaxatriol glycosides in man after oral administration of ginseng preparations. Using panaxatriol as internal standard, 20(S)-protopanaxadiol and 20(S)-protopanaxatriol (the aglucones of ginesenosides) could be determined at a detection level of a few ng per ml urine by GC-MS with selected-ion monitoring after their release from glycosides which occur in urine. The extraction recovery of ginsenosides from urine was more than 80% and the intra-assay coefficient of variation was less than 5.0%. The results after intake of single doses of ginseng preparations demonstrated a linear relation between the amounts of ginsenosides consumed and the 20(S)-protopanaxatriol glycosides excreted in urine. About 1.2% of the dose was recovered in five days.     

Exudation of low molecular weight organic acids by germinating seeds of two edaphic ecotypes of Silene nutans L.

Two parapatric ecotypes of Silene nutans, exhibiting distinct allozyme patterns, morphology and autecology were investigated for differences in exudation of low molecular weight organic acids from germinating seeds, and for differences in seed phosphorus content. The calcicolous ecotype is restricted to calcareous soils, and the silicicolous one predominantly occurs on acid soils, and sometimes, although less frequently, on neutral to alkaline soils. No clear difference was found between ecotypes. However, within the silicicolous ecotype seed samples showed marked differences in exudation pattern and seed phosphorus content depending on origin along the soil acidity gradient. Seeds of low-pH origin exuded more dicarboxylic acids (malic + succinic acid, oxalic acid) and had a lower phosphorus content than seeds of high pH origin. The exudation of dicarboxylic acids from seeds of low pH origin is probably an adaptation to adverse conditions (aluminium toxicity) on acid soils. The pattern is similar to that found among different cultivars of wheat. It is contrasted to the pattern found on comparison of a suite of calcifugous and calcicolous species, where exudation of di- and tricarboxylic acids is associated with solubilisation of recalcitrantly bound phosphorus and iron in calcareous soils.     

In vitro tests for screening of immuno-modulating mycobacterial strains in leprosy

There is an urgent need for the development of anin vitro assay for the initial screening of a large number of organisms from which potential candidates as vaccines can be identified. Our previous studies have demonstrated a crucial defect in the lepromatous macrophage. In this study by monitoring this defective macrophage response we have screened various mycobacteria for their ability to reverse the alterations induced byMycobacterium leprae. Among the limited Mycobacteria testedMycobacterium vaccae appears to be the most promising as an immunomodulator. Our results also indicate the need for caution in using the mouse model for this purpose     

A fast screening method for surface layers on Gram-positive bacteria

A two-step screening method is described to identify regularly arranged surface layers (S layers) on Gram-positive bacterial strains. A non-destructive release of S-layer sheets is achieved by enzymatic hydrolysis of the underlying peptidoglycan using lysozyme. The existence of regular S layers is then directly confirmed by scanning force microscopy or transmission electron microscopy. This method requires a minimal amount of bacterial cells and may be used as a `quick test' for demonstrating the presence of S layers.     

A peptide antibody for rapid screening of Streptomyces species producing phospholipase D

By examining the conserved regions in the protein sequences of eight different Streptomyces phospholipase Ds (PLD) reported so far and the X-ray crystallographic structure of a Streptomyces PLD, we designed a peptide sequence, DPANRGAVGSGGYSQIKSL, for the screening of microorganisms producing PLD. In the enzyme-linked immunosorbent assay using a mouse antibody raised against the designed peptide, we recovered seven producing strains out of 128 soil isolates.     

Nisin高产菌株的高通量筛选

【目的】乳酸链球菌素(nisin)是一种天然生物活性抗菌肽,对包括食品腐败菌和致病菌在内的许多革兰氏阳性菌具有强烈的抑制作用,而用作食品的防腐剂。本研究通过建立高通量筛选方法,实现高效快速省力的高产菌株筛选,为工业上筛选高产菌株提供研究方案。【方法】通过对Lactococcus lactis ATCC11454菌株进行紫外诱变,获得2511株突变株。利用Biomek FXP自动工作站建立96微孔板的高通量筛选方法,突变株经高通量挑选、菌种培养及菌液稀释后,加入到生长至对数中期的藤黄微球菌中,采用改进后的比浊法快速检测nisin生物活性。用此方法对突变株进行初筛、复筛后可得到nisin高产菌株,并通过摇瓶发酵评估高通量筛选方法。【结果】确定比浊法检测的条件为:nisin活性稀释在10–25 IU/m L范围内,与藤黄微球菌反应2 h后检测藤黄微球菌的菌体量(OD600)。2511株突变株经过2轮高通量筛选,最终获得约50株产量提升的菌株,对其中8株进行摇瓶精确测量,显示产量均有提高,并且其中一株产量提升了30%,成功建立了高通量筛选nisin高产菌株的方法。【结论】利用比浊检测法,在其基础上成功建立高通量筛选高产nisin菌的方法,经过初筛复筛,整个周期由1人耗时5 d即可完成2511株突变株的筛选工作。相较于传统的选育方法,高通量筛选具有快速、稳定、高效的特点,提高了筛选效率,缩短了选育周期,是工业上筛选高产nisin菌的有效手段。     

Effects of nutritional stress on brain tyramine concentration and dopamine turnover

This is an investigation of the effect of nutritional stress at various ages on the levels of thep- andm-isomers of tyramine in the caudate nucleus of the rat. For comparison, the effects of nutritional stress on the concentration and turnover of dopamine were also studied. Nutritional stress induced in pre-weaning (3 weeks of age) or post-weaning (up to 9 weeks of age) rats resulted in a decrease in the concentration ofp-tyramine and an increase in the concentration ofm-tyramine in the caudate nucleus. Dopamine concentration or turnover in the caudate nucleus was not affected by pre-weaning undernutrition; in the olfactory tubercles, however, a significant decrease was observed in dopamine turnover, calculated from the decrease in homovanillic acid levels after monoamine oxidase inhibition. The results suggest the changes observed are dependent on the availability of the amino acid precursorsp- andm-tyrosine and their competition towards aromatic-l-amino acid decarboxylase.     

贵州省野生火棘果营养成分与气候因子相关性分析

野生火棘在贵州省的资源分布广泛,由于省内各地区气候因子的不同,导致野生火棘果实的营养性成分存在差异。为探究贵州省野生火棘果的营养性成分差异,明确影响营养成分积累的气候因子,筛选出适宜生长区域,采用硫酸-苯酚法、全自动凯氏定氮仪、索氏提取法和福林-酚比色法对火棘果粗多糖、总蛋白、总脂肪和总多酚含量进行测定,并进行差异性分析、主成分分析和聚类分析;同时结合气候因子,进行灰色关联度的相关性分析。结果显示,不同产地之间4种营养成分含量存在明显差异,可将10个不同产地火棘分成3大类;灰色关联度分析表明,相对湿度是影响贵州野生火棘果营养成分含量的主要影响因子;降低相对湿度利于火棘果中营养性成分的积累。     

罗马直立生菜生长发育及营养指标对臭氧浓度升高和持续熏蒸的响应

环境臭氧(O₃)已成为影响植物生长发育的重要生态因子。为探究地面O₃污染对蔬菜形态学特征及营养指标的影响,选罗马直立生菜(Lactuca sativa var. roman)为实验材料,采用开顶式气室开展熏蒸实验。实验设置4个O₃熏蒸浓度(NF:未过滤的环境空气;NF40:环境空气+40 nmol/mol;NF80:环境空气+80 nmol/mol;NF120:环境空气+120 nmol/mol),每个处理设置3个重复组,分析评价O₃污染对植物造成的可见伤害、生产量、叶片解剖学特征以及食用部位营养指标的影响。研究表明:(1)O₃熏蒸对生菜叶片产生不可逆的可见伤害,叶片出现浅黄色斑点和棕色斑点,且随着熏蒸时间延长,叶片出现黄化,大面积的坏死斑块,衰老加速。(2)高浓度O₃胁迫显著降低了生长阶段的株高(P<0.05)。与NF组相比,NF40、NF80、NF120组分别使生物量下降5.90%、14.99%、39.21%。(3)随着O₃熏蒸浓度升高,气孔密度增加,气孔开度减小。叶片厚度、海绵组织厚度、栅栏组织厚度与O₃暴露剂量AOT40呈显著负相关关系(P<0.05)。(4)高浓度O₃暴露使蔬菜中Ca、Na、Fe、Zn、Mg等元素含量显著降低,脂肪和蛋白质含量增加,生菜的营养指标发生改变。研究表明,罗马直立生菜对环境O₃污染敏感,其生长发育及营养指标在O₃胁迫条件下发生明显变化。目前,关于O₃污染对蔬菜形态学特征影响的研究较少,研究系统探讨蔬菜的叶片厚度、栅栏组织、海绵组织、气孔密度及开度等形态学指标在臭氧污染条件下的变化。蔬菜的品质是关系到"三农"问题的重要方面,研究探讨了臭氧污染对蔬菜的产量及营养指标的影响,可为O₃污染条件下蔬菜的生产提供科学参考。     

Up-regulation of glutathione metabolism and changes in redox status involved in adaptation of reed (Phragmites communis) ecotypes to drought-prone and saline habitats

The glutathione (GSH) metabolic characteristics and redox balance in three ecotypes of reed (Phragmites communis), swamp reed (SR), dune reed (DR), and heavy salt meadow reed (HSMR), from different habitats in desert regions of northwest China were investigated. The DR possessed the highest rate of GSH biosynthesis and metabolism with the lowest levels of total and reduced GSH and its biosynthetic precursors, gamma-glutamylcysteine (gamma-EC) and cysteine (Cys), of the three reed ecotypes. This suggests that a higher rate of GSH biosynthesis and metabolism, but not GSH accumulation, might be involved in the adaptation of this terrestrial reed ecotype to its dry habitat. The HSMR shared this profile although it exhibited the highest reduced thiol levels of the three ecotypes. Two key enzymes in the Calvin-cycle possessing exposed sulfhydryl groups, NADP(+)-dependent glyceraldehydes-3-phosphate dehydrogenase (G3PD) and fructose-1,6-bisphosphatase (FBPase), and other two key enzymes in the pentose-phosphate pathway (PPP), glucose-6-phosphate dehydrogenase (G6PDH) and 6-phosphogluconate dehydrogenase (6-PGD), had very similar activities in the three reed ecotypes. Compared to the SR, the DR and HSMR had higher ratios of NADPH/NADP+ and NADH/NAD+, indicating that a more reduced redox status in the plant cells might be involved in the survival and adaptation of the two terrestrial reed ecotypes to long-term drought and salinity, respectively. These results suggest that changes of GSH metabolism and redox balance were important components of the adaptation of reed, a hydrophilic plant, to more extreme dune and saline habitats. The coordinated up-regulations of the rate of GSH biosynthesis and metabolism and reduction state of redox status of plant cells, conferred on the plant high resistance or tolerance to long-term drought and salinity.     

Rapid screening of <Emphasis Type="Italic">Aspergillus terreus</Emphasis> mutants for overproduction of lovastatin

Summary A novel rapid screening method is demonstrated for isolating lovastatin-overproducing strains of Aspergillus terreus. The screening methodology, based on the activity of lovastatin against the yeast Candida albicans, is nearly three times as fast as the selection methods used earlier. The new 6-h assay shows a linear correlation between the quantity of lovastatin generated by A. terreus isolates and the inhibition zones obtained on plates of C. albicans. The new technique is less expensive and requires less labour.     

In-vitro translation of different mRNA-containing fractions of Chlamydomonas chloroplasts

Starting from isolated chloroplasts of the Chlamydomonas reinhardii cw 15 mutant, several mRNA-containing chloroplast subfractions, i.e. thylakoid-bound polysomes, detached polysomes or isolated RNA, were prepared and incubated in homologous and heterologous translation systems. In the reticulocyte lysate these fractions gave rise to strikingly different product patterns. A most prominent difference concerned the in-vivo rapidly labelled 32,000-dalton thylakoid polypeptide. Neither this membrane protein nor its 34,000-dalton precursor was formed when membrane-containing or free polysomes were translated, while the 34,000-dalton precursor was a main product of the RNA isolated from the same membranes. The influence of thylakoid membranes during translation was also observed in homologous translation systems with lysed chloroplasts supplemented with ATP. Membrane and soluble fractions, when translated separately, yielded product patterns which differed from each other, although the RNAs extracted from the respective fractions gave the same product patterns when translated in reticulocyte lysate; the latter included a soluble protein, the large subunit of ribulose-1,5-bisphosphate carboxylase, and a membrane protein, the 34,000-dalton precursor of the 32,000-dalton membrane protein, as major labelled translation products. These results point to a regulatory role of thylakoid membranes in the expression of chloroplast mRNA and argue against compartmentation of the chloroplast mRNAs between the soluble and membrane fractions.Abbreviation SDS sodium dodecyl sulfate     

Berberine bridge enzyme catalyzes the six electron oxidation of (S)-reticuline to dehydroscoulerine

Berberine bridge enzyme catalyzes the stereospecific oxidation and carbon–carbon bond formation of (S)-reticuline to (S)-scoulerine. In addition to this type of reactivity the enzyme can further oxidize (S)-scoulerine to the deeply red protoberberine alkaloid dehydroscoulerine albeit with a much lower rate of conversion. In the course of the four electron oxidation, no dihydroprotoberberine species intermediate was detectable suggesting that the second oxidation step leading to aromatization proceeds at a much faster rate. Performing the reaction in the presence of oxygen and under anoxic conditions did not affect the kinetics of the overall reaction suggesting no strict requirement for oxygen in the oxidation of the unstable dihydroprotoberberine intermediate. In addition to the kinetic characterization of this reaction we also present a structure of the enzyme in complex with the fully oxidized product. Combined with information available for the binding modes of (S)-reticuline and (S)-scoulerine a possible mechanism for the additional oxidation is presented. This is compared to previous reports of enzymes ((S)-tetrahydroprotoberberine oxidase and canadine oxidase) showing a similar type of reactivity in different plant species.