测定λ原噬菌体诱导频率的新方法

本文报道两种测定λ原噬菌体紫外诱导频率的新方法 - 菌落计数法和平板诱导法.将溶源菌液经紫外诱导暗培养稀释后直接涂布在平板上培养,根据平板上菌落形成单位数计算λ噬菌体紫外诱导频率.另一种是将溶源菌与指示菌混合制备的平板用紫外线诱导,根据平板上噬菌体形成单位确定λ噬菌体紫外诱导频率.这两种方法不仅能准确测定噬菌体紫外诱导频率,而且操作简便,节省时间和用具,重复性好.本研究还将冬虫夏草浸出汁与溶源菌混合后进行紫外辐射,通过几种方法进行比较,结果证明建立的新方法确实可行,易操作;同时也表明冬虫夏草具有较强的抗紫外辐射作用.     

Identification of a Novel 1033‐Nucleotide Deletion Polymorphism in the Prophage Region of ‘Candidatus Liberibacter asiaticus’: Potential Applications for Bacterial Epidemiology

The prophage/phage region in the genome of ‘Candidatus Liberibacter asiaticus’, an alpha‐proteobacterium associated with citrus Huanglongbing, included many valuable loci for genetic diversity studies. Previously, a mosaic genomic region (CLIBASIA_05640 to CLIBASIA_05650) was characterized, and this revealed inter‐ and intracontinental variations of ‘Ca. L. asiaticus’. In this study, 267 ‘Ca. L. asiaticus’ isolates collected from eight provinces in China were analysed with a primer set flanking the same mosaic region plus downstream sequence. While most amplicon sizes ranged from 1400 to 2000 bp, an amplicon of 550 bp (S550) was found in 14 samples collected from south‐western China. Sequence analyses showed that S550 was the result of a 1033 bp deletion which included the previously known mosaic region. The genetic nature of the deletion event remains unknown. The regional restriction of S550 suggests that the ‘Ca. L. asiaticus’ population from south‐western China is different from those in eastern China. The small and easy‐to‐detect S550 amplicon could serve as a molecular marker for ‘Ca. L. asiaticus’ epidemiology.     

Screening of Marine Bacteria for the Production of Microbial Repellents Using a Spectrophotometric Chemotaxis Assay

A method for screening marine bacteria for the production of microbial repellents has been developed. The spectrophotometer provided quantitative information on bacterial chemotaxis in response to extracts from other strains of marine bacteria. Aqueous extracts were incorporated into an agar plug at the base of a cuvette, which was overlaid with a suspension of a motile strain. Negative chemotaxis of the motile strain in response to diffusion of repellent compounds from the agar could be measured by a fall in the optical density, allowing the direct screening of supernatants for repellent activity. Three strains producing metabolites with a repellent effect on a motile marine bacterium were identified. Antibiotic activity and the repellent effect of the supernatants were compared, with no significant correlation being found. The screening method will therefore allow the identification of bioactive metabolites that would be overlooked using traditional antibiotic screening strategies. Received March 4, 1998; accepted November 11, 1998.     

基于PCR的质粒快速定点诱变方法建立与评价

基因突变对生命的进化具有重要意义,针对质粒的DNA定点诱变技术也是基因工程、蛋白质工程研究中的重要手段之一。为了提高质粒定点诱变的效率,本研究利用引物部分重叠的设计方案,使用Tm值相对固定的引物设计模式,将同一引物分为重叠区（Tm=50±2℃）和非重叠区（Tm=60±2℃）,并在严格控制模板用量（2 pg/kb）的基础上,通过20个PCR循环对目标质粒进行定点诱变扩增,随后取0.5 μL产物直接用于转化。在FastPfu Fly酶系中,利用此法构建了6个含碱基替换、缺失和插入的质粒,均获得成功,突变效率可达96%以上,阳性克隆获得数达70个以上。此外,利用4种不同PCR酶系对该法的适用性进行了评价,结果表明突变效率均可达93%以上,阳性克隆获得数均在10个以上。通过适当增加PCR模板用量（10 pg/kb）并使用纯化后的PCR产物进行转化,该法可适用于转化效率大于106（cfu/μg）的任意感受态细胞,对应的突变效率可大于91%,阳性克隆获得数大于20。根据本法的作用原理,该方案适合质粒中10～20 bp（因重叠区GC含量及碱基序列的不同而改变）以内的任意碱基替换和插入,以及任意长度的DNA片段缺失。且具有通用性强、耗时少、诱变成功率高、成本低、对感受态及转化效率无特殊要求等优点,适合各实验室的日常研究使用。     

Origin of a Core Bacterial Gene via Co-option and Detoxification of a Phage Lysin

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Development and Preliminary Evaluation of a Multivariate Index Assay for Ovarian Cancer

Background

Most women with a clinical presentation consistent with ovarian cancer have benign conditions. Therefore methods to distinguish women with ovarian cancer from those with benign conditions would be beneficial. We describe the development and preliminary evaluation of a serum-based multivariate assay for ovarian cancer. This hypothesis-driven study examined whether an informative pattern could be detected in stage I disease that persists through later stages.

Methodology/Principal Findings

Sera, collected under uniform protocols from multiple institutions, representing 176 cases and 187 controls from women presenting for surgery were examined using high-throughput, multiplexed immunoassays. All stages and common subtypes of epithelial ovarian cancer, and the most common benign ovarian conditions were represented. A panel of 104 antigens, 44 autoimmune and 56 infectious disease markers were assayed and informative combinations identified. Using a training set of 91 stage I data sets, representing 61 individual samples, and an equivalent number of controls, an 11-analyte profile, composed of CA-125, CA 19-9, EGF-R, C-reactive protein, myoglobin, apolipoprotein A1, apolipoprotein CIII, MIP-1α, IL-6, IL-18 and tenascin C was identified and appears informative for all stages and common subtypes of ovarian cancer. Using a testing set of 245 samples, approximately twice the size of the model building set, the classifier had 91.3% sensitivity and 88.5% specificity. While these preliminary results are promising, further refinement and extensive validation of the classifier in a clinical trial is necessary to determine if the test has clinical value.

Conclusions/Significance

We describe a blood-based assay using 11 analytes that can distinguish women with ovarian cancer from those with benign conditions. Preliminary evaluation of the classifier suggests it has the potential to offer approximately 90% sensitivity and 90% specificity. While promising, the performance needs to be assessed in a blinded clinical validation study.     

土壤中毒死蜱降解菌的诱发和持效期研究

实验结果表明 ,施用毒死蜱 30d后 ,毒死蜱在灭菌土 (S土 )中的降解速度十分缓慢 ,在施过毒死蜱 30d后的土壤 (D土 )中的降解速度非常迅速 ,在未灭菌但未施过毒死蜱的土壤 (CK土 )中的降解速度则介于上述二者之间 ,说明微生物在毒死蜱的降解中起着十分重要的作用 ,同时也说明使用毒死蜱会诱发毒死蜱降解菌的产生。施用毒死蜱 5 0mg·kg-16 0d的毒死蜱在各种土壤中的降解速率与施过毒死蜱 30d后的土壤基本相同。 90d后 ,5 0mg·kg-1的毒死蜱在D土中的降解速率依然快于CK土和S土 ,但差别已经缩小。 12 0d后 ,毒死蜱在D土和CK土的降解速率基本相同。施药 30d后 ,分别以 0 1% ,1% ,10 %的D土加入S土 ,毒死蜱在其中的降解速率均比S土有不同程度的提高     

Bioprocess Intensification for Production of Novel Marine Bacterial Antibiotics Through Bioreactor Operation and Design

There is a lack of research into bioreactor engineering and fermentation protocol design in the field of marine bacterial antibiotic production. Most production strategies are carried out at the shake-flask level and lack a mechanistic understanding of the antibiotic production process, offering poor prospects for successful scale-up. This review shows that data need to be collated on media and physical optima differences between the trophophase and idiophase, along with investigations into the control mechanisms for biosynthesis, to allow implementation of novel fermentation protocols. Immobilization may play a part in bioprocess intensification of marine bacterial antibiotic production, through again this area is understudied. Similarly, mass transfer and shear stress data of fermentations are needed to provide the bioreactor design requirements to intensify antibiotic biosynthesis, with process scale-up in mind. The application of bioprocess intensification methods to the production of antibiotics (and other metabolites) from marine microbes will become an important strategy for improving supply of natural products, in order to assess their suitability as chemotherapeutic drugs. Received March 11, 1999; accepted May 4, 1999.     

以根为外植体建立大蒜的组织培养体系

以国内4个大蒜栽培品种为材料,建立了以根为外植体的再生体系。将蒜瓣去皮后灭菌消毒,萌发后选取苗龄为5~7 d的无菌苗的根接种到含不同激素配比的培养基中进行愈伤组织诱导,发现MS+2,4-D 1 mg/L+2ip 0.1 mg/L组合愈伤诱导效率最高,平均为56.06%;愈伤组织经过2~3次继代培养,选取胚性愈伤组织置于不同分化培养基上进行培养,2~3个月后可见小芽产生,分化培养基为MS+KT 1 mg/L时,植株再生效率最高,平均达到35.01 %。本研究建立了一种以根为外植体的高效的大蒜愈伤诱导和再生体系,为大蒜遗传转化体系的建立打下良好基础。     

Isolation and Characterization of a Fucoidan-Degrading Marine Bacterial Strain and Its Fucoidanase

A marine bacterial strain that degraded fucoidan from Kjellmaniella crassifolia (class Phaeophyceae, order Laminariales, family Laminariaceae) was isolated in our laboratory. The strain was gram-negative, ubiquinone 8 was the predominant respiratory quinone, and the GC-content of its genomic DNA was 36%. The cells of the strain were rod-shaped (2.0 m long × 1.0 m wide), and each cell was motile by means of one polar flagellum. Phylogenetic analysis of its 16S ribosomal DNA sequence indicated that it was a member of the family Alteromonadaceae. It produced a type of extracellular fucoidanase, an endosulfated fucan-digesting enzyme. The enzyme was purified with 3500-fold purity at 12.0% yield. Optimum conditions for the enzyme reaction were approximately pH 6.5 to 8.0 and temperature 30° to 35°C. The enzyme was activated by calcium ions, and maximum activity was observed in the presence of greater than 30 mM calcium ion.     

Quantitative Assay of Cyclin-Dependent Kinase Activity As a Sensitive Marker of Cell Proliferation in Marine Teleost Larvae

An evolutionarily conserved family of cyclin-dependent kinases (Cdks) regulates cell cycle progression in eukaryotes. Mitosis-promoting factor (MPF) is a transiently activated Cdk required for mitosis. We propose use of partially purified MPF kinase activity as a sensitive marker of cell proliferation in marine teleost larvae and describe a quantitative spin-filter assay of suc1-precipitated MPF activity. MPF extracted from embryos and larvae of red drum, Sciaenops ocellatus, phosphorylated histone H1 protein and a Cdk-specific peptide substrate. Kinetic analyses demonstrated that apparent K _m values of red drum MPF for adenosine triphosphate and the peptide substrate were 98.3 and 18.2 μM, respectively. A simple growth experiment showed that MPF activity was significantly greater in rapidly growing red drum larvae than in intermittently fed cohorts growing more slowly. Received July 23, 1998; accepted January 14, 1999     

真菌发育过程中的蓝光诱导

本文对多种真菌在发育过程中受蓝光诱导的现象进行了综述。蓝光可以诱导多种真菌的形态发生和发育,包括孢子的产生和菌丝的延伸等。此外,蓝光还可引起真菌的生理和生化改变,如细胞膜对离子的通透性的变化、类胡萝卜素的合成等。真菌也存在一个蓝光信号系统,通过蓝光受体接受蓝光信号,导致了真菌的一系列形态发生和生理生化的变化。     

Induction of Conjugation by Methyl Cellulose in Paramecium

ABSTRACT An improved method has been developed for the induction of selfing conjugation in Paramecium. Methyl cellulose induces selfing conjugation simply and efficiently in all species examined. Induction of conjugation by methyl cellulose was characterized in P. caudatum , where it occurred only in sexually mature, mating reactive cells. Conjugation produced sexual offspring and the time course of nuclear processes was substantially the same as that in natural conjugation between cells of complementary mating types. The method is useful for genetic studies in a wide variety of Paramecium species including P. caudatum, P. tetraurelia, P. multimicronucleatum and P. bursaria.     

Evaluation of Zebrafish Kidney Function Using a Fluorescent Clearance Assay

The zebrafish embryo offers a tractable model to study organogenesis and model human genetic disease. Despite its relative simplicity, the zebrafish kidney develops and functions in almost the same way as humans. A major difference in the construction of the human kidney is the presence of millions of nephrons compared to the zebrafish that has only two. However, simplifying such a complex system into basic functional units has aided our understanding of how the kidney develops and operates. In zebrafish, the midline located glomerulus is responsible for the initial blood filtration into two pronephric tubules that diverge to run bilaterally down the embryonic axis before fusing to each other at the cloaca. The pronephric tubules are heavily populated by motile cilia that facilitate the movement of filtrate along the segmented tubule, allowing the exchange of various solutes before finally exiting via the cloaca^2-4. Many genes responsible for CKD, including those related to ciliogenesis, have been studied in zebrafish⁵. However, a major draw back has been the difficulty in evaluating zebrafish kidney function after genetic manipulation. Traditional assays to measure kidney dysfunction in humans have proved non translational to zebrafish, mainly due to their aquatic environment and small size. For example, it is not physically possible to extract blood from embryonic staged fish for analysis of urea and creatinine content, as they are too small. In addition, zebrafish do not produce enough urine for testing on a simple proteinuria ‘dipstick’, which is often performed during initial patient examinations. We describe a fluorescent assay that utilizes the optical transparency of the zebrafish to quantitatively monitor the clearance of a fluorescent dye, over time, from the vasculature and out through the kidney, to give a read out of renal function^1,6-9.     

籼稻明恢63成熟种子愈伤组织的诱导及转基因水稻的抗性检测

利用不含附加营养成分的2,4-D培养基（2mg/L）诱导明恢63的成熟种子,9天预诱导后获得了大量的愈伤组织。利用基因枪辅助的土壤农杆菌转化法将天花粉蛋白（Trichosanthin, TCS）基因转入籼稻明恢63的愈伤组织,并通过再生（含有3mg/L 6-BA, 0.5mg/L ABA和1mg/L NAA的N6培养基）获得了转基因植物。 Southern blot分析和Western blot检测证明外源基因已经插入到T0代明恢63的基因组中并获得了表达。初步研究结果表明,转基因水稻对稻瘟病菌侵染具有抗性。     

Development of a Screening Assay for the In Vitro Evaluation of Thromboxane A2 Synthase Inhibitors

Abstract

Inhibitors of thromboxane A, (TxA₂) synthase are regarded as potentially useful agents in the treatment of cardiovascular diseases and in the prevention of tumour cell metastases. We report here a novel in vitro assay for the evaluation of TxA₂ synthase inhibitors. For the determination of inhibitory activity, malondialdehyde (MDA) formation by TxA₂ synthase in whole blood was utilized. After reaction with thiobarbituric acid MDA was quantified spectrofluorimetrically. The blank value was obtained by incubation with a selective TxA₂ synthase inhibitor. For the screening of compounds the simple MDA assay represents an alternative to the rather expensive and time consuming radioimmunoassay, HPLC and TLC methods. Only for compounds which have been shown to be good inhibitors in the MDA assay should a radioimmunoassay for selective inhibition of TxA₂ synthase be performed.     

Development in a Primary Cell Culture of the Marine Sponge Ircinia muscarum and Analysis of the Polar Compounds

We have established a primary cell culture of the marine demosponge Ircinia muscarum. The culture was started from a cell suspension obtained by a combination of mechanical chemical means. Microbial contamination was controlled by the use of a pool of antibiotics. Optical density, rather than hemocytometer count, is suggested to monitor the cellular growth. Analysis of the chemical composition of I. muscarum cells revealed absence of sterols, showing that the cells were unable to biosynthesize sterols. When the medium was supplemented with cholesterol an increase of about 70% in the number of cells was observed. These results suggest that the classic mammalian nutrient medium was not satisfactory for I. muscarum cell growth, and sterols were needed to satisfy the membrane requirements. Received November 11, 2000; accepted January 14, 2001