抗菌肽天蚕素B基因在纳豆芽胞杆菌中的表达

抗菌肽是生物体内经诱导产生的一类具有生物活性的小分子多肽。天蚕素B(Cecropin B)是最早从天蚕体内分离得到的一种热稳定的可溶性多肽,在已分离的众多抗菌肽中抗性较强。纳豆芽胞杆菌具有优良的益生特性,本研究选择枯草芽胞杆菌的一种表达载体p HT43,将抗菌肽天蚕素B基因导入纳豆芽胞杆菌中,验证其在目的菌中是否能够表达和稳定传代以及进行抗菌活性分析。结果表明,天蚕素B基因在纳豆芽胞杆菌中表达,并能稳定传代,能够提高纳豆芽胞杆菌的抑菌活性,抗金黄色葡萄球菌的活性优于干酪乳杆菌和枯草芽胞杆菌。本研究为该重组菌作为饲料添加剂的应用提供了技术基础。本文首次报道天蚕素B在纳豆枯草芽胞杆菌中表达。     

天蚕素类抗菌肽分子设计研究进展

天蚕素是一类由31~39个氨基酸残基组成的阳离子型线性α螺旋抗菌肽,具有抗细菌、抗真菌、抗病毒以及抑制肿瘤细胞等生物活性。天蚕素与传统抗生素作用机制不同,具有不易产生耐药性的特点,因此成为解决传统抗生素多重耐药性问题的一个新突破口。然而,天蚕素在抗菌活性、选择性、毒性以及稳定性等方面还存在诸多问题,并且天然天蚕素提取工艺复杂,成本较高,不适合大规模生产,其对细菌的高毒性也限制了原核工程菌的使用。近年来多肽分子设计的研究方法颇受青睐,为解决多肽物质诸多问题开辟了新的途径。针对天蚕素类抗菌肽研究过程中面临的主要问题综述了其分子设计的研究进展。     

昆虫抗茵肽的生理活性及其转基因应用前景

昆虫抗菌肽是昆虫免疫后存在于血淋巴中的一类活性肽.根据分子的结构可分为5类天蚕素类、昆虫防御素、富含脯氨酸或精氨酸的抗菌肽、富含甘氨酸的抗菌肽、抗真菌肽.且具有广谱的抗菌、抗病毒、抑制肿瘤的生物活性.概述了昆虫抗菌肽的基因的克隆与表达及转基因研究方面的进展,并展望了抗菌肽在基因工程中的应用前景.     

基于序列分析的天蚕素A串连融合表达载体的构建

天蚕素A(Cecropin A,CA)是来源于惜古比天蚕(Hyalophoracecropia)的含有37个氨基酸的抗菌肽,在非极性环境中形成α螺旋型结构,具有抗菌活性强、抗菌谱广等特点。为降低天蚕素A对宿主的毒性及提高其表达丰度和稳定性,在序列分析的基础上设计了3条天蚕素A氨基酸序列,分别为CA19、CA36和CA210。根据大肠杆菌密码子的偏爱性分别合成相应的3对寡核苷酸链,并通过重叠PCR方法合成成熟肽CA。将含有CA19、CA36、CA210和CA1～5个拷贝基因的质粒转化大肠杆菌BL21(DE3)和BLR(DE3)PlysS,成功构建了串连融合表达载体,为下一步的抗菌活性及免疫表位的分析打下基础。     

天蚕卵黄原蛋白cDNA的克隆及序列分析

天蚕卵黄原蛋白cDNA的全碱基序列由5 720个碱基构成,一个开读框有5 334个碱基序列,编码了包括由15个氨基酸组成的信号肽在内的共1 778个氨基酸的蛋白质前体。天蚕和柞蚕卵黄原蛋白的同源性非常高,氨基酸序列达到92.6%,碱基序列达到94%。天蚕、柞蚕和家蚕的卵黄原蛋白的糖链附加位点(N-linkedg-lycosylationsite):柞蚕有4个,家蚕有5个,天蚕和家蚕相同保存着5个。在N-末端区域,天蚕和家蚕有多聚丝氨酸区域和可被胰蛋白酶识别的RSRR部位;柞蚕虽然也具有多聚丝氨酸区域,但没有可被胰蛋白酶识别的RSRR部位。在C-末端区域里,大多数昆虫从G ICG功能部位至C-末端结尾具有7个~10个半胱氨酸(Cys)。鳞翅目的4种昆虫柞蚕、天蚕、家蚕和舞毒蛾(A.pernyi,A.yam am ai,B.m ori,L.dispar)都是7个半胱氨酸。     

两栖类皮肤抗菌多肽及其应用

抗菌多肽广泛分布于动物、植物,用于抵御细菌、真菌、病毒和原虫,在进化上是一类非常古老而有效的天然防御物质。两栖动物的后天获得性免疫系统与哺乳动物相比极为脆弱,它们在长期的进化历程中演化形成了一套非常有效的抵御微生物侵袭的防御系统,这套系统主要就是其裸露皮肤表面的抗菌多肽(又称初级免疫或者先天免疫系统)。本文结合本实验室近年的研究工作,对两栖类皮肤抗菌多肽的结构、功能及应用作一简要综述。     

植物多肽抗生素研究进展

植物多肽抗生素是一类对细菌、真菌等微生物及某些昆虫和动植物细胞具有抑制或杀灭作用的小分子多肽. 根据多肽抗生素的氨基酸序列及二级结构,可将植物多肽抗生素分为9类,包括硫素（thionins）、植物防御素（plant defensins）、转脂蛋白（lipid transfer proteins, LTPs）、橡胶素（heveins）、打结素（knottins）、凤仙花素(1b-AMPs)和新近发现的荠菜素（shepherdins）、蜕皮素（snakins）、环肽（cyclotides）. 对近年来植物多肽抗生素的分类、抗菌机理、生物活性及基因工程等方面的研究情况作一介绍,希望有助于我国在这一领域的研究与开发.     

昆虫抗菌肽的生理活性及其转基因应用前景

昆虫抗菌肽是昆虫免疫后存在于血淋巴中的一类活性肽。根据分子的结构可分为 5类 :天蚕素类、昆虫防御素、富含脯氨酸或精氨酸的抗菌肽、富含甘氨酸的抗菌肽、抗真菌肽。且具有广谱的抗菌、抗病毒、抑制肿瘤的生物活性。概述了昆虫抗菌肽的基因的克隆与表达及转基因研究方面的进展 ,并展望了抗菌肽在基因工程中的应用前景     

斜纹夜蛾两个天蚕素D基因的克隆及序列分析

天蚕素是昆虫抵御病菌入侵的一类抗菌肽家族。根据斜纹夜蛾Spodoptera litura天蚕素B基因设计特异引物,通过PCR扩增得到2个新的天蚕素基因部分序列,分别命名为cecD1和cecD2（GenBank登录号分别为EF555567和EF555568）。2个基因编码同一个天蚕素D蛋白,该蛋白的成熟肽与天蚕素B存在2个氨基酸残基差异。序列分析发现cecD1和cecD2中分别包含568 bp和377 bp的内含子序列,它们有相同的5′和3′拼接位点,A+T含量分别为59.7%和69.8%,符合大多数真核生物内含子高A+T含量的特征。     

融合基因Mdc-hly的构建及原核表达

构建家蝇天蚕素-人溶菌酶(Mdc-hly)融合基因,实现Mdc-hly基因在大肠杆菌中的表达。通过RT-PCR分别扩增出家蝇天蚕素和人溶菌酶的成熟肽基因序列,再利用Gene-SOEing技术构建融合基因,将融合基因克隆至pET32a表达载体,转化E.coli BL21(DE3),经IPTG诱导得到高效表达,融合蛋白分子量约为38kD。Western blotting杂交证实了表达蛋白的抗原活性。成功构建了融合其因并进行了原核表达,为进一步的生物活性研究打下基础。     

Heterogeneity of laser Doppler flowmetry in perfused muscle indicative of nutritive and nonnutritive flow

Laser Doppler flowmetry (LDF) signal responses have been compared with metabolic changes using both a surface macroprobe and randomly placed implantable microprobes in muscles of the constant-flow-perfused rat hindlimb. Changes in response to total flow and to vasoconstrictors that are known to increase (norepinephrine, NE) or decrease (serotonin, 5-HT) hindlimb oxygen uptake were assessed. The surface macroprobe (anterior end of biceps femoris) identified only one type of LDF response characterized by increased signal in response to NE and decreased signal in response to 5-HT. Implanted microprobes (tibialis, gastrocnemius, vastus, or bicep femoris) identified sites that gave three LDF responses of differing character. These responses were where the LDF signal increased with NE and decreased with 5-HT (56.7%), where the LDF signal decreased with NE and increased with 5-HT (16.5%), or where there was no net response to either vasoconstrictor (24.7%). The data are consistent with discrete regions of nutritive and nonnutritive flow in muscle where flow in each as controlled by vasoconstrictors relates directly to the metabolic behavior of the tissue.     

A Guide to Bivariate Ideas of Quantal Response Analysis,as Applied in Biometrics and Econometrics

A review is provided of the several bivariate generalisations of quantal response analysis that have appeared in the biometric and econometric literatures. There are three main types: (i) where a binary outcome is the result of two stimulants, and thus the bivariate distribution of the thresholds for response is relevant; (ii) where three or more alternative outcomes may arise from a single stimulant; and (iii) where the response itself is bivariate (i.e., two types of response may simultaneously be observed).     

Does Climate or Habitat Affect the Frequency of Cooperative Brood Rearing in Canada Geese?

Canada geese ( Branta canadensis ) are unusual because individuals use either of two different brood-rearing behaviors: cooperative broods (two or more merged broods attended by more than three parents) or two-parent families. We tested whether cooperative broods form in response to habitat or climatic conditions by examining variation in cooperative brood frequencies among Canada geese nesting in Connecticut from 1982 to 1996. Percent of goslings raised in cooperative broods ranged from 0 to 100% at a given site in different years, but the pattern of annual variation was different at each site. The sites were in close proximity to each other and had similar climates; thus, the differences in annual variation among sites was not likely to be a response to climatic conditions. Cooperative brood frequencies also varied among sites in each individual year, but sites with the most gang brooding in one year often had the least the next. Such would not be expected if gang brooding occurred in response to non-ephemeral habitat characteristics. Sites where gang brooding occurred and where it did not have similar food resources and predation risks. These findings failed to support the hypotheses that gang broods form in response to food competition or predation. Sites where gang broods occurred had more parent geese and more goslings than sites where they did not occur. Furthermore, the proportion of goslings raised in gang broods was correlated with the number of goslings and parents at the site. Our results support the hypothesis that gang broods form from the inadvertent mixing of goslings. This single factor, however, was not sufficient to account for all of the observed variation in gang brooding frequencies.     

Is there evidence for the post‐invasion evolution of increased size among invasive plant species?

Many plant species grow taller and have higher reproductive capacity where they are nonindigenous invaders than where they are native components of the flora. Traditionally, it has been accepted that this is a plastic response to a benign environment, though recently this assumption has been challenged and a genetic basis for increased plant size has been invoked. We tested the hypothesis that the increased size of certain weed species is genetically, rather than environmentally, based. A common environment growth experiment revealed no significant differences in the size of Carduus nutans , Digitalis purpurea , Echium vulgare or Senecio jacobaea sampled from alien (Australia and New Zealand) or native (Britain and continental Europe) habitats. We conclude that post-invasion genetic changes associated with increased size may be unusual and that the phenomenon, where it occurs, generally reflects a plastic response to a novel environment.     

Regression modeling of ordinal data with nonzero baselines

This paper develops regression models for ordinal data with nonzero control response probabilities. The models are especially useful in dose-response studies where the spontaneous or natural response rate is nonnegligible and the dosage is logarithmic. These models generalize Abbott's formula, which has been commonly used to model binary data with nonzero background observations. We describe a biologically plausible latent structure and develop an EM algorithm for fitting the models. The EM algorithm can be implemented using standard software for ordinal regression. A toxicology data set where the proposed model fits the data but a more conventional model fails is used to illustrate the methodology.     

Undrugged rotational response in nigro-striatal system-lesioned rats is related to the previous early response to apomorphine when repeatedly administered

Development and time-course characteristics of undrugged rotational response weeks or months after repeated apomorphine administration in 6-hydroxydopamine-lesioned rats were explored. In one series of experiments, all groups received several drug doses two-three weeks post-lesion and remained undrugged at different intervals from the last drug injection. Weeks or months later, they were injected with saline in the same environment where they previously had received apomorphine. In this way we studied acquisition, time-course and extinction of the rotational response after saline. Furthermore, we related this undrugged response which does not fully develop until two weeks after treatment, and which previously had not been related to a specific parameter of the rotational response to the drug, to a critical point of the time-course response to the drug, i.e., the early rotational response in the first minute after injection. This early response is a learning phenomenon based on the environmental cues where drug has been repeatedly administered. Finally, we state the concept of pharmacological conditioning using this animal model of Parkinson's disease, in agreement with our own results and the previous results of Silverman and Ho (1981).     

Quantitative Analysis of Pedogenic Thresholds and Domains in Volcanic Soils

Ecosystems - Pedogenic thresholds describe where soil properties or processes change in an abrupt/nonlinear fashion in response to small changes in environmental forcing. Contrastingly, soil...     

Effects of spatial grouping on the functional response of predators.

A unified mechanistic approach is given for the derivation of various forms of functional response in predator-prey models. The derivation is based on the principle of mass action but with the crucial refinement that the nature of the spatial distribution of predators and/or opportunities for predation are taken into account in an implicit way. If the predators are assumed to have a homogeneous spatial distribution, then the derived functional response is prey-dependent. If the predators are assumed to form a dense colony or school in a single (possibly moving) location, or if the region where predators can encounter prey is assumed to be of limited size, then the functional response depends on both predator and prey densities in a manner that reflects feeding interference between predators. Depending on the specific assumptions, the resulting functional response may be of Beddington-DeAngelis type, of Hassell-Varley type, or ratio-dependent.     

Distribution of California stream fishes: influence of environmental temperature and hypoxia

Synopsis Metabolic rates of seven fish species were used to assess the importance of temperature and dissolved oxygen as factors affecting longitudinal distributions of stream fish within California drainages. Metabolic rates of all species generally increased at higher acclimation temperatures and with abrupt temperature increases. In response to low dissolved oxygen, four species showed no change in metabolic rates up to a threshold temperature where hypoxia-induced metabolic depression was apparent. These threshold temperatures were near the lethal temperatures for each species. In contrast, two species showed metabolic depressions at every temperature, whereas one showed no depression at any temperature. In general, species occupying similar longitudinal positions in California streams behaved similarly in their metabolic responses. For most species, there was good correspondence between metabolic response and relevant field observations of occurrence. In cases where our analysis predicted species presence in waters where they did not exist, other abiotic factors, such as flow rate, or biotic factors, such as predation or competition, must be considered.     

Fertility of the early postpartum, lactating domestic rabbit

Sixty-four crossbred primiparous lactating does each suckling six pups were allocated at random into four groups and were mated on either Day 1, 2, 3, or 4 post partum (where Day 0 = the day of parturition). They were subsequently killed on Day 10 post coitum (where Day 0 = the day of mating) to assess fertility. There were no significant differences between treatment groups in their mating response (97% overall), ovulation response (77% overall), implantation response (83% overall), implantation rate (8.7 overall), or preimplantation mortality rate (24% overall). Ovulation rate was significantly increased in does mated on Days 3 and 4 (13.3 and 13.1, respectively), compared with those mated on Day 1 (10.2, P<0.05) and Day 2 (9.6, P<0.01) post partum. From these results we conclude that fertility is high throughout the early postpartum period in the lactating rabbit.