家蝇β-葡萄糖苷酶基因的克隆及重组表达

克隆家蝇内源性β-葡萄糖苷酶(beta-glucosidase,BG)基因并建立原核表达体系,检测其表达产物的活性,了解家蝇内源性BG酶特点,为进一步解释家蝇极强环境适应能力和发现新的种群控制措施提供分子生物学基础。以草地贪夜蛾等结构信息相对完整且研究较为透彻的6种代表性昆虫的BG酶基因序列为参照对象,利用同源克隆结合RACE-PCR的方法,从家蝇cDNA中克隆得到BG酶基因的全长cDNA序列并对其进行生物信息学分析。分别采用原核表达载体pET-28a(+)构建原核表达体系并在大肠杆菌E.coli BL21(DE3)中诱导表达BG酶基因的融合蛋白。采用七叶苷平板显色法和DNS法检测表达体系融合蛋白的酶活性,并对其性质进行初步的分析。家蝇体内克隆得到了长度为1933 bp的家蝇BG酶基因全长cDNA序列,推导其为562个氨基酸残基组成的蛋白多肽。重组原核质粒经诱导表达后,在65 kDa附近均出现特异性蛋白条带,证实重组质粒成功表达。重组表达的家蝇BG酶兼具内切β-1,4-葡聚糖酶、外切β-1,4-葡聚糖酶和BG的酶活性。家蝇BG酶是一种新的多功能纤维素酶,是昆虫纤维素酶研究的重要补充。     

两歧双歧杆菌中serpin基因的克隆、表达与功能分析

从Bifidobacterium bifidum WBBI02基因组中克隆了serpin基因片段,构建了重组Serpin蛋白的原核表达体系,实现了Serpin的表达与纯化。纯化的Serpin蛋白进行了抑制肠道蛋白酶活性检测,以及对双歧杆菌粘附作用影响的显微观察研究。结果表明:WBBI02中长度为768 bp的serpin基因序列,与GENEBANK中Bifidobacterium longum NCC2705 serpin序列同源性为99. 9 %。原核表达载体pBX₂-WBBI02表达的Serpin能有效地抑制糜蛋白酶和胰弹性蛋白酶的活性,最高抑制率分别为90%和97%,显微观察结果证实Serpin能促进双歧杆菌对HT-29细胞的粘附。     

不同载体表达的家蝇β-葡萄糖苷酶生物学活性比较

克隆家蝇内源性β-葡萄糖苷酶(beta-glucosidase,BG)基因,建立两种原核表达体系和一种真核表达体系,分别检测表达产物的活性,比较不同表达体系对其表达水平及重组蛋白酶学性质的影响,为进一步研究和利用家蝇BG酶奠定基础。本研究根据BG酶基因的cDNA序列设计引物扩增BG酶基因成熟肽的基因片段,分别采用表达载体pET-28a(+)和pEGX-4T-1构建原核表达体系并在大肠杆菌BL21(DE3)中诱导表达。在65 kDa附近均出现特异性蛋白条带,进行Western Blotting鉴定证实重组质粒在其宿主菌E.coli BL21(DE3)中成功表达,命名为MDBG-1蛋白和MDBG-2蛋白。同时选用真核表达载体pPICZa A构建酵母分泌型表达体系在酵母细胞GS115中获得稳定的表达,将其命名为MDBG-3蛋白。采用七叶苷平板法和DNS法对三种表达体系所重组表达的蛋白进行酶活性鉴定和检测,显示3种表达体系所表达的融合蛋白均具有BG酶活性,其酶活力有所不同,且真核表达体系所表达的蛋白酶活性最高。本研究对家蝇β-葡萄糖苷酶基因表达的重组蛋白特性进行初步研究,为发现新的有效纤维素酶体系提供基础数据。     

家蝇3种丝氨酸蛋白酶抑制剂Serpin基因克隆、序列分析及表达模式

对家蝇3种丝氨酸蛋白酶抑制剂Serpin基因SP2、SP13和SP16进行克隆、序列分析和时空表达模式检测。运用瑞士生物信息学研究所的蛋白分析专家系统（ExPASy）和美国国家生物技术信息中心（NCBI）等有关的生物信息学分析工具,预测和分析3条Serpin基因编码蛋白质的结构与生物学功能。以RPS18（核糖体S18蛋白）为内参基因,采用实时荧光定量PCR（Real-Time PCR）技术检测3条 Serpin 基因在家蝇不同发育时期,以及家蝇3龄幼虫中脂肪体、唾液腺、中肠、马氏管和体壁的表达特征。SP2、SP13、SP16基因序列的开放阅读框ORF全长分别为1191 bp、1137 bp和1212 bp,分别编码含396、378和403个氨基酸残基的蛋白质,其理论分子量分别为45315.3 Da、43701.5 Da和45011.5 Da,等电点分别为9.01、5.98和6.04,蛋白质的N端都含有一段信号肽,并具有Serpin基因的保守结构域,属于Serpin家族。SP2和SP16在蛋白质的C端含反应中心环（Reactive center loop,RCL),而SP13 C端不含RCL。时空表达模式分析结果显示,SP2基因在2龄幼虫中的表达量最高,而雄蝇的表达量与卵期相比有所下调;SP13基因在蛹中的表达量最高,1龄幼虫和雌蝇中的表达量与卵期相近;SP16基因在2龄幼虫和3龄幼虫中的表达量最高,雄蝇与雌蝇的表达量与卵期相比有所下调。在家蝇3龄幼虫的各主要组织中,以体壁为参照,SP2基因在唾液腺表达水平最高,其次是脂肪体,中肠和马氏管均低于体壁;SP13基因在马氏管和脂肪体中的表达水平最高,中肠和唾液腺的表达量低于体壁;SP16基因在唾液腺、中肠、脂肪体和马氏管的表达量均低于体壁。推测Serpin基因在家蝇个体发育和免疫调节中起不同作用。     

家蝇3种丝氨酸蛋白酶抑制剂(Serpin)基因的克隆、序列分析及表达模式

对家蝇3种丝氨酸蛋白酶抑制剂Serpin基因SP2、SP13和SP16进行克隆、序列分析和时空表达模式检测。运用瑞士生物信息学研究所的蛋白分析专家系统(Ex PASy)和美国国家生物技术信息中心(NCBI)等有关的生物信息学分析工具,预测和分析3条Serpin基因编码蛋白质的结构与生物学功能。以RPS18(核糖体S18蛋白)为内参基因,采用实时荧光定量PCR(Real-Time PCR)技术检测3条Serpin基因在家蝇不同发育时期,以及家蝇3龄幼虫中脂肪体、唾液腺、中肠、马氏管和体壁的表达特征。SP2、SP13、SP16基因序列的开放阅读框ORF全长分别为1191 bp、1137 bp和1212 bp,分别编码含396、378和403个氨基酸残基的蛋白质,其理论分子量分别为45315.3 Da、43701.5 Da和45011.5 Da,等电点分别为9.01、5.98和6.04,蛋白质的N端都含有一段信号肽,并具有Serpin基因的保守结构域,属于Serpin家族。SP2和SP16在蛋白质的C端含反应中心环(Reactive center loop,RCL),而SP13 C端不含RCL。时空表达模式分析结果显示,SP2基因在2龄幼虫中的表达量最高,而雄蝇的表达量与卵期相比有所下调;SP13基因在蛹中的表达量最高,1龄幼虫和雌蝇中的表达量与卵期相近;SP16基因在2龄幼虫和3龄幼虫中的表达量最高,雄蝇与雌蝇的表达量与卵期相比有所下调。在家蝇3龄幼虫的各主要组织中,以体壁为参照,SP2基因在唾液腺表达水平最高,其次是脂肪体,中肠和马氏管均低于体壁;SP13基因在马氏管和脂肪体中的表达水平最高,中肠和唾液腺的表达量低于体壁;SP16基因在唾液腺、中肠、脂肪体和马氏管的表达量均低于体壁。推测Serpin基因在家蝇个体发育和免疫调节中起不同作用。     

家蝇肽聚糖识别蛋白(PGRP-SA)的克隆表达及细菌结合研究

对家蝇PGRP-SA基因进行克隆表达以及研究其重组蛋白与细菌结合能力。从构建的家蝇(Musca domestica)幼虫cDNA质粒文库中筛选到PGRP-SA基因,以cDNA质粒为模板设计引物,通过PCR扩增,获得PGRP-SA基因完整编码序列。运用生物信息学方法对该基因及其编码蛋白进行预测和分析。构建pET-28a(+)-PGRP-SA重组质粒,转化到大肠杆菌BL21(DE3)中进行诱导表达及蛋白纯化。利用半定量RT-PCR检测PGRP-SA在家蝇3龄幼虫不同组织中的表达量差异。PGRP-SA重组蛋白进行微生物结合实验。结果表明,PGRP-SA基因ORF全长615 bp,编码204个氨基酸,理论分子量22.8 k D,等电点9.11,具有保守的PGRP结构域。成功构建了pET-28a(+)-PGRP-SA重组质粒,蛋白经IPTG诱导后在大肠杆菌中获得表达,经亲和层析柱纯化获得目的蛋白,利用Western blot检测证明纯化蛋白与预期大小相符。PGRP-SA在家蝇3龄幼虫血淋巴、脂肪体、前肠、中肠、气管、马氏管都有表达,血淋巴组织中表达量最高,后肠无表达,由此说明PGRP-SA基因的表达具有一定的组织性。PGRP-SA重组蛋白能与金黄色葡萄球菌和大肠杆菌结合,与白色念珠菌不能结合。成功表达及纯化家蝇PGRP-SA蛋白,证实家蝇PGRP-SA能与金黄色葡萄球菌和大肠杆菌结合。     

家蝇变应原Tropomyosin基因的克隆、序列分析及诱导表达

对家蝇变应原原肌球蛋白(Tropomyosin)基因进行同源克隆,序列分析;构建原核表达载体并在大肠杆菌中表达。从家蝇幼虫cDNA文库中筛选获得Tropomyosin基因。以该基因的cDNA文库质粒为模板,进行PCR扩增,获得家蝇Tropomyosin完整编码序列。运用生物信息学方法对该基因及其编码蛋白的基本理化性质、信号肽、二级结构、三级结构、抗原表位和亚细胞定位等方面进行预测和分析。构建pEASY-E1-Tropomyosin重组质粒,转化到大肠杆菌OrigamiB(DE3)中进行诱导表达。Tropomyosin基因ORF全长828 bp,编码275个氨基酸,理论分子量31.6 kD;等电点为4.65,具有Tropomyosin家族的蛋白保守结构域。成功构建重组原核表达pEASY-E1-Tropomyosin并诱导表达重组蛋白。     

硫氰酸生成酶样基因在家蝇高温和重金属镉应激中的作用

硫氰酸生成酶同源结构域普遍存在于古细菌、真细菌以及真核生物的多种蛋白分子内,这些蛋白构成一个功能多样的硫氰酸生成酶超家族。本研究根据家蝇转录组序列信息,分拣到一条硫氰酸生成酶样基因的EST序列,经分析发现其编码蛋白C末端含有典型的硫氰酸生成酶同源结构域,属于硫氰酸生成酶超家族,将其命名为家蝇硫氰酸生成酶样基因(Md-Rhodlike)。应用实时定量RT-PCR技术,分析了Md-Rhodlike在热激、镉胁迫条件下的表达量变化。Md-Rhodlike的mRNA水平在高温和镉处理后上调,提示该基因可能参与家蝇对高温及镉金属等环境胁迫因子的耐受,是一种新的家蝇应激相关蛋白。     

丝氨酸蛋白酶抑制剂Serpin1对东亚飞蝗酶学免疫的影响

Serpins是东亚飞蝗Locusta migratoria manilensis体内具有免疫调节功能的一类丝氨酸蛋白酶抑制剂.前期研究发现Serpin1能够降低绿僵菌Metarhizium对蝗虫的杀虫效果,本研究旨在从酶学角度明确Serpin1蛋白抑制绿僵菌毒力的原因,进一步揭示Serpins的功能与作用机制.本实验采用饵剂饲喂的方法进一步明确Serpin1蛋白对绿僵菌侵染东亚飞蝗的抑制效果;测定绿僵菌侵染东亚飞蝗过程中,添加Serpin1蛋白对东亚飞蝗体内保护酶(超氧化物歧化酶SOD、过氧化物酶POD、酚氧化酶PO)、解毒酶(多功能氧化酶MFO、谷胱甘肽转移酶GSTs、乙酰胆碱酯酶AchE)共6种酶的影响,以明确Serpin1对东亚飞蝗酶学免疫的调节作用.结果表明,Serpin1能够显著降低绿僵菌对蝗虫的杀虫效果;将Serpin1与绿僵菌混合后处理东亚飞蝗,12 d后其死亡率为63.5％,显著低于绿僵菌单独处理(死亡率为80.6％).酶活测定结果显示,将绿僵菌IMI330189与Serpin1蛋白混合处理后,与绿僵菌处理组相比,东亚飞蝗体内保护酶SOD和PO的活力总体表现为上调,而POD的活力呈现降低的趋势;解毒酶MFO、GSTs的活性呈现升高趋势,AChE的活力呈现先升高后降低的趋势.上述结果表明,Serpin1蛋白能够增强东亚飞蝗体内解毒酶和保护酶的活性,提高东亚飞蝗的酶学免疫,增强对绿僵菌侵染的抵御能力,从而降低东亚飞蝗的死亡率.本研究为进一步揭示Serpins的功能提供了参考.     

家蝇核糖体蛋白S18基因的克隆及表达模式研究

旨为证实核糖体蛋白S18(Ribosomal protein S18,RPS18)基因在家蝇体内的表达稳定性。从构建家蝇幼虫c DNA文库中筛选到核糖体蛋白S18基因,以c DNA为模板,通过PCR扩增,获得RPS18基因完整编码序列(登录号:KT006855),运用生物信息学方法对该基因及其编码蛋白进行预测和分析。构建p ET28a/RPS18重组质粒,转化到大肠杆菌Transetta(DE3)中进行诱导表达及蛋白纯化,制备多克隆抗体并进行抗血清特异性分析。应用RT-PCR、q PCR及Western-blot从转录、翻译水平上分析RPS18基因在家蝇不同发育时期和三龄幼虫不同组织中的表达情况。结果表明,RPS18基因ORF全长459 bp,编码152个氨基酸,理论分子量为17 590.5 Da,等电点为10.48;将构建的重组质粒转入大肠杆菌Transetta(DE3)中,纯化得到RPS18蛋白;将该纯化蛋白免疫大白兔获得多克隆抗体,His单抗鉴定及抗血清特异性分析显示,其均可见单一条带;RT-PCR、q PCR及Westernblot分析表明,RPS18基因在家蝇不同发育时期及幼虫不同组织均能够稳定表达;综合分析表明,该基因在家蝇不同发育时期及幼虫不同组织均保持较好的表达稳定性。     

Definitions,measurements, and classifications of stimuli,situations, and environments

A review is presented of issues relevant to the definition, measurement, and classification of stimuli, situations, and environments. Problems such as the lack of adequate definitions of concepts, error and bias in measurement procedures, confusion between measurement of a concept and measurement of its behavioral effects, and the lack of agreement among alternative measures are emphasized. It is suggested that concepts be defined in terms of objective characteristics while allowing for the study of the transactional relationship between organism and environment. The work of the ethologists in defining stimuli while studying their relationship to different organismic states and situational contexts is emphasized in this regard. Following Brunswik, it is also suggested that wherever possible there be a representative sampling of variables in natural settings. Note from the editors: From time to time, Human Ecology will publish a review article. Our first in this series is a review by a psychologist of basic definitional and conceptual problems in environmental studies.This paper was prepared while the author was a Visiting Research Fellow at the Educational Testing Service. The support of ETS and my colleagues in the Division of Psychological Studies is gratefully acknowledged. The review was also supported in part by a grant from the Rutgers University Research Council.     

Synthesis,tautomerism, and antimicrobial,anti-HCV,anti-SSPE,antioxidant, and antitumor activities of arylazobenzosuberones

2-Dimethylaminomethylene-1-benzosuberone 1 was coupled with diazotized aniline derivatives to afford a series of the hitherto unreported 2-arylazo-1-benzosuberones 3a–i. The tautomeric structure and the effect of substituents on the tautomeric form (s) of the products 3a–i were discussed. Similar coupling of the enaminone 1 with diazonium salts of heterocyclic amines gave the respective fused azolotriazino-benzosuberones. Some of the newly synthesized compounds showed potent antimicrobial, anti-HCV, antioxidant, antitumor (as topoisomerase I inhibitors), and antimicrobial activities.     

The effect of malathion,diazinon, and various concentrations of zinc,copper, nickel,lead, iron,and mercury on fish

Acute and chronic toxicity tests for malathion, diazinon, copper (Cu), mercury (Hg), lead (Pb), zinc (Zn), nickel (Ni), and iron (Fe) were conducted. Mortalities ofBarilius vagra andCyprinus carpio (common carp) were variable but LC50-96 hr were similar for pesticides. AdultB. vagra seem to be more sensitive to malathion than juvenile carp. Both juvenile carp and adultB. vagra were extremely sensitive to diazinon. Long-term exposure to pesticides modified morphology and behavior. The LC50-96 values for Cu, Hg, and Pb were 0.3, 0.16, and 0.44, respectively, for smaller fish and 1.0, 0.77, and 1.33, respectively, for larger fish. Replicate LC50 values for Zn, Ni, and Fe were somewhat variable, and for these metals, the size of the fish seemed to affect response because LC50 values increased as fish size increased. Cooper, Pb, Zn, and Fe residues following exposure to sublethal concentrations of these metals for 15 d were significantly greater in whole juvenile common carp than in controls.     

Low-frequency dynamics and Raman scattering of crystals, of B-, A-, and Z-DNA, and fibers of C-DNA

Normal modes of vibration of DNA in the low-frequency region (10-300 cm-1 interval) have been identified from Raman spectra of crystals of B-DNA [d(CGCAAATTTGCG)], A-DNA [r(GCG)d(CGC) and d(CCCCGGGG)], and Z-DNA [d(CGCGCG) and d(CGCGTG)]. The lowest vibrational frequencies detected in the canonical DNA structures--at 18 +/- 2 cm-1 in the B-DNA crystal, near 24 +/- 2 cm-1 in A-DNA crystals, and near 30 +/- 2 cm-1 in Z-DNA crystals--are shown to correlate well with the degree of DNA hydration in the crystal structures, as well as with the level of hydration in calf thymus DNA fibers. These findings support the assignment [H. Urabe et al. (1985) J. Chem. Phys. 82, 531-535; C. Demarco et al. (1985) Biopolymers 24, 2035-2040] of the lowest frequency Raman band of each DNA to a helix mode, which is dependent primarily upon the degree of helix hydration, rather than upon the intrahelical conformation. The present results show also that B-, A-, C-, and Z-DNA structures can be distinguished from one another on the basis of their characteristic Raman intensity profiles in the region of 40-140 cm-1, even though all structures display two rather similar and complex bands centered within the intervals of 66-72 and 90-120 cm-1. The similarity of Raman frequencies for B-, A-, C-, and Z-DNA suggests that these modes originate from concerted motions of the bases (librations), which are not strongly dependent upon helix backbone geometry or handedness. Correlation of the Raman frequencies and intensities with the DNA base compositions suggests that the complex band near 90-120 cm-1 in all double-helix structures is due to in-plane librational motions of the bases, which involve stretching of the purine-pyrimidine hydrogen bonds. This would explain the centering of the band at higher frequencies in structures containing G.C pairs (greater than 100 cm-1) than in structures containing A.T pairs (less than 100 cm-1), consistent with the strengths of G.C and A.T hydrogen bonding.     

Synthesis and properties of N-, O-, and S-phospho derivatives of amino acids, peptides, and proteins

The literature on chemical (i.e., nonenzymic) phosphorylation of amino acids, peptides, and proteins is reviewed through 1982. The review covers synthetic methods, chemical reactions, and physical properties, with emphasis on the techniques used for separation and characterization of the products. Synthetic methods are classified by reagent rather than product, and are illustrated by experimental procedures for the most important methods. Chemical reactions are classified into four groups depending on whether the reaction site is the phospho group, the amino group, the carboxyl group, or in the case of serine the hydroxyl group. Physical data are given for all of the known N-, O-, and S-phospho derivatives of the amino acids, peptides, and proteins, within certain limitations, and are discussed in detail in the section on physical properties. Emphasis is given to the techniques used for separation of the products, such as chromatography and electrophoresis, and for characterization of the products, particularly spectroscopy. Medical and other uses of the products are mentioned.     

Cultural, Biochemical, and Immunological Properties of Mima, Herellea, and Flavobacterium Species

From study of cultural and biochemical characteristics of 40 strains of Herellea, Mima, or Flavobacterium species, a proposed schema for identification was developed. The reactions observed by agglutination, gel diffusion, and immunofluorescence suggest antigenic heterogeneity of this group of organisms.     

Pet Loss and Representations of Death,Attachment, Depression,and Euthanasia

Studies that have examined pet loss hypothesize that attachment, representations of death, and the belief in an afterlife for animals may influence owners’ bereavement and depressive outcomes. The following instruments were administered to 159 Italian participants recruited through snowball sampling: the Lexington Attachment to Pets Scale (LAPS), the Pet Bereavement Questionnaire (PBQ), the Testoni Death Representation Scale (TDRS), and Beck’s Depression Inventory II (BDI-II). Questions concerning pet euthanasia-related issues and the relationship between owners and veterinarians were also submitted to the participants. A path model was conducted, showing that the representation of death and the attachment to a pet had a direct effect on pet grief, which in turn had a direct effect on depression. The results show a positive correlation between the LAPS and PBQ factors, particularly with the PBQ factor Grief. The LAPS factors positively correlated with the TDRS representation of Death as a Passage and negatively correlated with the TDRS representation of Death as Annihilation. The LAPS People Substituting factor positively correlated with the total score and the Cognitive-Affective factor of the BDI-II. The PBQ factors positively correlated with the BDI-II, whereas only the TDRS Death as Annihilation factor positively correlated with the BDI-II. Belief in a transcendent dimension was associated with higher scores on the PBQ Guilt factor and the TDRS factors of Death as a Passage and Death as Change, whereas these beliefs were associated with lower scores on the TDRS factor Death as Annihilation.

The results indicated that the sensitivity of the veterinarian and a veterinarian who helps owners make conscious and informed decisions for their pet and choose the right time to perform euthanasia are important variables in the management of pet loss. However, these factors are not sufficient and psychological support should be improved to help owners better cope with grief.     

Count data, detection probabilities, and the demography, dynamics, distribution, and decline of amphibians

The evidence for amphibian population declines is based on count data that were not adjusted for detection probabilities. Such data are not reliable even when collected using standard methods. The formula C = Np (where C is a count, N the true parameter value, and p is a detection probability) relates count data to demography, population size, or distributions. With unadjusted count data, one assumes a linear relationship between C and N and that p is constant. These assumptions are unlikely to be met in studies of amphibian populations. Amphibian population data should be based on methods that account for detection probabilities.