鱼类同工酶和蛋白质的聚丙烯酰胺梯度凝胶电泳法

同工酶分析一般常用淀粉凝胶电泳进行,但它常受淀粉质量的制约,在淀粉的水解和制胶过程较难标准化,因而不易掌握,其机械强度不够也不易操作。       

肺癌患者与肺结核患者血清蛋白的电泳分析

采用SDS聚丙烯酰胺凝胶电泳银染色的方法,对58例肺癌患者和32例肺结核患者的血清蛋白进行比较分析,发现有五种特异蛋白,分别存在于A区、C区、F区和G区。肺癌组和肺结核组比较,存在极显著性差异(P<0.01),提示有早期诊断肺癌的价值,有可能成为临床诊断肺癌的一个重要参考指标。     

FOUR PROTEIN POLYMORPHISMS IN PIGEON BRAIN EXTRACTS DETECTED BY TWO-DIMENSIONAL GEL ELECTROPHORESIS

Abstract— Proteins of the brain extracts of 85 individual pigeons (Columba livia) were mapped by two-dimensional gel electrophoresis. The method is a modification of O'Farrell 'S technique and separates proteins first by charge and then by molecular weight. There were three proteins, A, B and D which had each a variant form. The positions of these six proteins on the gel corresponded to the following pH values and molecular weight values: protein A₁, 6.4/43,000; A₂, 6.6/43,000; B₁, 5.7/41,000; B₂, 5.8/40,000; D₁, 6.2/22,000; D₂, 6.2/21,000. The variants are genetically determined, since protein A, B and D each occurred in three phenotypes (A₁, A₁A₂ and A₂; B₁, B₁B₂ and B₂; D₁, D₁D₂ and D₂) corresponding to the three possible genotypes. From the observed frequencies of the phenotypes the following allele frequencies were calculated: allele A₁, 72%; A₂, 28%; B₁, 15%; B₂, 85%; D₁, 74%; D₂, 26%. A fourth protein named C occurred in four different forms (C₁, 7.2/37,000; C₂, 7.2/36,000; C₃, 7.1/37,000; C₄, 7.1/36,000) and six phenotypes (C₁, C₁C₂, C₂, C₁C₃, C₂C₃ and C₄C₃). This polymorphism is also interpreted as being genetically determined. The four alleles coding for the four protein C forms had the following frequencies: allele C₁, 62%; C₂, 27%; C₃, 10.5%; C₄, 0.5%.     

AM真菌DNA的提取与PCR-DGGE分析

分别从丛枝菌根(AM)真菌的单孢、宿主植物根系及土壤样品中提取DNA,对AM真菌的18SrDNA中NS31/Glol区进行Nested PCR特异性扩增,表明Nested PCR能很好地以微量DNA为模板扩增出目标产物;对扩增产物进行DGGE电泳,3种样品表现出不同的DGGE指纹图谱特征。本文认为,将Nested PCR与DGGE技术相结合,可以成为AM真菌分子生态学研究的有效途径。     

六种疫霉菌体可溶性蛋白质和酯酶的聚丙烯酰胺凝胶平板电泳研究*

本文对槐生疫霉(Phytophthora robinicola)、掘氏疫霉(P.drechsleri)、樟疫霉(P.cinnamomi)、烟草疫霉(P.nicotianae)、棕榈疫霉(P.palmivora)及辣椒疫霉(P.capsici)等6种疫霉81株菌菌体可溶性蛋白质和酯酶进行了聚丙烯酰胺凝胶平板电泳研究。结果指出,种间蛋白质图谱差异明显,种内菌株间基本一致。就酯酶图谱来说,种内菌株间存在一定差异,但种间差异更为显著。因此,本试验进一步支持槐生疫霉新种的建立。同时表明,菌体可溶性蛋白质电泳图谱在疫霉种的鉴定和分类上具有重要意义,而酯酶电泳图谱在一定程度上也有助于疫霉种的研究。     

POLYACRYLAMIDE GEL ELECTROPHORESIS OF RAT BRAIN ACETYLCHOLINESTERASE: ISOENZYME CHANGES FOLLOWING PARATHION POISONING1

Brain acetylcholinesterase (EC 3. 1. 1. 7) isoenzymes of 15- and 30-day-old rats were found to be inhibited by 2.5 mg/kg and 1.25 mg/kg dosage levels of intraperitoneally administered parathion (E-605; O, O-diethyl-p-nitrophenyl phosphorothionate). With 2.5 mg/kg dose level, the response of isoenzymes in 15- and 30-day-old rats was similar. At both ages, there was no significant sex difference in the degree of depression of the isoenzymes. There were no significant regional differences in the degree of inhibition of acetylcholinesterase isoenzymes in the rat brain. At 1.25 mg/kg dosage level, a differential isoenzyme inhibition was evident, with the major isoenzyme (isoenzyme 3) exhibiting the greatest sensitivity to the inhibitor in all brain areas examined. The course of isoenzyme depression and recovery following the administration of parathion differed in brain, serum and skeletal muscle. Whereas brain isoenzymes exhibited most marked inhibition at 2 h after injection, inhibition of serum and skeletal muscle isoenzymes was more prolonged. At 4 h after injection, these isoenzymes were still inhibited while brain isoenzymes had recovered to a substantial degree. Twenty four h following the injection of parathion, when brain and serum acetylcholinesterase isoenzymes had returned to control activity levels, isoenzymes of skeletal muscle demonstrated only minimal recovery.     

六种疫霉菌体可溶性蛋白质和酯酶的聚丙烯酰胺凝胶平板电泳研究

本文对槐生疫霉(Phytophthora robinicola)、掘氏疫霉(P.drechsleri)、樟疫霉(P.cinnamomi)、烟草疫霉(P.nicotianae)、棕榈疫霉(P.palmivora)及辣椒疫霉(P.capsici)等6种疫霉81株菌菌体可溶性蛋白质和酯酶进行了聚丙烯酰胺凝胶平板电泳研究。结果指出,种间蛋白质图谱差异明显,种内菌株间基本一致。就酯酶图谱来说,种内菌株间存在一定差异,但种间差异更为显著。因此,本试验进一步支持槐生疫霉新种的建立。同时表明,菌体可溶性蛋白质电泳图谱在疫霉种的鉴定和分类上具有重要意义,而酯酶电泳图谱在一定程度上也有助于疫霉种的研究。     

MOLECULAR GENETIC-DISTANCE ESTIMATES AMONG THE URSIDAE AS INDICATED BY ONE- AND TWO-DIMENSIONAL PROTEIN ELECTROPHORESIS

Evolutionary relationships among eight species of Ursidae (including the giant panda) relative to two Procyonidae species (raccoon and red panda) were estimated based on the extent of electrophoretic variation of 289 radiolabelled fibroblast proteins resolved by two-dimensional gel electrophoresis and among 44 isozyme loci resolved by one-dimensional electrophoresis. Allelic differences among these species were converted to genetic distances, and phenetic trees were constructed. In addition, the electrophoretic data were coded as unit characters, and minimum-length trees were derived based on the Wagner method using maximum parsimony. Regardless of the tree-building method employed, the data sets agreed on the following branching sequence: between 22.4 and 32.3 million years (MY) ago, the ancestors of the procyonids and the ursids split into two lineages. Within 10 MY, the red panda split from the line that led to the raccoon. An ancestor of the giant panda split from the ursid line 18–22 MY ago, and the South American spectacled bear split from the line leading to ursine bears 10.5–15.0 MY B.P. A group of six closely related ursine bears (brown bear, polar bear, Asiatic black bear, Malayan sun bear, American black bear, and sloth bear) diverged from a common ancestor during the past 4–8 MY. Much of this ursine radiation was not resolved by our results, with the exception of a recent (2–3 MY B.P.) divergence of brown bear and polar bear. The topological concordance of the data sets from one- and two-dimensional electrophoresis supports the usefulness of these procedures for evolutionary inference and provides additional precision to the reconstruction of divergence nodes of this carnivore group.     

STARCH GEL ELECTROPHORESIS OF PLANT ISOZYMES: A COMPARATIVE ANALYSIS OF TECHNIQUES

The use of genetic information expressed in isozymic form has enhanced our understanding of heritable variation within and among plant populations. For over three decades now, protein electrophoresis coupled with histochemical staining has provided data on the concordance of observable features in plants (e.g., morphology, cytology, ecological adaptation) with isozyme phenotypes. Yet inadequate dissemination of procedural details and varied investigative approaches have limited the comparative value of electrophoretic data and have made it difficult for novices to apply the technique to other experimental systems. Information gathered from over 25 laboratories utilizing enzyme electrophoresis is treated comparatively, providing access to protocols that often are either unpublished or widely dispersed in the literature. Analyzed in the review are methods and guidelines for enzyme extraction, effective storage of plant samples and buffers, efficient screening of taxa for enzymatic activity, and interpretation of diploid and polyploid banding patterns.     

桶形芋螺毒液蛋白质组分析及二硫键异构酶的鉴定

蛋白质二硫键异构酶(PDI)是内质网新生肽链折叠中一个重要的折叠酶。在热带药用海洋生物芋螺的毒液中,富含PDI酶,该酶对于毒液中芋螺毒素神经肽的体内氧化折叠至关重要。研究上样量、水化方式与时间、除盐步骤、聚焦电压和时间、平衡时间、凝胶电泳方法和染色方法等方面,优化并建立了较为理想的芋螺毒液蛋白样品双向电泳的实验条件与方法;通过双向电泳和MALDI-TOF-MS质谱分析技术,从海南产桶形芋螺(Conus betulinus Linnaeus)毒液蛋白中成功分离鉴定出PDI等9种蛋白;通过双向电泳和PDQuest软件分析了并比较了三种不同大小桶形芋螺毒液总蛋白的差异性,并质谱鉴定出5个明显的差异蛋白点。研究建立了桶形芋螺毒液蛋白双向电泳分析及质谱鉴定的技术方法,为后续大量分离纯化出天然的芋螺PDI酶以及利用蛋白质组学技术方法深入研究芋螺毒液特征提供了重要的基础。     

ANALYSIS OF GENOME AND SPECIES RELATIONSHIPS IN THE POLYPLOID WHEATS BY PROTEIN ELECTROPHORESIS

Protein spectra from 4n and 6n species of Triticum were obtained by electrophoresis of seed extracts on polyacrylamide gels. Homologies among the species with regard to protein bands were tested by comparing each species with the standard T. dicoccum in a protein mixture spectrum. By reference to two pairs of homologous bands, each spectrum was then adjusted to the migration velocity of the standard by photographic enlargement. The homologies were found to be consistent with evidence from conventional methods regarding genome relationships among the Triticum polyploids. T. dicoccoides and other known AABB tetraploids showed nine fast-moving albumin homologues, while T. timopheevi and other known AAGG tetraploids showed seven. The two genomic groups had five albumin bands in common. The hexaploid (AABBDD) subspecies showed 12 albumin homologues, 9 of which were also homologous with the 9 of the AABB tetraploids and 3 of which were attributed to the D-genome donor. Differences among species within each of the tetraploid genomic groups and among the hexaploid subspecies were largely confined to the slow-moving bands of the gliadin series.     

ANALYSIS OF SOUNDS PRODUCED BY MALE PADOGOBIUS MARTENSI (PISCES,GOBIIDAE) AND FACTORS AFFECTING THEIR STRUCTURAL PROPERTIES

ABSTRACT

In this paper a detailed analysis of the physical structure of sounds produced by male Padogobius martensi is reported. Sound production occurs during courtship and inter-male agonistic encounters. Both aggressive and courtship calls are made up of rapidly repeated pulses, with a pulse repetition rate decreasing through the course of the emission. By means of computerized analysis, the pulse repetition rate, its modulation and sound duration were determined. The water temperature was found to exert a marked and significant effect on the above parameters. In particular, the temperature directly affects the pulse rate and its decrease through the course of the emission (i.e. frequency modulation) and inversely affects sound duration. By contrast, size of the calling animal does not significantly influence the sound parameters considered. Aggressive sounds last longer and have a lower pulse repetition rate than the courtship sounds. Moreover, aggressive sounds appear more variable than the courtship ones as far as pulse rate and duration are concerned.     

应用微核试验和单细胞凝胶电泳技术来检测农药对青蛙蝌蚪及成体的遗传毒性

应用青蛙红细胞微核试验和单细胞凝胶电泳试验研究了两种新型杀虫剂 -吡虫啉和抑食肼对青蛙蝌蚪和成体的遗传毒性 ,结果表明 :当吡虫啉为 2mg/L时 ,蝌蚪红细胞微核率与对照组相比 ,无显著性差异 (p >0 .0 5) ;浓度升高到 8mg/L时 ,微核率与对照组相比 ,有显著性差异 (p <0 .0 5) ;当浓度为 3 2mg/L时 ,微核率与对照组相比 ,有极显著性差异 (p <0 .0 1) ;并有明显的剂量 -效应关系 (r =0 .9843 )。而抑食肼在浓度为 2 .5mg/L和 10mg/L时 ,微核率与对照组相比 ,无显著性差异 (p >0 .0 5) ;当浓度增至 40mg/L时 ,微核与对照组相比 ,有极显著性差异 (p <0 .0 1) ;吡虫啉与抑食肼各浓度组对青蛙红细胞的DNA损伤与阴性对照组相比 ,都有极显著性差异 (p <0 .0 1) ,且具有明显的剂量 -效应关系 (r =0 .960 ,r=0 .990 )。     

PULSED‐FIELD GEL ELECTROPHORESIS ANALYSIS OF GENOME SIZE AND STRUCTURE IN PAVLOVA GYRANS AND DIACRONEMA SP. (HAPTOPHYTA)1

Information regarding genome size and structure is a prerequisite for selecting model organisms and for facilitating the most efficient study of their chromosomal DNA. The goal of this study was to identify future candidates for complete‐genome‐sequencing projects among economically or evolutionarily important species of haptophyte algae. Using pulsed‐field gel electrophoresis (PFGE), we identified relatively small genomes and chromosome sizes in two haptophyte species from the class Pavlovophyceae, Pavlova gyrans Butcher and Diacronema sp. The basal position of Pavlovophyceae in the Haptophyta; the key position of this group in the chromalveolates; and their economic and potential biomedical importance, ease of culturing, and small genome size make these taxa ideal models for complete‐genome sequencing.     

A NOVEL METHOD FOR ASSESSMENT OF 16S RRNA GENE COPY NUMBER IN BACTERIAL GENOMES BY PULSED-FIELD GEL ELECTROPHORESIS AND PCR AMPLIFICATION

16S rRNA gene ( rrn ) copy number in bacterial genomes is indicative of ecological strategies of bacteria and is critical for quantification of bacterial abundance in mixed populations using rrn- based approaches. For accurate assessment of rrn copies, a novel technical strategy by means of pulsed-field gel electrophoresis and polymerase chain reaction amplification analysis was introduced. Experimental and in silico analysis on a test bacterial culture Caulobacter crescentus proved it to be simple, effective, accurate and a good alternative to traditional time-consuming methods.

PRATICAL APPLICATIONS

This method can be used for routine determination of gene copy number in most bacteria whose full genome sequences are not available. Moreover, the pulsed-field gel electrophoresis bands containing a target gene fragment can be determined and therefore constructing an expected fragments oriented genomic library is possible.     

家蚕核型多角体病毒及核衣壳的纯化和其结构蛋白的凝胶电泳分析

 用差速离心和蔗糖密度梯度离心提纯了家蚕核型多角体病毒及其核衣壳,并用SDS聚丙烯酰胺凝胶(均一胶和梯度胶)电泳分析了它们的结构蛋白。测量使用游标卡尺,计算使用微型计算机。凝胶用很染色方法染色。 结果表明,家蚕核型多角体病毒长381.2±17.81纳米,直径88.86±9.605纳米;核衣壳长328.9±5.917纳米,直径41.36±1.167纳米。两种凝胶电泳所得数据显示病毒粒子至少含有20组分子量不同的结构多肽,含量较多的是P31、P40、P28和P20。P45和一些小分子多肽看来主要存于囊膜上。P31的分子量与多角体蛋白相同,我们的实验表明它可能是病毒粒子和核衣壳的结构组分。     

The Incorporation of 14C-Proline into the Proteins of Growing Cells: II. A NOTE ON EVIDENCE FROM CULTURED CARROT EXPLANTS BY ACRYLAMIDE GEL ELECTROPHORESIS

The method of acrylamide gel electrophoresis has been appliedto the separation of the proteins of growing carrot explantswhich are soluble in a buffer solution at pH 8.3. At least ninedistinct bands were detectable in this way. When carrot tissuehad absorbed ¹⁴C-proline it entered into the composition ofall these bands and, in all except one, it was converted tohydroxyproline to different degrees which characterized theband in question. Thus, in the soluble protein the ¹⁴C-hydroxyproline:¹⁴C-proline ratio varied from 0 to 0.53. The bulk of the proteinin the tissue was insoluble in buffer at pH 8.3; it containedthe bulk of the radioactivity absorbed from proline (84.8 percent.); and its average ¹⁴C-hydroxyproline : ¹⁴C-proline ratiowas the highest of all (1.28). A particulate protein preparation,separated at 25,000 g. from the soluble protein, had an intermediateratio (0.643) of ¹⁴C-hydroxyproline to ¹⁴C-proline. Therefore,there are in cultured carrot explants many distinct proteinmoieties which incorporate ¹⁴C-proline, and they convert itto ¹⁴C-hydroxyproline to very different degrees. The evidenceis consistent with the incorporation of the proline, first intothe various soluble proteins which are electrophoretically separableand subsequently, with progressively greater hydroxylation,into the more insoluble protein that constitutes the bulk ofthe protein of the cell and its organelles. It is, therefore,quite incorrect to say (as some have done) that all of the hydroxyproline-containingprotein is in very close association with the cell wall, forpart of it is present in the cell in soluble and electrophoreticallyseparable forms.     

Lipopolysaccharide Engineering in Neisseria meningitidis: STRUCTURAL ANALYSIS OF DIFFERENT PENTAACYL LIPID A MUTANTS AND COMPARISON OF THEIR MODIFIED AGONIST PROPERTIES*

Engineering the lipopolysaccharide (LPS) biosynthetic pathway offers the potential to obtain modified derivatives with optimized adjuvant properties. Neisseria meningitidis strain H44/76 was modified by expression of the pagL gene encoding lipid A 3-O-deacylase from Bordetella bronchiseptica and by inactivation of the lgtB gene encoding the terminal oligosaccharide galactosyltransferase. Mass spectrometry analysis of purified mutant LPS was used for detailed compositional analysis of all present molecular species. This determined that the modified LPS was mainly pentaacylated, demonstrating high efficiency of conversion from the hexaacyl to the 3-O-deacylated form by heterologous lipid A 3-O-deacylase (PagL) expression. MS analyses also provided evidence for expression of only one major oligosaccharide glycoform, which lacked the terminal galactose residue as expected from inactivation of the lgtB gene. The immunomodulatory properties of PagL-deacylated LPS were compared with another pentaacyl form obtained from an lpxL1⁻ mutant, which lacks the 2′ secondary acyl chain. Although both LPS mutants displayed impaired capacity to induce production of the pro-inflammatory cytokine IL-6 in the monocytic cell line Mono Mac 6, induction of the Toll-interleukin-1 receptor domain-containing adaptor-inducing interferon-β-dependent chemokine interferon-γ-induced protein 10 was largely retained only for the lgtB⁻/pagL⁺ mutant. Removal of remaining hexaacyl species exclusively present in lgtB⁻/pagL⁺ LPS demonstrated that these minor species potentiate but do not determine the activity of this LPS. These results are the first to indicate a qualitatively different response of human innate cells to pentaacyl lpxL1⁻ and pagL⁺ LPS and show the importance of detailed structure-function analysis when working with modified lipid A structures. The pagL⁺ LPS has significant potential as immune modulator in humans.