Electrophoretic Analysis of Isoenzymes in the Identification of Trypanosomatids of the Genus Phytomonas1

Five strains of trypanosomatids of the genus Phytomonas, isolated from different species of Euphorbia {Euphorbia heterophylla, E. characias, E. pinea, E. hyssopifolia) and from Manihot escutenta, were cultured and compared through the electrophoretic mobility of isoenzymes of six enzymes: aspartate aminotransferase (EC 2.6.1.1), alanine aminotransferase (EC 2.6.1.2), phosphoglucomutase (EC 2.7.5.1), glucose-6-phosphate dehydrogenase (EC 1.1.1.49), glucosephosphate isomerase (EC 5.3.1.9), and malate dehydrogenase (EC 1.1.1.40). The strains could be distinguished from one another by their respective isoenzyme profiles.     

Electrophoretic Analysis of Isoenzymes of Mycoplasma Species

The purpose of this paper is to further characterize and determine relatedness among some Mycoplasma species or serogroups of significant practical veterinary interest. Twenty-four strains were examined for the presence of 35 enzymes by horizontal starch gel electrophoresis, revealing a total of 127 different electromorphs of 30 enzymes. Inter- as well as intraspecific differences were found demonstrating the application of isoenzyme studies in classification as well as epidemiology. It is concluded that the F38 group of MacOwan and group 7 of Leach constitute 2 new species, The elevation of the 2 subspecies of M. mycoides (mycoides and capri) to species level is favoured, but it is suggested that a decision be taken internationally, considering the practical consequences of a given nomenclature. Three alternative possibilities for classification are presented. Regarding identification the results suggest that the presence or absence of maltase and ornithine transcarbamylase indicate whether an isolate is related to the agent of contagious bovine pleuropneumonia or merely a representative of either the caprine LG type of M. mycoides subsp. mycoides or the classical caprine subspecies, M. mycoides subsp. capri.     

Guidelines for the Description of New Species of Lower Trypanosomatids1

Morphological, cultural, and biochemical criteria that have been used in describing lower trypanosomatids, genera Blastocrithidia, Crithidia, Leptomonas, Herpetomonas, Rhynchoidomonas, and Phytomonas are reviewed. Kinetoplast structure, carbohydrate utilization, electrophoretic mobilities of isoenzymes, and kDNA fingerprinting are among the recommended criteria for species differentiation. Temperature, pH, and osmolarity tolerance are useful growth parameters. Generic placement may be assisted by the determination of nitrogenous excretion products and ornithine-arginine cycle enzymes.     

Electrophoretic analysis of endonuclease-generated fragments of k-DNA, of esterase isoenzymes, and of surface proteins as aids for species identification of insect trypanosomatids

In order to verify the applicability of biochemical methods for species identification of Trypanosomatidae, 13 species of monoxenic trypanosomatids plus the heteroxenous Trypanosoma cruzi were comparatively analyzed by three different biochemical methods. Insect trypanosomatids examined were: Crithidia acanthocephali, C. fasciculata (three varieties), C. luciliae luciliae, C. luciliae thermophila, C. deanei, C. oncopelti, Herpetomonas muscarum muscarum, H. megaseliae, H. samuelpessoai, H. mariadeanei, Leptomonas seymouri, L. collosoma, L. samueli, and Blastocrithidia culicis. Also included in the survey were aposymbiotic strains of C. deanei and C. oncopelti. Methods used were: electrophoretic profiling of endonuclease-generated fragments of k-DNA, esterase isoenzymes profiling, and polyacrylamide-gel electrophoresis (SDS-PAGE) of radioiodinated cell surface proteins. Interspecific but not intraspecific differences were detected by all three methods among the 13 monoxenic species examined. Thus, it is concluded that these methods can be successfully used, in addition to classical criteria, for species identification of insect trypanosomatids.     

Ultrastructural Differences Between Species of Trypanosomatids With and Without Endosymbionts1

Species of trypanosomatids without endosymbionts (Leptomonas seymouri, L. collosoma, L. samueli, Crithidia fasciculata, C. luciliae, C. acanthocephali, Herpetomonas megaseliae, H. mariadeanei, H. samuelpessoai, H. muscarum muscarum, Trypanosoma cruzi) and species of trypanosomatids with endosymbionts (Crithidia deanei, C. oncopelti, Blastocrithidia culicis) were comparatively studied by means of electron microscopy. Artificially aposymbiotic strains derived from species with symbiont were also included in the survey. Species with symbiont were found to differ in some ultrastructural aspects from the group of species without symbiont. Paraxial rods of flagella or intraflagellar structure were found exclusively in species without symbiont. Peripheral branching of mitochondria, accompanied by absence of subpellicular microtubules in sites where the mitochondrial branches are appressed to the cell membrane, were found exclusively in species with symbiont. Networks of kinetoplast DNA fibrils were found to be larger and looser in species with symbiont. Symbiont-free strains of species with symbiont retained the same morphological characteristics of their parental species.     

Esterase Isoenzymes as Markers for the VA 1 Gene of Zea mays and for the B Linkage Group of Tripsacum dactyloides

It is suggested that cathodal esterase isoenzyme (E₁) might be used as a marker for the VA 1 gene on 7 S maize chromosome and for the B linkage group of Tripsacum dactyloides in Zea mays L. ×T. dactyloides L. hybrids. The latter genic zones have a regulatory effect on fertility and on the apomictic mode of reproduction.     

Comparative Electrophoretic Analyses of Soluble Proteins from Heterodera glycines Races 1-4 and Three Other Heterodera Species

Modified polyacrylamide gel and SDS-polyacrylamide gel electrophoretic systems using a low molarity tris-HCl buffer and equal pH of homogenizing buffer and stacking gel provided improved stacking for separation of soluble proteins from Heterodera schachtii, H. trifolii, H. lespedezae, and H. glycines races 1, 2, 3, and 4, compared with previous studies with cyst nematodes, The four Heterodera species were easily distinguished using the polyacrylamide gel system, but H. trifolii and H. lespedezae had similar protein patterns. H. glycines races were not separable by that system. The SDS-polyacrylamide gel system produced different protein patterns for all four Heterodera species although H. trifolii and H. lespedezae differed by only a single band, suggesting that these two may be subspecifically related. A protein band unique to H. glycines races 3 and 4 was not detected in SDS-polyacrylamide gel profiles from races 1 and 2. Molecular weight determinations were 55,000 for distinctive proteins in profiles of H. trifolii and 75,000 for H. glycines races 3 and 4.     

Comparative Electrophoretic Studies of the Seed Proteins of Certain Species of Brassica and Sinapis

The saline-soluble seed proteins of Brassica campestris var.autumnalis, B. nigra, B. oleracea, B. campestris var. rapa andSinapis alba have been investigated using serological methodsand detection of enzymes after electrophoresis in starch gels.These methods were used to determine the generic status of Sinapisalba and the varietal or specific status of B. campestris var.rapa. The results of protein analysis agree with establishedtaxonomy and suggest that both methods help to resolve sometaxonomic problems.     

Synthetic Peptide Fragments as Probes for Structure Determination of Potassium Ion-Channel Proteins

Potassium channels are a diverse class of transmembrane proteins that are responsible for diffusion of potassium ion across cell membranes. The lack of large quantities of these proteins from natural sources, is a major hindrance in their structural characterization using biophysical techniques. Synthetic peptide fragments corresponding to functionally important domains of these proteins provide an attractive approach towards characterizing the structural organization of these ion-channels. Conformational properties of peptides from three different potassium channels (Shaker, ROMK1 and minK) have been characterized in aqueous media, organic solvents and in phospholipid membranes. Techniques used for these studies include FTIR, CD and 2D-NMR spectroscopy. FTIR spectroscopy has been a particularly valuable tool for characterizing the folding of the ion-channel peptides in phospholipid membranes; the three different types of potassium channels all share a common transmembrane folding pattern that is composed of a predominantly -helical structure. There is no evidence to suggest the presence of any significant -sheet structure. These results are in excellent agreement with the crystal structure of a bacterial potassium channel (Doyle, D. A. et al. (1998) Science 280:69–77), and suggest that all potassium channel proteins may share a common folding motif where the ion-channel structure is constructed entirely from -helices.     

A Method for the Electrophoretic Analysis of Proteins and RNAs of Single Cells*

SYNOPSIS A method is described for the electrophoretic analysis of proteins or RNAs from individual amebae. The method is based on fluorographic autoradiography of semi-micro polyacrylamide gels in which [³⁵S]methionine or [³H]uridine materials from single cells have been subjected to electrophoresis. The method is more sensitive and provides better resolution than previous methods for single cells. It is suitable, also, for quantitation of the separated components.     

藏北3种裸鲤同工酶的电泳分析及物种分化的探讨

对藏北高原３种裸鲤的乳酸脱氢酶（ＬＤＨ）、苹果酸脱氢酶（ＭＤＨ）和脂酶（ＥＳＴ）进行电泳分析的结果表明,３种裸鲤酶谱均表现出种间的差别,而且在同一种群个体之间也存在着明显的分化,但无性别差异。３种裸鲤被检测的３种同工酶均有沉默基因表达的现象,重复基因ＬＤＨ－Ａ^2、ＬＤＨ－Ｂ^2、s-MDH-A^2和m-MDH-B^2也在部分个体中表达。遗传距离分析表明,色林错裸鲤（Ｇ.selincuoensis)与错鄂裸鲤（Ｇ．cuoensis)之间较之于与纳木错裸鲤（Ｇ．namensis)有更近的亲缘关系。与其他四倍体鱼类相比,裸鲤鱼类同工酶在重复基因和沉默基因上都有较高的表达频率,这种情况说明裸鲤鱼类目前可能还外于多倍化后进化的早期过程并早于胭脂鱼类所处的相应时期,这与裂腹鱼类起源较晚以及青藏高原业已存在的恶劣环境条件直接相关。     

Screening of Lysine-rich Plant Species and Identification of the Purified Proteins

The nutritional quality of seed proteins from cereals, such as wheat and rice, is comparatively low due to its deficency in lysine and some essential amino acids. In this research extensive varieties of plant seed samples were collected and screened by analysis of amino acid composition. Three lysing-rich species which contain more than 6.7% of lysine in total seed proteins were found. 31 kinds of proteins were purified from a species which contains 7.9% of lysine using the modified methods of IEF and SDS electrophoresis. One protein with PI 6.1 and 18 kD was identified which contains 11.4% of lysine and was rich in threonine, valine and isoleucine. This is the first example of the protein which could complement several limiting amino acids of wheat or rice. Further research on the structural gene encoding this protein would have great potential value for improvement of protein quality of these cereals.     

离子束介导大豆DNA导入小麦后其蛋白质和酯酶同工酶分析

对由离子束介导大豆DNA导入豫麦52获得的第四代两个转化株系9004和9002中的可育株籽粒和矮秆不育株杂交籽粒以及受体豫麦52籽粒,进行酯酶和可溶性蛋白的聚丙烯酰胺凝胶电泳分析。结果表明,可育株籽粒可溶性蛋白电泳在谱带上与受体存在差异,矮秆不育株材料的酯酶和可溶性蛋白电泳图谱谱带差异更明显。由此推测,外源大豆DNA导入受体后某些片段有可能插入受体基因组,从而导致基因突变或受体基因表达发生改变。     

Spectrum Analysis of Esterase Isozymes of culti-species and wild Species in Arachis

The electrophoresis analysis of isozymes of Arachis show a very close relationship among four types of cultispecies (A. hypogaea) and A. monticola, the tetraploid wild species is a related species. Among the five diploid wild species of Arachis Section, both A. cardenasii with A genome and A. batizocoi with B genome are found to be relatively nearer to cultis'pecies than A. correntina, A. stenosperma and A. villosa, while A. rigonii of Erectoides Section and A. pusilla of Triseminala Section are the distant species.     

Otolith Shape Analysis as a Tool for Species Identification and Studying the Population Structure of Different Fish Species

An analysis and a comparison of the methods of geometric morphometrics as applied to fish species identification and to studies on the population structure of fish stocks based on peculiarities of the otolith shape are performed. A review of the geometric morphometric methods used in studies on fish otoliths is provided. The results of our own research on possible utilization of elliptical Fourier analysis for species identification are also described.     

Electrophoretic Mobility Patterns of Ribosomal Proteins as an Aid to Phenoset Classification of Amicronucleate Strains of Tetrahymena1

Comparison of the electrophoretic migration patterns of proteins of active 40S and 60S ribosomal subunits isolated from nine amicronucleate strains of Tetrahymena of known phenoset revealed strain dependent differences which correlated with the phenoset classification of these strains as determined by Borden, Whitt & Nanney, who compared isoenzyme patterns.     

Identification of Peptidoglycan-Associated Proteins as Vaccine Candidates for Enterococcal Infections

Infections by opportunistic bacteria have significant contributions to morbidity and mortality of hospitalized patients and also lead to high expenses in healthcare. In this setting, one of the major clinical problems is caused by Gram-positive bacteria such as enterococci and staphylococci. In this study we extract, purify, identify and characterize immunogenic surface-exposed proteins present in the vancomycin resistant enterococci (VRE) strain Enterococcus faecium E155 using three different extraction methods: trypsin shaving, biotinylation and elution at high pH. Proteomic profiling was carried out by gel-free and gel-nanoLC-MS/MS analyses. The total proteins found with each method were 390 by the trypsin shaving, 329 by the elution at high pH, and 45 using biotinylation. An exclusively extracytoplasmic localization was predicted in 39 (10%) by trypsin shaving, in 47 (15%) by elution at high pH, and 27 (63%) by biotinylation. Comparison between the three extraction methods by Venn diagram and subcellular localization predictors (CELLO v.2.5 and Gpos-mPLoc) allowed us to identify six proteins that are most likely surface-exposed: the SCP-like extracellular protein, a low affinity penicillin-binding protein 5 (PBP5), a basic membrane lipoprotein, a peptidoglycan-binding protein LysM (LysM), a D-alanyl-D-alanine carboxypeptidase (DdcP) and the peptidyl-prolyl cis-trans isomerase (PpiC). Due to their close relationship with the peptidoglycan, we chose PBP5, LysM, DdcP and PpiC to test their potential as vaccine candidates. These putative surface-exposed proteins were overexpressed in Escherichia coli and purified. Rabbit polyclonal antibodies raised against the purified proteins were able to induce specific opsonic antibodies that mediated killing of the homologous strain E. faecium E155 as well as clinical strains E. faecium E1162, Enterococcus faecalis 12030, type 2 and type 5. Passive immunization with rabbit antibodies raised against these proteins reduced significantly the colony counts of E. faecium E155 in mice, indicating the effectiveness of these surface-related proteins as promising vaccine candidates to target different enterococcal pathogens.     

Immunological Characterization of Honey Proteins and Identification of MRJP 1 as an IgE-Binding Protein

We encountered a fourth case of honey allergy in Japan. We characterized and identified the IgE-binding proteins in honey using the serum of a honey-allergenic patient. Immunoblot analysis revealed that IgE in the patient serum specifically bound to four proteins in each honey sample. At least three of these IgE-binding proteins were N-linked glycoproteins. To identify the 60-kDa IgE-binding protein in dandelion honey, the N-terminal sequences of the fragmented protein were analyzed, revealing the protein to be major royal jelly protein 1 (MRJP 1). Three IgE-binding proteins removed of N-linked oligosaccharide showed a large reduction in IgE-binding activity as compared with the intact protein. This suggests that the carbohydrates in the IgE-binding proteins are a major epitope for patient IgE.