An immunochemical testing of pathophysiological reactions of several PSTVinfected tomato (Lycopersicon esculentum L.) cultivars

Several tomato cultivars were infected with a severe strain of PSTV and a pathophysiological reaction was characterized by means of enzyme-linked immunosorbent assay with serum containing IgGs to disease-associated host-specific leaf proteins. A strong expression of disease-associated symptoms (stunting, epinasty, leaf blade malformation and rugosity) and strong immunochemical reaction was found for cultivars Bizon, Linia, Revermun and Rutgers. The immunochemical assay revealed appearence of a major antigenic protein having a molecular mass of about 70 kD in these cultivars. The immunochemical reaction with disease-associated proteins reached a maximum four weeks after inoculation of PSTV. A weak immunochemical reaction was observed, if proteins from cvs. Sonato, Harzfeuer and Karlik were analyzed. Cvs. Sonato and Harzfeuer did not show characteristic symptoms such as stunting, leaf blade malformation and rugosity. Except for stunting, the same was true for cv. Karlik. On the other hand, no significant difference in PSTV accumulation was observed among the cultivars analyzed. Reciprocal hybrids obtained by crossing cvs. Revermun and Karlik showed strong symptoms of the disease (Revermun-type) and increased activity of nuclease due to PSTV infection. On the contrary, an immunochemical analysis revealed a low level of the disease-associated antigens in these hybrid tomatoes, suggesting rather recessive genetic background determining their expression during PSTV-caused pathogenesis.     

Immunochemical studies of the plasma and cultured fibroblast media fractions containing the cystic fibrosis ciliary inhibitor.

The cystic fibrosis ciliary inhibitor (CFCI) has been fractionated from plasma of cystic fibrosis (CF) homozygotes and from the media of cultured fibroblasts derived from CF homozygotes. Plasma and fibroblast media from normal controls have been fractionated in an identical manner. Fractions from plasma and fibroblast culture media that demonstrate ciliary inhibitory activity contain several proteins in a molecular weight range of approximately 5,000-11,000. These proteins have been partially characterized by immunochemical analysis with antisera to 33 human serum proteins. Immunological determinants of albumin, C3 (but not C3a), C4, C5, alpha1-lipoprotein, beta-lipoprotein, beta2-microglobulin and immunoglobulin light chains have been detected by hemagglutination in fractions of CF plasma that inhibited ciliary activity and in analogous fractions from normal sera. None of the proteins were detected in media of cultured fibroblasts from either genotype. Since the same proteins and protein fragments were identified in both CF and normal plasma fractions, and were not detected in CF fibroblast media, it appears that none of these proteins can be identified as the CFCI. Identification of these proteins will permit further purification of the CFCI by immunochemical methods.     

Variability of some seed proteins of the speciesPhaseolus vulgaris and their relationship to phytohaemagglutinating activity

The variability of some seed proteins ofPhaseolus vulgaris was followed in individual seeds of 26cultivars of this species. Polymorphism was established with two proteins showing completely different immunochemical specificities which have previously been classified as the protein I and the protein II of the specificity Veltruská Saxa and Krupnaya sakhamaya respectively. Various combinations of these proteins occur in several cultivars. It was further found that the protein II of the specificity Veltruská Saxa had phytohaemagglutinating activity.     

Conditions that allow for effective transfer of membrane proteins onto nitrocellulose membrane in Western blots

A major hurdle in characterizing bacterial membrane proteins by Western blotting is the ineffectiveness of transferring these proteins from sodium dodecyl sulfate -- polyacrylamide gel electrophoresis (SDS-PAGE) gel onto nitrocellulose membrane, using standard Western blot buffers and electrophoretic conditions. In this study, we compared a number of modified Western blotting buffers and arrived at a composition designated as the SDS-PAGE-Urea Lysis buffer. The use of this buffer and specific conditions allowed the reproducible transfer of highly hydrophobic bacterial membrane proteins with 2-12 transmembrane-spanning segments as well as soluble proteins onto nitrocellulose membranes. This method should be broadly applicable for immunochemical studies of other membrane proteins.     

Cellular origin of prealbumin in the rat

The synthetic rate of prealbumin and albumin in primary monolayer cultures of rat hepatocytes was measured by immunochemical methods. The isolated hepatocytes synthesized these proteins in the same ratio as that previously found for the whole body synthesis in vivo. It is concluded that the hepatocytes synthesize the main part of prealbumin in the rat.     

A detergent-induced charge shift as a prerequisite for the electrochemical analysis of the adenine nucleotide translocator, a basic membrane protein

The adenine nucleotide translocator is a hydrophobic, basic protein of the inner mitochondrial membrane which is solubilized by the non-ionic detergent Triton X-100. For immunochemical characterization of this membrane-protein by crossed immunoelectrophoresis a charge shift of the protein-Triton X-100 micelle by the introduction of an ionic detergent (deoxycholate) was necessary as a prerequisite to avoid unspecific precipitation of the protein. Beside the charge shift of the protein-detergent micelle, the selection, concentration and ratio of the detergents used and the choice of the agarose with different degrees of electroendosmosis should be carefully considered. The principle derived from these results provides a new methodological possibility for the immunochemical characterization of hydrophobic, basic membrane proteins.     

Immunological polyfunctionality of proteins and its meaning for the analysis of an allergen-active bacterial substrate]

It was shown with the aid of immunosorption of an allergen-active substrate of E. coli 020: K84 (No. 2-rII) that protein substances taking part in the phenomenon of cell hypersensitivity were active in the humoral immunity reactions. The allergenic and immunochemical activity served as functions of the same molecules of bacterial proteins, this substantiating the use of immunochemical analysis for the study of an allergen-active bacterial substrate. By protein denaturing it is possible to obtain immunochemical inert allergen-active preparations capable of detecting the cell hypersensitivity to crude bacterial proteins. The problem of immunological polyfunctionality of proteins is discussed from the aspect of nonhomogeneity of their antigenic determinant groups.     

Application of the Serological Method for Evaluation of Relations between Gymnospermous and Dicotyledonous Plants

An attempt has been undertaken to evaluate interrelations of gymnospermous and dicotyledonous plants on the basis of immunochemical studies of seed proteins. For this purpose, 12 antisera were raised to proteins of taxa representing four gymnosperm classes: Ginkgoopsida, Cycadopsida, Coniferopsida, and Gnetopsida. Seed proteins of eight dicotyledonous subclasses (after Takhtadzhyan, 1987) were used. The representatives of all dicotyledonous subclasses gave immunochemical reactions with those of all gymnospermous classes. The data obtained suggest the presence of sufficiently close immunochemical relations between gymnosperms and dicotyledons. Samples were found among the representatives of subclasses Dilleniidae, Hamamelididae, and Rosidae, which gave satisfactory reactions with eight to ten antisera to proteins of dicotyledonous seeds. Analysis of the data we obtained suggests that gymnospermous and dicotyledonous plants took their origin from a common pragymnospermous ancestor and later evolved independently or that dicotyledons separated from gymnosperms at an early stage of their evolution before divergence of the latter into several phyletic lineages.     

High mobility group proteins 1 and 2 are present in simian virus 40 provirions, but not in virions.

Viral particles at the late stages of SV40 morphogenesis were examined for the presence of HMG proteins 1 and 2, by an immunochemical method involving the transfer of proteins from polyacrylamide gels to nitrocellulose membranes. It was found that these proteins are present in SV40 provirions, in which histone H1 is still associated with viral chromatin, but absent in mature SV40 virions.     

Proteomic identification of oxidatively modified proteins in Alzheimer's disease brain. Part I: creatine kinase BB,glutamine synthase,and ubiquitin carboxy-terminal hydrolase L-1

Oxidative alterations of proteins by reactive oxygen species (ROS) have been implicated in the progression of aging and age-related neurodegenerative disorders such as Alzheimer's disease (AD). Protein carbonyls, a marker of protein oxidation, are increased in AD brain, indicating that oxidative modification of proteins is relevant in AD. Oxidative damage can lead to several events such as loss in specific protein function, abnormal protein clearance, depletion of the cellular redox-balance and interference with the cell cycle, and, ultimately, to neuronal death. Identification of specific targets of protein oxidation represents a crucial step in establishing a relationship between oxidative modification and neuronal death in AD, and was partially achieved previously in our laboratory through immunochemical detection of creatine kinase BB and beta-actin as specifically oxidized proteins in AD brain versus control brain. However, this process is laborious, requires the availability of specific antibodies, and, most importantly, requires a reasonable guess as to the identity of the protein in the first place. In this study, we present the first proteomics approach to identify specifically oxidized proteins in AD, by coupling 2D fingerprinting with immunological detection of carbonyls and identification of proteins by mass spectrometry. The powerful techniques, emerging from application of proteomics to neurodegenerative disease, reveal the presence of specific targets of protein oxidation in Alzheimer's disease (AD) brain: creatine kinase BB, glutamine synthase, and ubiquitin carboxy-terminal hydrolase L-1. These results are discussed with reference to potential involvement of these oxidatively modified proteins in neurodegeneration in AD brain. Proteomics offers a rapid means of identifying oxidatively modified proteins in aging and age-related neurodegenerative disorders without the limitations of the immunochemical detection method.     

Distinctive cationic proteins of the human eosinophil granule: major basic protein, eosinophil cationic protein, and eosinophil-derived neurotoxin

The human eosinophil granule contains a number of cationic proteins that have been identified and purified to homogeneity, including the major basic protein (MBP), the eosinophil cationic protein (ECP), and the eosinophil-derived neurotoxin (EDN). Because of confusion in the literature regarding the distinctiveness of MBP and ECP, we investigated the immunochemical and physicochemical properties of these purified proteins by electrophoresis on sodium dodecyl sulfate-polyacrylamide gels (SDS-PAGE), by specific double antibody radioimmunoassays (RIA) for MBP and ECP, and by fractionation of acid-solubilized eosinophil granules on Sephadex G-50 columns. Analysis of a mixture of the three purified proteins by SDS-PAGE showed that they migrated as three distinct bands with differing m.w. Comparison by specific RIA for MBP and ECP did not demonstrate any appreciable immunochemical cross-reactivities among the three proteins. Sephadex G-50 column fractions of acid-solubilized eosinophil granules were analyzed by RIA and by SDS-PAGE analysis of individual column fractions. MBP, ECP, and EDN eluted at different volumes from Sephadex G-50 columns as determined by RIA and SDS-PAGE. Soluble extracts of eosinophil granules from patients with the hypereosinophilic syndrome contained between six and 64 times more MBP than ECP on a weight basis. These observations demonstrate that MBP, ECP, and EDN are distinctive cationic proteins of the human eosinophil granule and that eosinophil granules from patients with eosinophilia contain considerably greater quantities of MBP than ECP.     

Relationships between tobacco mosaic virus and the non-virus proteins

1. The non-virus proteins, A4, B3, and B6, characteristically found in tobacco leaf infected with TMV exhibit specific immunochemical cross-reactions with serum prepared against the virus. The close immunochemical relations which occur among these proteins do not extend to any normal tobacco leaf proteins. 2. The rate of appearance of the non-virus proteins in newly infected cultured leaf tissue at various times after inoculation has been determined by immunochemical techniques and by direct isolation of the proteins. Both methods give comparable results. The non-virus proteins appear abruptly at about 220 hours after inoculation, when the TMV content is about one-third its final value. The amount of A4 rises rapidly and then levels off. The B6 content rises rapidly and continuously over the course of the experiment. B3 appears last, and increases in amount considerably more slowly than A4 and B6. 3. The isotope contents of TMV, B3, and B6 which appear in given intervals after inoculation in newly infected leaf cultured in nutrient containing N¹⁵H₄ have been compared. The isotope levels of concurrent TMV, B3, and B6 are identical within the experimental error. The isotope conditions employed in this experiment lead to the conclusion that this coincidence of N¹⁵ levels means that the virus and non-virus proteins are probably synthesized at about the same time from the same non-protein source of nitrogen. 4. Possible interpretations of the available data on the non-virus proteins are discussed. It is likely that one or more of these proteins represents small protein units which occur in the TMV nucleoprotein.As they exist in the infected leaf, the non-virus proteins are probably no longer available to the biochemical processes which lead to TMV synthesis. They are probably not precursors of TMV protein in a temporal sense.     

Immunological relations of proteins from four salmonellae sharing an "O" factor 8.

The proteins from S. virginia, with the sole 8 "O" factor, precipitated against homologous and related heterologous sera by a conspicuous, homogenous line of serological identity, with proteins from: S. newport (6.8), S. blockley (6.8), S. emek (8.20). The proteins, however, were not involved in the bacterial agglutinations since the absorptions, which removed the common precipitins from the sera, did not modify the homologous agglutinations. The results with anti-S. newport and anti-S. blockley sera sharing identical "O" factors while displaying different immunochemical compositions as well as the strong immunochemical relations found between S. virginia and S. newport belonging to the different subgroups C2 and C3 underline the non-relatedness of agglutinins and precipitins.     

Selection of cell wall antigens for the rapid detection of bacteria by immunological methods.

Some proteins extracted from the cell wall of Gram-positive and Gram-negative bacteria that affect the time for which meat can be stored are antigens produced by the strains from our laboratory collection or from field samples. Moreover, the rapid detection of bacteria (within 8 h for Gram-positive or 5 h for Gram-negative) that influence the quality of meat is made possible by immunochemical techniques such as ELISA or by introducing simultaneous detection of these antigens, as no cross-reactions have been observed.     

Assessment of snake antivenom purity by comparing physicochemical and immunochemical methods

Purity is a characteristic that, together with effectiveness and safety, must be tested to determine the quality of biopharmaceutical products. In therapeutic immunoglobulins, such as human intravenous immunoglobulin (IVIG), purity is evaluated on the basis of physicochemical properties, and is usually assessed by chromatography and electrophoresis. However, in the case of antivenoms these methods fail to discriminate between antibodies towards venom antigens, which constitute the active substance, and antibodies towards non-venom antigens, which are the major impurities in most of the current formulations. The assessment of this aspect of purity requires the use of the immunochemical methods. In this study, it was demonstrated that antivenoms showing physicochemical purity higher than 90% might present immunochemical purity lower than 40%. It is proposed that a comprehensive analysis of antivenom purity should combine physicochemical and immunochemical parameters. In addition, these results are crucial to decide the more appropriate strategies to improve antivenom purity. Taking into account that the current methods of antivenom purification remove most non-antibodies proteins, we propose that efforts must be primarily directed to the improvement of immunization protocols to enhance the antibody response towards venom components in hyperimmunized animals, and secondarily, in the realm of immunoglobulin purification technology.     

Phylogenetic conservation of antigenic determinants in archaebacterial elongation factors (Tu proteins)

By using affinity chromatography methods, we have purified elongation factor Tu (EF-Tu) proteins from a host of archaebacteria covering all known divisions in the archaebacterial tree except halophiles, and from such distantly related eubacteria as Thermotoga maritima and Escherichia coli. Polyclonal antibodies were raised against the Tu proteins of Sulfolobus solfataricus, Thermoproteus tenax, Thermococcus celer, Pyrococcus wosei, Archaeoglobus fulgidus, Methanococcus thermolitotrophicus, Thermoplasma acidophilum, and Thermotoga and used to probe the immunochemical relatedness of elongation factors both within and across kingdom boundaries. A selection of the results, presented here, indicates that (i) every archaebacterial EF-Tu is closer (immunochemically) to every other archaebacterial EF-Tu than to the functionally analogous proteins of eubacteria and eukaryotes, with only one possible exception concerning the recognition of eukaryotic (EF-1 alpha) factors by Thermococcus EF-Tu antibodies, and (ii) within the archaebacteria there appears to be a correlation between EF-Tu immunochemical similarities and the phylogenetic relatedness of the organisms inferred from other (sequence) criteria. On the whole, immunochemical similarity data argue against the proposal that the archaebacterial taxon should be split and redistributed between two superkingdoms.     

Comparison of the structures of L-glutamate decarboxylases from human and rat brains

Human and rat L-glutamate decarboxylases have been purified to electrophoretic homogeneity. These two enzymes were compared using an immunochemical method, amino acid analysis and tryptic fingerprinting. Structural studies revealed several differences in the primary structure of the two enzymes, but the immunochemical method used did not distinguish between the antigenicity of the two proteins.     

Composition and immunochemical properties of the cell surface proteins of Vibrio cholerae

The composition and immunochemical properties of cell surface proteins of Vibrio cholerae belonging to both the biotypes (classical and El Tor) and the serotypes (Ogawa and Inaba) were investigated. Proteins were isolated by extraction with EDTA/NaCl. When the extract was further treated with sodium deoxycholate, a product significantly enriched with the major protein was obtained. The surface localization of these proteins was confirmed by immunoelectron microscopy using protein A-colloidal gold particles as probes. Antisera to these proteins possessed complement-mediated bactericidal activities towards V. cholerae strains belonging to both the biotypes and the serotypes, and upon crossed immunoelectrophoresis produced several immunoprecipitation reactions towards whole-cell sonicates belonging to all types of V. cholerae. These proteins were immunogenic in the rabbit intestine, as antibodies of two classes (IgG and IgA) were detected in the intestinal fluids. The intestinal immune response was greatly enhanced when cell surface proteins were administered with liposomes. These results suggest that cell surface proteins represent common antigens of V. cholerae and can be explored as vaccine candidates against cholera.     

Conservation of antigenic determinants among different seed lectins.

Immunochemical techniques were employed to examine seed lectins for structural similarities. Antisera raised against eight homogeneous lectins were used to test for cross-reacting material in crude seed extracts as well as highly purified lectins. The data provide immunochemical evidence that lectins isolated from different species may be structurally related proteins. As structurally related proteins, the cross-reacting lectins may also possess a similar function. In addition, antisera raised against Ulex agglutinin I or Bandeiraea agglutinin, appeared to recognize an identical set of determinants on those lectins showing cross-reactivity. This highly conserved region of the lectin molecule may be important for the proper functioning of these proteins.