Testing of Arg-8-gonadotropin-releasing hormone-directed antisera by immunological and immunocytochemical methods for use in comparative studies

Three polyclonal antisera raised in rabbits against the mammalian molecular form of gonadotropin-releasing hormone (GnRH) were tested in enzyme-linked immunosorbent assays for crossreactivity with naturally occurring GnRHs and with GnRH analogues. Antisera were then tested immunocytochemically in order (i) to identify amino acids essential for the binding of each antiserum, and (ii) to evaluate the specificity of the immunocytochemical reaction in brain sections from various species of cyclostomes, amphibians, reptiles, and birds. Antiserum GnRH 80/1, recognizing mainly a discontinuous determinant including the NH2- and COOH-termini, crossreacts with GnRHs the molecular bending of which enables the spatial approach of both terminal amino acid residues. Antiserum GnRH 80/2, by requiring the COOH-terminus for binding and not tolerating substitutions by aromatic amino acids in the middle region of the molecule, recognizes chicken I GnRH, however, not the salmon form. The use of this antiserum is appropriate in species synthesizing the mammalian and/or the chicken I form of GnRH. GnRH antiserum 81/1 is specific mostly for mammalian GnRH. The results obtained by ELISAs are confirmed by immunocytochemical studies. A comparison between the results obtained in ELISA and in immunocytochemistry involving mammalian-, chicken I-, chicken II-, salmon-, and lamprey-directed GnRH antisera resulted in the following conclusions: (1) An antiserum recognizing the discontinuous antigen determinant including both NH2- and COOH-termini may be reactive in most vertebrate brain sections thus being appropriate for phylogenetically directed immunocytochemical studies. (2) Moreover, this discontinuous determinant seems to be immunocytochemically reactive in all parts of the neurons in the GnRH system, whereas, in some species, determinants located in the middle region of the molecule(s) tend to become reactive only during the axonal transport. (3) A crossreaction between tissue-bound antigen and antibodies recognizing the above cited discontinuous determinant indicates an appropriate bending of the molecule even in case of severe molecular differences, e.g., in lamprey form of GnRH. (4) It follows that in phylogenetic studies, an immunologically well characterized antiserum can be substituted for a species-directed antiserum.     

The use of xenoantigenic antisera for the identification of tilapiine species: comparative laboratory and field studies

Rapid and reliable identification of tilapiine taxa and strains is essential for selective breeding purposes and the conservation of natural genetic resources. There is evidence that antisera‐mediated erythrocyte agglutination assays can fit these requirements. We evaluated the applicability of agglutination tests by studying the capacity of species characteristic antisera to recognize erythrocytes from individuals of 10 natural Ghanaian populations of Oreochromis niloticus and Sarotherodon melanotheron. The vast majority of the 218 tested individuals could be identified based on antisera‐mediated erythrocyte recognition. Controls indicated the specificity of these reactions. Still, erythrocytes from 16% of all tested specimens did not respond to any antiserum (zero responders), indicating the possible existence of blood group properties in tilapias. We discuss the specificity of the antisera, the relevance of zero responses and the applicability of these tests in aquaculture and field studies.     

Testing lineage and comparative methods for inferring adaptation

The competing lineage (homology) and comparative (convergence) methods for inferring adaptation are distinguished by their falsification and verification practices, respectively. Although these are substantially different paradigms of historical inference, their theoretical and philosophical presuppositions can be tested in a rigorous and comparable manner. Several such tests were performed. Sufficient outright failures were recorded for both methods to call into question their validity as sources of increased knowledge of adaptation.     

福氏志贺群7因子血清免疫方法的探讨

为了提高福氏志贺菌免疫的群7因子的血清效价和优化免疫方法,以两种免疫原成分,按两种浓度、剂量免疫动物后定期采集血清。用凝集试验检测血清抗体效价,比较新法和传统方法免疫家兔体内的抗体水平。结果显示,传统方法免疫的家兔所产生的抗体1∶1280,而新法免疫中高剂量组可使福氏志贺群7因子血清效价达到1∶2560的水平,完全可以用于常规生产。     

Specific antisera and immunological procedures for characterization of methanogenic bacteria.

Specific antisera were raised in rabbits to 19 methanogenic bacteria representing the species available in pure culture at the present time. The antisera were characterized, labeled, and organized in a bank to serve as a source of material for preparation of antibody probes and thus provide standardized reagents for immunological analysis of methanogens. An indirect immunofluorescence procedure was standardized for optimal staining of homologous and heterologous bacterial strains. Two immunoenzymatic assays were developed: (i) a simple slide assay, useful for rapid antibody detection in small samples, antibody titrations, and disclosure of cross-reactions among methanogens, and (ii) a quantitative method. The latter is useful for quantification of antigenic relatedness. Procedural details were developed to obtain optimal bacterial preparations for use as immunogens to raise antibodies in vivo, and as antigens for antibody assay in vitro.     

Antibody to thymus myosin: its immunological characterization and use for immunocytochemical localization of myosin in vertebrate nonmuscle cells

Thymus myosin differs immunologically from smooth muscle and striated muscle myosin isoenzymes. In the enzyme linked immunosorbent assay a moderate degree of cross reaction was observed between anti-thymus myosin and myosin from chicken gizzard (about 50% of the titer of the homologous reaction). In contrast, the cross reactivity between thymus myosin and antibodies to gizzard myosin was very low (about 5%) and no significant cross reaction was observed between thymus myosin and antibodies to striated muscle myosin and vice versa (below 1%). Antibodies to thymus myosin were further distinguished from antibodies to gizzard and striated muscle myosin by their reaction with both smooth muscle and a very broad spectrum of vertebrate nonmuscle cells. Nonmuscle cells reacting with anti-thymus myosin included (1) cell types which did not display any detectable affinity for anti-gizzard myosin (e.g. lymphocytes, polymorphonuclear leucocytes, vascular endothelium, adrenal chromaffine cells) and (2) cell types which reacted with anti-gizzard myosin as well (e.g. intestinal epithelial brush border, thymic epithelial cells, liver cells and stress fibres of cultured cells). These results illustrate, that anti-thymus myosin is a potent tool for investigating the intracellular localization of myosin in most if not all vertebrate nonmuscle cells. With respect to lymphatic tissue the present findings indicate that lymphocyte maturation appears to be accompanied by an increased level of expression of myosin and filamentous actin (the latter was visualized by labelled phalloidin). On the ultrastructural level, gold labelled antibodies to thymus myosin bound preferentially to the head region of in vitro assembled thymus myosin filaments. In cultured cells (PtK1) the antibodies showed a particular affinity for stress fibre densities, and in lymphocytes the anti-myosin label (immunoperoxidase) displayed a more or less diffuse distribution which was similar to the distribution of actin filaments (identified by decoration with heavy meromyosin).     

Relatedness among coagulase-negative staphylococci: Deoxyribonucleic acid reassociation and comparative immunological studies

DNA-DNA-homology values were determined under restrictive to relaxed reassociation conditions with type strains and some additional strains of coagulase-negative staphylococci belonging to ten different species. The immunological relationship of the catalases present in the type strains of these species was also determined by applying double immunodiffusion and microcomplement fixation. The results of these studies support the previous proposal to subdivide the coagulase-negative staphylococci into at least ten separate species. However, it is evident that some of the species are more closely related than others and can form species groups. According to the results presented in this study, the coagulase-negative staphylococci can be combined into five species groups: The Staphylococcus saprophyticus group is composed of S. saprophyticus, S. xylosus and S. cohnii. The S. epidermidis group comprises S. epidermis, S. capitis and S. warneri. The S. hominis group which exhibits a significant relationship to S. epidermidis includes S. hominis and S. haemolyticus. The species group S. sciuri consists of S. sciuri ssp. sciuri and S. sciuri ssp. lentus and the species group S. simulans is presently represented by the corresponding single species.Abbreviations G+C guanosine + cytosine - SSC standard saline citrate buffer This work was supported by Deutsche Forschungsgemeinschaft     

The use of immunological methods to detect and identify bacteria in the environment

Immunological detection methods have become increasingly important in microbial ecology for the tracking of specific microorganisms and for community analysis. For a reliable application of these techniques, the monoclonal antibodies or polyclonal antisera used have to fulfill several quality criteria. Cross reactivity, cellular localization of the antigenic determinant, affinity characteristics and the expression of the antigenic determinant at environmental conditions have to be determined. Immunological methods can be used for the identification, quantification and enrichment of specific bacteria in extracts as well as for the visualization of cells in situ. The sensitivity of advanced immunological methods can be compared to PCR techniques. Using image processing of epifluorescence micrographs or confocal laser scanning microscopy, the immunofluorescence approach can now be applied to study complex environmental samples.     

Modulation of cystathionine beta-synthase activity by the Arg-51 and Arg-224 mutations

Human cystathionine beta-synthase (CBS) catalyzes a pyridoxal 5'-phosphate (PLP) dependent beta-replacement reaction to synthesize cystathionine from serine and homocysteine. The enzyme is unique in bearing not only a catalytically important PLP but also heme. In order to study a regulatory process mediated by heme, we performed mutagenesis of Arg-51 and Arg-224, which have hydrogen-bonding interactions with propionate side chains of the prosthetic group. It was found that the arginine mutations decrease CBS activity by approximately 50%. The results indicate that structural changes in the heme vicinity are transmitted to PLP existing 20 A away from heme. A possible explanation of our results is discussed on the basis of CBS structure.     

Factor Va is inactivated by activated protein C in the absence of cleavage sites at Arg-306, Arg-506, and Arg-679

Activated protein C (APC) exerts its anticoagulant activity via proteolytic degradation of the heavy chains of activated factor VIII (FVIIIa) and activated factor V (FVa). So far, three APC cleavage sites have been identified in the heavy chain of FVa: Arg-306, Arg-506, and Arg-679. To obtain more insight in the structural and functional implications of each individual cleavage, recombinant factor V (rFV) mutants were constructed in which two or three of the APC cleavage sites were mutated. After expression in COS-1 cells, rFV mutants were purified, activated with thrombin, and inactivated by APC. During this study we observed that activated rFV-GQA (rFVa-GQA), in which the arginines at positions 306, 506, and 679 were replaced by glycine, glutamine, and alanine, respectively, was still inactivated by APC. Further analysis showed that the inactivation of rFVa-GQA by APC was phospholipid-dependent and sensitive to an inhibitory monoclonal antibody against protein C. Inactivation proceeded via a rapid phase (kx1=5.4 x 10(4) M(-1) s(-1)) and a slow phase (kx2=3.2 x 10(3) M(-1) s(-1)). Analysis of the inactivation curves showed that the rapid phase yielded a reaction intermediate that retained approximately 80% of the original FVa activity, whereas the slow cleavage resulted in formation of a completely inactive reaction product. Inactivation of rFVa-GQA was accelerated by protein S, most likely via stimulation of the slow phase. Immunoblot analysis using a monoclonal antibody recognizing an epitope between Arg-306 and Arg-506 indicated that during the rapid phase of inactivation a fragment of 80 kDa was generated that resulted from cleavage at a residue very close to Arg-506. The slow phase was associated with the formation of fragments resulting from cleavage at a residue 1.5-2 kDa carboxyl-terminal to Arg-306. Our observations may explain the unexpectedly mild APC resistance associated with mutations at Arg-306 (FV HongKong and FV Cambridge) in the heavy chain of FV.     

Localization of phosphoprotein C23 in nucleoli by immunological methods

Antiserum to a major phosphorylated nucleolar protein. C23 (MW 103000, pI 5.2) from Novikoff hepatoma was produced in rabbits. By immunodiffusion analysis, the antiserum produced precipitin bands and with various crude extracts of nucleoli, but not with extranucleolar or cytosol fractions. The specificity of the antibody was assessed using acid-urea polyacrylamide gel electropherograms of acid-soluble nucleolar proteins in which the separated proteins were transferred to nitrocellulose sheets. The purified antibody reacted predominantly with protein C23 as visualized by the immunoperoxidase procedure. By the indirect immunofluorescence technique, protein C23 was localized predominantly to nucleoli of Novikoff hepatoma or normal rat liver cells. In Novikoff hepatoma cells, traces of fluorescence were seen near the inner layer of the nuclear envelope. Additional narrow regions of fluorescence extended from the nucleoli into the extranucleolar areas of some Novikoff cells. The nucleolar areas of fluorescence were smaller but brighter in the normal liver than in Novikoff hepatoma, consistent with the small size of rat liver nucleoli. These data indicate that the major location of protein C23 is the nucleolus.