Effects of amino acid substitutions outside an antigenic site on protein binding to monoclonal antibodies of predetermined specificity obtained by peptide immunization: Demonstration with region 113–120 (antigenic site 4) of myoglobin

Immunochemical cross-reactivity of protein variants has been very frequently used to map protein antigenic sites. The approach is based on the assumption that amino acid substitutions affecting the binding of a protein to its antibody, particularly when monoclonal antibodies (mAbs) are used, must be part of the antigenic site and not far from it. The assumption was investigated in this study by determining the effects of amino acid substitutions outside the antigenic site on the reactivity of six myglobin (Mb) variants with three mAbs of predetermined specificity prepared by immunization with a free synthetic peptide representing region 113–120 (antigenic site 4) of Mb. Two of the Mb variants used had no substitutions within residues 113–120 (the region to which the specificity of the mAbs is directed) and yet exhibited markedly decreased cross-reactions and binding affinities, relative to the reference antigen, sperm-whale Mb. The other three Mb variants possessed substitutions within, as well as outside, region 113–120 and showed very little cross-reactivities. The results of this study, particularly with the Mbs that have no substitutions within the indicated antigenic site, clearly show that substitutions outside the site, and which by design are not part of the site, can influence very markedly the reactivity of the protein variant with the anti-site mAbs. The approach can, therefore, lead to serious errors if used to identify residues of protein antigenic sites.     

Use of synthetic peptides for the detection and quantification of autoantibodies

Summary and conclusions The rapid progress made over the last 10 years in the identification of individual autoantigens and in the localization of the epitopes involved, has resulted in a parallel reduction in the complexity of the antigen required for the detection of autoantibodies. The ability to use synthetic peptides as antigens is a remarkable culmination of this process considering that many antigenic particles contain multiple proteins (eg. Sm consist of 8 or more individual proteins).Despite the fact that patients with SLE have a polyclonal hypergammaglobulinemia, excellent correlations between ELISAs utilizing the P2 or SmB/B synthetic peptides, ELISAs utilizing r proteins and immunoblotting were obtained [28, 38, 50]. However, false positive/non-specific binding to a P2-BSA-glutaraldehyde conjugate has been observed with serum from old MRL/lpr mice (unpublished observations). In addition, some of the results obtained in human autoimmune diseases suggest that non-specific binding may be problematic in some instances. It is difficult, at present, to know whether the higher frequencies of detection of autoantibodies to certain synthetic peptide antigens reflect increased sensitivity or decreased specificity.Synthetic peptide antigens have beeen used to detect autoantibodies in both organ specific and multisystem autoimmune diseases. In only a small number of cases have these reagents been rigorously tested for sensitivity and specificity. Despite this, synthetic peptides have been shown to be valuable for detection and quantification of autoantibodies in certain clinical situations. Undoubtedly, further progress in epitope mapping of autoantigens coupled with technological advances in protein synthesis and improved prediction of protein structure will lead to a large number of synthetic peptide antigens for research and clinical applications. It is unlikely that short synthetic peptides will substitute for native proteins in all instances since some autoantibodies show a striking preference for conformational epitopes.Abbreviations r recombinant - SLE systemic lupus erythematosus     

Complete nucleotide sequence of the mitochondrial DNA from a liverwort,Marchantia polymorpha

Libraries of cosmid and plasmid clones covering the entire region of mtDNA from the liverwortMarchantia polymorpha were constructed. These clones were used for the determination of the complete nucleotide sequence of the liverwort mtDNA totally 186,608 bp (GenBank no. M68929) and including genes for 3 species of ribosomal RNAs, 29 genes for 27 species of transfer RNAs, and 30 genes for functionally known proteins (16 ribosomal proteins, 3 subunits of cytochromec oxidase, apocytochromeb protein, 3 subunits of H⁺-ATPase, and 7 subunits of NADH ubiquinone oxidoreductase). The genome also contains 32 unidentified open reading frames. Thus the complete nucleotide sequences from both chloroplast and mitochondrial genomes have been determined in the same organism. Plasmid clones are available upon the request. Gene names are represented according to Lonsdale and Leaver (1988) with modifications recommended by Lonsdale (personal communication).     

Stoichiometry of interaction between soluble (—)-dopa melanin and enantiomers of ephedrine by NMR spectroscopy

Synthetic soluble (—)-dopa melanin was prepared in deuteriated buffer, pH 8, by autooxidation of the precursor. At 6 mM of the precursor, the incorporation was over 90%. The changes in the line width measurements of N-CH₃ protons of enantiomers of ephedrine in the soluble melanin were quantified by NMR spectroscopy. The dissociation constants of (—)-1R,2S-ephedrine, (+)-1S,2R-ephedrine, (—)-1R,2R-ψ-ephedrine, and (+)-1S,2S-ψ-ephedrine were 11.7, 4.20, 3.60, and 4.80 mM, respectively. Since the concentration of (—)-dopa was known and since the conversion of (—)-dopa to indole units of melanin was considered as 1:1, the stoichiometry of the interaction between the drug and the indole unit was calculated. Based on the dissociation constants of the enantiomers, it appears that up to four molecules of (—)-ephedrine can interact with one indole unit of the melanin, while such a ratio for other isomers appear to be 2:1. The preference by indole units of melanin is stereoselective. © 1992 Wiley-Liss, Inc.     

Conservative and variable regions of homologous snake phospholipases A2 sequences: Prediction of the taxonspecific peptides structure

Homologous amino acid sequences of phospholipases A₂ (PLA₂) of snakes belonging to the families Elapidae, Viperidae, and Colubridae were considered in order to study the conservative and variable regions location. The PLA₂ sequences were divided into two groups (taxons) according to the phylogenetic tree reconstructed from the pair similarity matrix. Results of the intergroup comparison were plotted to facilitate the identification of significant conservative and variable regions. It was shown that the results of the comparison between two phylogenetic groups of snake PLA₂ did not much depend on the number of each group representatives and did not markedly change if one of the groups was represented by the single sequence. The knowledge of the number and location of conservative and variable regions and their dependence on the phylogenetic relations between compared taxa may be used to predict a synthetic peptide structure to obtain specific antibodies against PLA₂ of one of these taxons. Such prediction is possible if there is a specific region conservative for one taxon but variable for two of them.     

Larval development ofMacrophthalmus erato De Man, 1887 (Brachyura ocypodidae)

The zoeal and megalopal stage ofMacrophthalmus erato were obtained under laboratory conditions and are described and figured. Five zoeal and one megalopal stages were reared at 25; salinity and temperature of 29°C. Their features are compared with those of known species of the genus.     

Epeiric sedimentation and sea level: synthetic ecostratigraphy

Carbonate strata from the central parts of epicontinental seas are ideal for detailed biostratigraphic study of eustatic sea level change. Using a model for epeiric seas in which carbonate accumulation rate is depth-dependent, we derive synthetic stratigraphies for sea level histories simulating post-glacial transgression and constant and sinusoidally fluctuating ridge volume increase. These sea level histories give distinctively different trends for water depth as a function of stratigraphic position in sections' bathymetric curves. In general, depth is proportional to the rate of sea level rise. Depth-dependent sedimentation leads to a time lag between sea level fluctuation and corresponding depth fluctuation which, as examples show, can approach 10⁶ years for depth fluctuations of only a few meters – a fundamental consideration in reconstructing sea level curves, time-correlating sections by their bathymetric curves, and attempting to relate bathymetric history on continents to mechanisms driving sea level change. Bathymetric curves based on gradient analysis of fossil assemblages (coenocorrelation curves) for Middle Ordovician sections in New York and the American Midwest approximate patterns for sinusoidally increasing sea level. The model's predictions are tested in an ‘artificial experiment’ that takes advantage of differential subsidence between the craton's middle and its edge to make a difference in the bathymetric histories of sections that otherwise record the same sea level history. Depth fluctuations of no more than a few meters over million year spans are potentially useful in time-correlation to within fractions of a cycle's period. The depth-dependence in sedimentation was that, above wave base, net accumulation per year was very roughly three-millionths the water depth. Association of volcanic ash layers with transitory sea level minima on the craton, and with onset of more rapid subduction in the Taconic are - continent collision zone, suggests interrelationship on a million year scale between sea level and large-scale tectonic phenomena.     

Effects of piretanide on plasma fibrinolytic activity,platelet aggregation and platelet factor-4 release in man

Piretanide, 4-phenoxy-3-(pyrrolidinyl)-5-sulphamoyl benzoic acid, apart from being an efficient diuretic, enhances endogenous plasma fibrinolytic activity after a single dose of 6 mg administered by oral route. After ingestion of the drug, acceleration of fibrinolytic acitivity became manifest within 1 h, reached its peak in 3 h and was associated with a fall in fibrinogen and diminished urokinase excretion. Piretanide did not cause lysis of fibrinin vitro. Primary platelet aggregation, induced by adenosine-diphosphate, was inhibited by piretanide. Inin vitro experiments piretanide led to effective inhibition of adenosine-diphosphate-induced platelet aggregation with complete inhibition at 5 mM concentration. Piretanide led to a highly significant decrease of platelet factor-4 release.     

Inhibition of ornithine decarboxylase induces embryonal carcinoma cell differentiation

Murine embryonal carcinoma cells can be induced to differentiate in vitro by various physical and chemical means. We report here that inhibition of ornithine decarboxylase activity with a specific enzyme-activated inhibitor, alpha-difluoromethylornithine, can induce differentiation in embryonal carcinoma cells. The differentiated phenotype can be distinguished from undifferentiated embryonal carcinoma cells by altered cellular morphology, biochemical and cell surface antigenic properties. These results suggest that alterations in the levels of cellular polyamines may play a role in embryonal carcinoma cell differentiation.