... WITH A VIBRIO ANGUILLARUM BACTERIN: INDICATIONS FOR A COMMON MUCOSAL IMMUNE SYSTEM Jan WHM Rombout, Leen J. Blok, Cor HJ Lamers, Egbert Egberts Department of Experimental Animal Morphology and Cell Biology, Agricultural University, PO... more
... WITH A VIBRIO ANGUILLARUM BACTERIN: INDICATIONS FOR A COMMON MUCOSAL IMMUNE SYSTEM Jan WHM Rombout, Leen J. Blok, Cor HJ Lamers, Egbert Egberts Department of Experimental Animal Morphology and Cell Biology, Agricultural University, PO Box 338 ...
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Research Interests: Immunology, Immunogenetics, Protein Structure and Function, Phylogeny, Sequence alignment, and 15 moreAnimals, Polymerase Chain Reaction, Atlantic Salmon, Genome Organization, Genomic DNA, Major histocompatibility complex, Genetic Polymorphism, Carps, Amino Acid Sequence, Base Sequence, Recombinant Protein, Cyprinus Carpio, Fusion Protein, Full Length Movies, and Molecular Sequence Data
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Major histocompatibility complex (MHC) class II protein polymorphism is maintained in allelic lineages which evolve in a trans-specific manner, passing from one species to descendant species. Selection pressure on peptide binding residues... more
Major histocompatibility complex (MHC) class II protein polymorphism is maintained in allelic lineages which evolve in a trans-specific manner, passing from one species to descendant species. Selection pressure on peptide binding residues should be greatest during speciation, when organisms move into new environments and their MHC molecules encounter new pathogens. The isolation ofMHC genes from teleost fishes, the most diverse
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Major histocompatibility complex (MHC) class II protein polymorphism is maintained in allelic lineages which evolve in a trans-specific manner, passing from one species to descendant species. Selection pressure on peptide binding residues... more
Major histocompatibility complex (MHC) class II protein polymorphism is maintained in allelic lineages which evolve in a trans-specific manner, passing from one species to descendant species. Selection pressure on peptide binding residues should be greatest during speciation, when organisms move into new environments and their MHC molecules encounter new pathogens. The isolation ofMHC genes from teleost fishes, the most diverse group of vertebrates, has created possibilities for testing this hypothesis. The large barbels of Lake Tana have undergone an adaptive radiation within the last 5 million years, producing 14 morphotypes which inhabit different ecological niches within the lake. We studied the variability in class II beta chain-encoding genes of four of these morphotypes using polymerase chain reaction amplification and DNA sequencing. The sequences obtained were orthologous to four of the known class II genes from the common carp, from which barbels diverged approximately 32 million years ago. When subjected to phylogenetic analysis, the 48 sequences clustered into groups which represent allelic lineages. A comparison of nonsynonymous and synonymous substitutions between the peptide binding region codons and non-peptide binding region codons of these sequences revealed that they are under strong selective pressure.
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Antibody affinity is of major significance in immunoassays. Since affinity may be influenced by the immunoassay methodology it is important to determine this parameter under the conditions of the assay used. Here a method is described for... more
Antibody affinity is of major significance in immunoassays. Since affinity may be influenced by the immunoassay methodology it is important to determine this parameter under the conditions of the assay used. Here a method is described for the determination of binding constants (K) in a direct ELISA with the use of the computer program LIGAND. Five of the antibodies studied bound to their antigen with two classes of antigen binding site, while all the other antibodies studied reacted with only a single class of antigen binding site. The accuracy of the method and the implications for antigen-antibody reactions are discussed.
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We investigated the ribonucleolytic breakdown of poly(U), poly(A), RNA trascribed from calf thymus DNA with E. coli RNA polymerase, ribosomal RNA, tRNA and mengovirus RNA by an enzyme fraction obrained from a postribosomal supernatant of... more
We investigated the ribonucleolytic breakdown of poly(U), poly(A), RNA trascribed from calf thymus DNA with E. coli RNA polymerase, ribosomal RNA, tRNA and mengovirus RNA by an enzyme fraction obrained from a postribosomal supernatant of Ehrlich ascites tumor cells. The single-stranded homopolyribonucleotides are preferentially degraded by the enzyme fraction with the production of ribonucleoside 5'-monophosphates. The RNase activity is completely dependent on the presence of Mg2+ ions and is highest at Mg2+ and K+ concentrations optimal for cell-free protein synthesis. Ribonucleoside 5'-monophosphates, ribonucleoside 2'(3')-monophosphates, ribonucleoside 2'(3'),5'-bisphosphates and transition state analogs consisting of vanadyl sulfate and either ribonucleosides or ribonucleoside 5'-monophosphates in a molar ratio 1:1 inhibit the ribonucleolytic activity of the enzyme fraction. The ribonucleoside 2'(3'),5'-bisphosphates and the transition state analogs are the most effective inhibitors. However, only in the presence of ribonucleoside 2'(3'),5'-bisphosphates a concomitant stimulation by 50 to 60% of poly(U)-directed polyphenylalanine synthesis is observed; all the other RNase inhibitors tested also inhibit polypeptide synthesis. The results of preliminary experiments show that poly(U) and ribonucleoside 2'(3'),5'-bisphosphates are well suited as ligands for affinity chromatography of ribonucleases from Ehrlich ascites tumor cells.
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The effect of mengovirus infection on the protein synthetic capacity of Ehrlich ascites tumor cells cultured in vitro was studied in vivo and in vitro employing postnuclear supernatants prepared at various times post-infection in the... more
The effect of mengovirus infection on the protein synthetic capacity of Ehrlich ascites tumor cells cultured in vitro was studied in vivo and in vitro employing postnuclear supernatants prepared at various times post-infection in the absence and in the presence of 1% Triton X-100. The amino acid incorporating activities of extracts obtained in the presence of the detergent were reduced by about 30% compared with the capacities of the corresponding postnuclear supernatants prepared in the absence of Triton X-100; but the course of the activity vs. time curve was not influenced by the detergent. Under the conditions employed, the postnuclear supernatants were unable to reinitiate protein synthesis once elongation of nascent polypeptide chains concomitant with ribosome runoff was completed. After mengovirus infection, a gradual disappearance of polysomes from postnuclear supernatants and a simultaneous accumulation of monosomes was observed. The protein-synthesizing activities of normal and infected cells were inversely proportional to the monosome concentrations of their corresponding extracts. Qualitatively, protein synthesis in intact cells and in postnuclear supernatants responded similarly to mengovirus infection. In both cases an initial reduction of host-specific amino acid incorporation was followed by a burst of viral protein synthesis. However, the two activity vs. time curves showed the following significant differences: 1) The activities of extracts from control cells and from mengovirus-infected cells nearly in the infectious cycle were low compared with the activities observed in vivo. 2) In the middle of the infectious cycle, the peak of viral protein synthesis occurred later and the activity was higher in vitro. 3) Finally, in the late period of the infectious cycle the postnuclear supernatants had considerable protein synthesizing activity, at a time when protein synthesis in vivo was nil.
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The histone synthesizing capacity of mengovirus-infected Ehrlich ascites tumor cells and of their corresponding postnuclear supernatants was investigated as a funcion of time post-infection. In addition, histone synthesis was compared... more
The histone synthesizing capacity of mengovirus-infected Ehrlich ascites tumor cells and of their corresponding postnuclear supernatants was investigated as a funcion of time post-infection. In addition, histone synthesis was compared with the synthesis of other basic host proteins under identical conditions. In the scope of mengovirus infection of Ehrlich ascites tumor cells the less complex fraction comprising basic protein, separated from the acidic proteins by carboxymethyl cellulose chromatography, can be regarded as a representative of total host protein. Histones and the remaining basic host proteins therefore are well suited as easily identifiable indicators of the host protein synthesizing potential of mengovirus-infected Ehrlich ascites tumor cells. The cessation of histone synthesis proceeds faster than the arrest of the synthesis of other basic host protein.
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Antibody affinity is of major significance in immunoassays. Since affinity may be influenced by the immunoassay methodology it is important to determine this parameter under the conditions of the assay used. Here a method is described for... more
Antibody affinity is of major significance in immunoassays. Since affinity may be influenced by the immunoassay methodology it is important to determine this parameter under the conditions of the assay used. Here a method is described for the determination of binding constants (K) in a direct ELISA with the use of the computer program LIGAND. Five of the antibodies studied bound to their antigen with two classes of antigen binding site, while all the other antibodies studied reacted with only a single class of antigen binding site. The accuracy of the method and the implications for antigen-antibody reactions are discussed.
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In this study, experiments are described that were designed to investigate whether fish have an immune regulatory systems similar to the major histocompatibility complex (MHC) in higher vertebrate species. From combinations of gynogenetic... more
In this study, experiments are described that were designed to investigate whether fish have an immune regulatory systems similar to the major histocompatibility complex (MHC) in higher vertebrate species. From combinations of gynogenetic carp showing either slow or fast rejection of skin transplants, the latter were chosen for alloantiserum production by hyperimmunization with peripheral blood leucocytes. The resulting alloantisera were analyzed for hemagglutinating reactivity with gynogenetic siblings and proved to be operationally monospecific in absorption experiments. The serologically determined carp erythrocyte specificities were shown to correspond to two codominantly expressed allelic products of a single locus and were designated K1 and K2, respectively. Flow cytometer analysis revealed that the same products are also present on leucocytes from peripheral blood, thymus, spleen, and pronephros. K1- and K2-homozygous second-generation gynogenetic siblings were used to study the histocompatibility nature of the K locus products. Skin transplants between K-allogeneic gynogenetic siblings were rejected significantly faster than within K-syngeneic combinations. Taken together, these data suggest that the K locus incorporates MHC class I-like characteristics.
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B cell and immunoglobulin (Ig) heterogeneity was demonstrated in carp, Cyprinus carpio L., using two monoclonal antibodies (MAbs; WC14, WCI12) produced against carp serum Ig. Immunochemical results showed that both WCI4 and WCI12 react... more
B cell and immunoglobulin (Ig) heterogeneity was demonstrated in carp, Cyprinus carpio L., using two monoclonal antibodies (MAbs; WC14, WCI12) produced against carp serum Ig. Immunochemical results showed that both WCI4 and WCI12 react with a protein determinant on the heavy chain of Ig (relative molecular mass approximately 70,000). Immunofluorescence microscopic and flow cytometric analyses of lymphoid cells suggest three distinct subpopulations of B cells and plasma cells: WCI4+12- cells, WCI4-12+ cells, and WCI4+12+ cells. WCI4-12+ and WCI4+12+ anti-DNP antibody-secreting cells were also demonstrated with the ELISPOT assay in pronephros and spleen cell suspensions from primary immunised carp. Affinity chromatography of carp serum and sequential immunoprecipitation of 125I-labelled peripheral blood leucocyte (PBL) membrane proteins only indicated the presence of two antigenically different Ig molecules, i.e., WCI4-12+ and WCI4+12+ molecules. WCI4+12- molecules could not be detected by affinity chromatography or immunoprecipitation. During ontogeny, a shift in percentages of WCI4+12- and WCI4-12+ cells was found in the spleen and the pronephros. In these organs, WCI4+12- cells formed the majority of B cells at 2 weeks of age, but the percentages of this cell type decreased during ontogeny. On the other hand, the percentages of WCI4-12+ cells increased during development, and these cells became the major population of B cells from 13 weeks onward. The proportion of WCI4+12+ cells remained almost constant during ontogeny. The distribution of B cell subpopulations in blood was more or less stable at all ages. The functional significance of Ig heterogeneity in fish and in particular carp is discussed.
Research Interests: Immunology, Flow Cytometry, Fluorescence Microscopy, Immunohistochemistry, Western blotting, and 14 moreSpleen, Animals, Monoclonal Antibodies, Affinity chromatography, Developmental, Molecular Mass, Carps, Monoclonal Antibody, Membrane Protein, Cyprinus Carpio, Comparative Government Developmental Economics, Lymphoid Tissue, Immunoglobulin, and Hemocyanin
The advent of polymerase chain reaction technology has provoked a large amount of progress in the field of fish major histocompatibility complex (MHC) research. Many new teleost sequences have been reported in the last four years,... more
The advent of polymerase chain reaction technology has provoked a large amount of progress in the field of fish major histocompatibility complex (MHC) research. Many new teleost sequences have been reported in the last four years, including representatives of all classes of MHC genes. While the intron-exon structure of teleost MHC genes is now becoming clear, the organisation of the genes within the teleost MHC is still unclear. The sequences reported to date have been used for phylogenetic analysis and, due to their evolutionary position, are discussed in relation to hypotheses regarding the origin of the MHC. Teleost MHC gene sequences are also examined to see if conserved features of the both the nucleotide and amino acid sequences of higher vertebrate MHC genes are present. Differences in these features will reflect functional differences between teleost and mammalian MHC genes and may also have evolutionary implications.
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In all vertebrates studied to date, the expression of MHC class II genes is known to be restricted to a limited number of tissues and cell types. In order to have a better understanding of the function of the equivalent genes in teleost... more
In all vertebrates studied to date, the expression of MHC class II genes is known to be restricted to a limited number of tissues and cell types. In order to have a better understanding of the function of the equivalent genes in teleost fish, the distribution of MHC class II beta transcripts (Cyca-DAB) in the common carp (Cyprinus carpio L.) was investigated. RNA was isolated from tissues and leucocytes, cDNA was produced, and amplification of the Cyca-DAB genes was carried out by PCR. Of the organs with known immunological function, the highest level of Cyca-DAB transcription was found in the thymus. Despite their expected different cellular organization, total blood, head kidney, spleen and the second segment of the gut had similar Cyca-DAB expression levels. No class II transcripts were detected in the skeletal muscle. The studies carried out with leucocytes isolated from the lymphoid tissues point to a direct correlation between the levels of expression and the amounts of surface immunoglobulin positive (sIg+) cells present in the different cell fractions. However, thymus leucocytes did not follow this correlation since the highest level of class II expression was found in a thymocyte fraction that contained very low numbers of Ig+ cells. In PBL the Ig+ cells were highly positive whereas the Ig- were weakly positive. Adherent leucocytes shown to be class II positive, although adherent cells from PBL show a lower level of expression compared to those from the spleen and head kidney.
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Mouse anti-carp thymocyte monoclonal antibodies were used to investigate the appearance of lymphoid cell determinants in young carp. Determinants detected by clone WCT 4 were present on thymocytes as soon as the thymus was visible... more
Mouse anti-carp thymocyte monoclonal antibodies were used to investigate the appearance of lymphoid cell determinants in young carp. Determinants detected by clone WCT 4 were present on thymocytes as soon as the thymus was visible histologically, day 4 after fertilization (a.f.) at 21 degrees C, but WCT 4-positive lymphocytes were not present in the pronephros until day 7 a.f.. In contrast, determinants detected by monoclonal antibodies from clones WCT 8 and WCT 10 appeared on thymocytes from day 7 a.f. at 21 degrees C, and WCT 8-positive and WCT 10-positive lymphocytes were not detected in the pronephros until days 16 and 12 a.f., respectively. These results are discussed in relation to the appearance of determinants detected by anti-carp serum immunoglobulin antisera.