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C J Lobb

Publications and source records attributed to C J Lobb.

At least 37 records · Page 2Linked to original sources
PubMed1992

Organization of immunoglobulin heavy chain constant and joining region genes in the channel catfish.

A channel catfish genomic lambda library was screened with CH and JH probes which were derived from our earlier sequence analyses on different full-length heavy chain cDNA clones. One clone, designated C7, contained a genomic insert of about 18 kb and hybridized with specific probes for each of the four domains of the known C region gene as well as with different oligonucleotides specific for JH gene segments. Southern blot hybridization analysis identified a cluster of JH gene segments which are closely linked to the CH gene. Sequence analysis of the CH-proximal JH element, located about 1.9 kb upstream from the CH1 domain, showed that this element contains 5'-recombination signals typical of JH elements defined in higher vertebrates, i.e. a nonamer, a 24 bp spacer, and a heptamer. The coding region of this JH element was identical to that contained in the variable region sequence of a cDNA clone previously reported. Sequence analysis of the catfish JH-CH intron suggests that several sequences are present which appear similar to important transcriptional regulatory elements found within JH-CH introns of higher vertebrates. These features include sequences similar to higher vertebrate enhancer elements and regulatory octamers. An additional feature reminiscent of some higher vertebrate heavy chain switch regions is a repetitive sequence area composed of tandemly repeated simple sequences. Lastly, several restriction length polymorphisms were identified and mapped within a 1 kb region located immediately upstream from the JH cluster. This finding suggests that polymorphisms within the IgH locus should be useful in the analyses of channel catfish populations. These combined studies provide further evidence that the genomic organization of heavy chain genes in bony fish shares common organizational features with those known from higher vertebrates.

Amino Acid Sequence↗
PubMed1991

Heavy chain variable region gene families evolved early in phylogeny. Ig complexity in fish.

The V regions of channel catfish H chain cDNA clones have been analyzed. Based upon sequence relationships and hybridization analyses, five different groups of VH genes are identified whose definition is consistent with that of five different VH families. Genomic Southern blots indicate that as many as 100 different germ-line VH genes are likely represented by these families. The sequence diversity between identified members of these different families is similar in magnitude to the divergence represented between members of different human or mouse VH families. The FR regions are the most conserved regions when members of different catfish VH families are compared; specific amino acid positions appear to be highly conserved in phylogeny. Equally important is that diversity is represented in complementarity-determining regions CDR1 and CDR2 in members of the different families as well as in members of the same VH family. These results suggest that an extensive repertoire of VH genes can contribute to antibody diversity in this lower vertebrate. Sequence comparisons indicate that one of the catfish VH families shares considerable structural similarity to several higher vertebrate VH gene families--a relationship which suggests that this VH family may be ancestral to some VH gene families of higher vertebrates. Characteristic of the genomic organization of higher vertebrate H chains, catfish appear to have different VH families wherein a VH gene likely undergoes functional recombination with putative DH gene segments and one of apparently several different JH segments. The recombined V region is expressed with the same C region gene. These combined results suggest that bony fishes are the earliest known phylogenetic representatives to have evolved extensive V region gene families.

Animals↗
PubMed1990

Channel catfish as an unconventional model for immunological studies.

The channel catfish, Ictalurus punctatus, is an economically important species which is readily available, acclimates well to the laboratory setting, and is amenable to considerable experimental manipulation. Although the channel catfish is still a relatively circumscribed species in terms of comprehensive physiologic and/or endocrinologic studies, our current understanding of the basic immunobiology and immunochemistry of the channel catfish is significantly further advanced than for any other teleost species. In this respect the channel catfish is not only proving useful in the general areas of comparative immunology but it is also showing considerable promise as a model system for definitive studies on problems which bridge the fields of immunology and endocrinology, i.e., understanding the effects of environmental temperature and stress on the immune system.

Animals↗
PubMed1989

Nucleotide sequence of channel catfish heavy chain cDNA and genomic blot analyses. Implications for the phylogeny of Ig heavy chains.

Our prior analyses defined the cDNA sequence on part of the CH2 domain, the complete CH3 and CH4 domains, and the 3'-untranslated region of a catfish H chain. To complete the catfish H chain mRNA sequence, a primer-extended H chain cDNA library was constructed. Analysis of this library has resulted in the definition of full-length clones encoding a 61-bp 5' untranslated region, a 51-bp leader sequence, the V region and the complete CH1 and CH2 domains. The high similarity defined with other vertebrate V regions readily allowed the catfish sequence to be divided into FR and CDR regions. Sequence comparisons with mammalian VH and JH genes strongly suggest that the catfish V region is the product of multiple genes. Using a catfish VH cDNA probe, at least 25 different genomic VH members were defined. Because this probe does not hybridize with other full-length H chain cDNA clones, additional VH families will likely be defined in catfish. Phylogenetic sequence comparisons of the catfish C region domains indicated that the CH1 and CH4 were the most highly conserved. In addition several important features were defined in genomic Southern blot analyses of catfish DNA. Gene titration experiments established that the catfish CH gene is represented by a single genomic copy. This finding provides clear evidence that the genomic organization of H chain genes in catfish must be different from that defined in sharks and suggests that the phylogeny of single copy CH genes may have been established at the level of the bony fishes. It is also likely that there is an additional CH gene in catfish. This gene is also represented by a single genomic copy, and based upon its relative signal intensity when compared with the known CH gene it appears to share higher similarity with the known CH1 domain than it does with the CH2 domain.

Amino Acid Sequence↗
PubMed1989

Cloning and sequence analysis of channel catfish heavy chain cDNA indicate phylogenetic diversity within the IgM immunoglobulin family.

Catfish cDNA libraries were constructed using the poly(A+) RNA obtained from in vitro stimulated catfish leukocytes. Antigenic analysis with different antisera to catfish Ig resulted in the definition of cDNA clones encoding the catfish H chain. Sequence analysis confirmed that the catfish H chain was definitively identified, based on its similarities with chicken and mouse mu chains. Two clones were each shown to encode part of the CH2 domain, the complete CH3 and CH4 domains, the C-terminus, and a 184-bp 3' untranslated region before the poly(A+) tail. The conservation of domain size and structure is clearly evident. The two cysteines forming the intradomain disulfide bridge, as well as the tryptophans located within each domain, are absolutely conserved. There are four carbohydrate acceptor sites in the catfish H chain, only one of which is phylogenetically conserved. Of the six sequenced H chain clones, one was found to differ in a single base in the CH3, which results in the loss of a carbohydrate acceptor site. Whether this difference indicates isotypic variation between closely related genes or somatic mutation is unresolved. Amino acid sequence comparisons indicate that there is a approximately 24% similarity when the catfish H chain is aligned with mouse mu chains. This is considerably less than the approximately 40% amino acid conservation found between the chicken and mouse mu chain. The amino acid sequence of the catfish H chain is most conserved in the C-terminus (approximately 30%) and the CH4 (approximately 26%); there is less conservation in the CH3 (approximately 20%) when comparisons are made with mouse mu chain. The CH3 domain of the catfish H chain also has different hydropathy properties, when compared with the CH3 domain of the higher vertebrate mu chains. Finally, the sequence of the catfish H chain indicates an unusual arrangement of the cysteines that likely participate in intersubunit and inter-H chain disulfide linkages. The disulfide linkage of these cysteines during Ig polymerization may account for the unusual covalent architecture associated with the catfish tetramer.

Amino Acid Sequence↗
PubMed1989

Activation of complement by different immunoglobulin heavy chain isotypes of the channel catfish (Ictalurus punctatus).

The approximately 750,000 mol. wt tetrameric Ab population from catfish immunized with sheep erythrocytes (E) was purified and shown to sensitize E to the hemolytic activity of catfish serum complement. Catfish complement was heat-labile at 45 degrees C and was hemolytically inactivated by both EGTA and EDTA. Hemolytic activity of EDTA treated serum was restored by the addition of both Ca+ and Mg2+ ions. In comparative assays, the CH50 titers of catfish sera were similar to the CH50 titers of human sera. Catfish complement was hemolytically active at incubation temps ranging from 3 to 40 degrees C, which suggests that complement should be functional throughout the catfish's normal environmental temps. Human complement exhibited considerable cross-reactivity when assayed on catfish Ab sensitized E at 25 degrees C; however, at 3 degrees C, no hemolytic activity was observed. They findings suggest that there may be structural differences between the complement component(s) of these two systems. Mouse mAbs to the recently described H chain isotypes of catfish Ig were used to fractionate catfish anti-sheep E antisera. Each of the purified isotypes contained Ab which sensitized E to the hemolytic activity of catfish complement. Dose-response analysis of the average number of hemolytic sites per cell, as a function of relative Ab concn, predicts that the catfish tetramer conforms to the one-hit theory of immune hemolysis. Purified catfish anti-fluorescein Ab of each isotype was also reacted with fluorescein-labeled sheep E and the level of complement-mediated hemolysis determined. Dose-response analysis indicated that similar numbers of Ab molecules bound to haptenated E resulted in similar levels of complement-mediated lysis. Lastly, the effect of cell surface hapten density of target cells was examined. These studies, similar to those reported with human IgM, showed that the binding of the catfish tetramer to a cell surface hapten may not be sufficient to activate lytic complement.

Animals↗
PubMed1988

Immunoglobulin heavy H chain isotypes in a teleost fish.

Three mouse mAb were derived which identified different populations of the approximately 700,000-Da tetrameric Ig of channel catfish (Ictalurus punctatus). Immunoprecipitation analyses using the three mAb in various combinations showed that the catfish Ig population identified by each mAb was antigenically distinct. Each Ig population contained both classes of catfish L chains (F and G). Solid-phase binding assays indicated that the mAbs preferentially identified H chains rather than L chains. Comparative peptide mapping analyses of the H chains defined by each mAb indicated that H chains were different, although each had the same apparent mass (approximately 70,000 Da). This structural distinction was not based upon allotypic variation because the analysis of the serum from individual catfish showed the presence of each H chain in fish examined. Therefore, the different H chains represent different isotypes; each representing approximately 20% of the total serum Ig. There is at least one undefined additional H chain isotype found in the serum Ig population. Partial sequence information was obtained on the first variable framework region (FR1) of two of the three H chain isotypes. These results indicated that the variable region associated with each isotype was heterogeneous; two and sometimes three different residues were represented in the majority of positions. The sequence information also showed that the FR1 of one H chain was distinct from the other; there was less than a 50% match of the primary residues. The analyses suggest that the V region genes which code for the FR1 are differently associated with each H chain isotype. Finally, the relative levels of the three H chain isotypes were monitored during the temporal immune response of individual catfish to the dinitrophenyl hapten. These results indicated that one H chain isotype was preferentially expressed early in the immune response (approximately 3 wk) and remained the predominantly expressed isotype during the humoral immune response.

Amino Acid Sequence↗
PubMed1987

Secretory immunity induced in catfish, Ictalurus punctatus, following bath immunization.

Individual adult channel catfish were immunized by immersion in an antigen bath containing dinitrophenylated-horse serum albumin. Anti-DNP hemagglutination titers of serum and cutaneous mucus were determined following both primary and secondary bath immunization. The results showed that five of the six fish had a cutaneous mucosal anti-DNP titer following the bath immunizations. In contrast, only one of the six catfish was shown to have any demonstrable change in its serum anti-DNP titer following the bath immunizations. The mucous anti-DNP hemagglutinin was shown to be antibody (Ab). The affinity-purified mucous Ab was found to have the same complex tetrameric architecture as well as the same molecular weight heavy and light chains as serum anti-DNP Ab. Histological studies showed that the catfish epidermis was richly vascularized. Within the epidermis there were numerous lymphocytes which were predominantly associated with the basal layer. These studies indicate that the secretory immune system of catfish can be stimulated by external antigens. Secondly, these studies show that bath immunization can differentially effect the relative antibody response of the catfish secretory and systemic immune systems.

Animals↗
PubMed1987

Rapid plasmid analysis for identification of Edwardsiella ictaluri from infected channel catfish (Ictalurus punctatus).

Eighteen different strains of Edwardsiella ictaluri isolated from infected channel catfish (Ictalurus punctatus) were screened to determine whether plasmid DNA was present. Two plasmids of 5,700 and 4,900 base pairs were identified. Restriction enzyme analysis showed that each of the strains harbored these same two plasmids. Restriction maps of the separated plasmids indicated that these plasmids were not closely related to each other. A rapid screening technique was developed that would allow the presence of these plasmids from either broth cultures or single colonies of E. ictaluri to be determined within 2 to 3 h by agarose gel electrophoresis. These results suggest that plasmid fingerprinting of E. ictaluri should become a useful tool in the presumptive identification of this bacterium from infected channel catfish.

Animals↗
PubMed1986

Preferential expression of catfish light chain immunoglobulin isotypes in anti-dinitrophenyl antibodies.

Previous studies indicated that immunoglobulins of the channel catfish, Ictalurus punctatus, contained two distinct isotypes of light chains. These different light chains, designated F and G, are reminiscent of kappa and lambda light chains of higher animals. This study was undertaken to establish whether or not there was preferential expression of one of the light chains in antibodies induced to the dinitrophenyl moiety during the course of the catfish humoral antibody response. The results indicated that, although antibody produced very early (1-2 weeks) after primary immunization contained significant amounts (approximately 20%) of G light chains, the vast majority (approximately 90%) of antibody produced later (3 weeks through 1 year) was of the F isotype. Thus fish, as well as higher vertebrates, can express different isotypes of antibody during the immune response.

Animals↗
PubMed1985

Covalent structure and affinity of channel catfish anti-dinitrophenyl antibodies.

Serum antibody (Ab) of channel catfish, Ictalurus punctatus, is predominantly a tetrameric, approximately 700,000-dalton protein. This tetrameric Ab dissociates into 8 distinctive subpopulations, each composed of equimolar heavy and light chains, when in the presence of denaturing solvents. The present studies utilizing purified anti-DNP Abs obtained from catfish monitored for either short (1-2 months) or long (1-2 years) periods post-primary immunization with dinitrophenylated horse serum albumin showed no demonstrable differences in the ratio of the covalent Ab subpopulations. Although significant humoral Ab levels were sustained during the 1-2 year monitoring period, there were no significant changes in the binding affinities or heterogeneity of the induced Ab.

Animals↗
PubMed1984

Temperature-mediated processes in teleost immunity: differential effects of in vitro and in vivo temperatures on mitogenic responses of channel catfish lymphocytes.

The in vitro mitogenic responses of channel catfish peripheral blood leucocytes to ConA and LPS were differentially affected by both in vitro and in vivo temperatures. The magnitude of the response to LPS was relatively independent of both in vitro culture temperature and in vivo acclimation temperature. The magnitude of the response to ConA was suppressed at lower in vitro temperatures although this suppression could be reduced by lower in vivo acclimation temperatures. In vitro temperature-shift experiments indicated that channel catfish PBL could respond to ConA at a lower in vitro temperature if first stimulated with ConA at a higher in vitro temperature. The converse, however was not true in that channel catfish PBL did not respond at a higher in vitro temperature after an initial stimulation with ConA at a lower in vitro temperature. This latter failure to respond could not be attributed to the induction of a suppressor cell (or factor) by exposure to ConA at a lower temperature. These studies, when coupled with other available data on channel catfish PBL subpopulations, are interpreted as supporting the hypothesis that low temperature immunosuppression in fish may result from preferential inhibitory effects on T cells rather than B cells.

Animals↗
PubMed1984

A new method for the cytochemical staining of cells immobilized in agarose.

A quick, simple and inexpensive technique utilizing an isotonic matrix of semi-solid agarose for immobilizing channel catfish (Ictalurus punctatus) peripheral blood leucocytes onto microscope slides before cytochemical staining is described. The technique was demonstrated to be superior to the more conventional means of immobilizing cells for procedures requiring either fixed (Sudan Black B and nonspecific esterase stains) or viable (Nitro-Blue Tetrazolium stain) cells. Although the agarose immobilization technique was developed with channel catfish leucocytes, its utility with other fragile cell populations seems likely.

Animals↗
PubMed1984

Immunoglobulin light chain classes in a teleost fish.

Analysis of predominant, approximately 700,000 dalton serum antibody (Ab) of the channel catfish by SDS-PAGE revealed only one molecular mass species of heavy (H) chain (approximately 70,000 daltons), whereas a marked heterogeneity in the relative mobilities of the light (L) chains was evident. Three molecular mass L chain variants of approximately 26,000, approximately 24,000, and approximately 22,000 daltons were observed. The relationships between the L chain variants were not clear until mouse monoclonal Ab (mAb) reactive with catfish immunoglobulin (Ig) were developed. Two of these mAb, designated 3F12 and 1G7, were found to independently recognize different populations of catfish Ig. In addition, when these two mAb were used in combination they immunoprecipitated greater than 95% of specifically purified catfish anti-DNP or anti-fluorescein Ab. The L chains of catfish Ig recovered from immunoabsorbent affinity matrices conjugated with either mAb 3F12 or 1G7 were found to be clearly distinct. Monoclonal Ab 3F12 reacted with a subpopulation of catfish Ig that contained the approximately 24,000 and approximately 22,000 dalton L chain variants, whereas mAb 1G7 reacted with another subpopulation of catfish Ig that contained only the approximately 26,000 dalton L chain variant. Solid phase plate binding assays with the use of mildly reduced catfish Ig H and L chains showed that both of these mAb preferentially reacted with catfish L chains. Subsequent analysis of the two antigenically distinct L chain populations by peptide mapping procedures demonstrated that these L chains were structurally different. Furthermore, analysis of the serum from individual catfish showed the presence of both L chain types in each of 25 catfish examined. These studies strongly suggest that distinct L chain classes have evolved in fish.

Animals↗
PubMed1984

Phylogeny of lymphocyte heterogeneity: the cellular requirements for in vitro mitogenic responses of channel catfish leukocytes.

Cell separation and enrichment techniques were employed to isolate three distinct leukocyte subpopulations present in channel catfish (Ictalurus punctatus) peripheral blood. Surface immunoglobulin-positive (sIg+) and sIg- lymphocytes were separated by an indirect "panning" technique employing monoclonal antibodies reactive with channel catfish Ig. A third cell population composed of macrophages was isolated by adherence to baby hamster kidney cell microexudate-coated surfaces. Functional features of these three subpopulations were assessed by in vitro mitogenic responses to lipopolysaccharide (LPS) and concanavalin A (Con A). The results that were obtained indicated that the sIg+ cells responded only to LPS stimulation regardless of the presence or absence of macrophages. The sIg- subpopulation, however, responded to neither LPS nor Con A unless macrophages were present, in which case responses were obtained to both mitogens. The accessory cell nature of the macrophages was shown by experiments utilizing fixed numbers of one cell type mixed with varying numbers of another cell type. Furthermore, the accessory cell function was abrogated by passage through Sephadex G-10 and preincubation with L-leucine methyl ester. These studies provide further evidence that teleosts not only contain B and T cells akin to those in mammalian systems, but contain accessory cells (macrophages) as well.

Animals↗
PubMed1983

Distinctive subpopulations of catfish serum antibody and immunoglobulin.

High molecular weight antibodies and immunoglobulin of the channel catfish were found to be composed of heterogeneous mixtures of covalent subpopulations. Although these proteins appeared tetrameric in physiological buffers (mol. wt approximately 700,000), molecular weight estimates in the presence of the detergent sodium dodecyl sulfate indicated that they dissociated into eight distinct subpopulations. Each dissociative subpopulation was found to be stable under the conditions of electrophoretic analysis, and furthermore each was apparently composed of equimolar H and L chains. Based upon relative mobilities it seems probable that the catfish antibody was composed of various combinations of covalent linkages between covalently-linked H-L chain pairs, i.e. 8, 7, 6, 5, 4, 3, 2 and 1. Further studies suggest the presence of size heterogeneity in catfish light chains. Whether or not this distinction represents isotypic L chain differences is not yet known but these findings coupled with the complex architecture of the physiological tetramer suggest an unusual and previously unappreciated level of complexity present in fish antibody.

Animals↗
PubMed1982

Fish lymphocytes differ in the expression of surface immunoglobulin.

Catfish peripheral blood and splenic lymphocytes were assayed for surface immunoglobulin using fifteen different mouse hybridoma antibodies to catfish immunoglobulin (Ig). These studied showed that this battery of monoclonal antibodies did not detect significant amounts of Ig on all lymphocytes. Unlike polyclonal antisera which demonstrated nearly 100% surface Ig+ cells, the monoclonal antibodies detected approximately 40% surface Ig+ cells. Furthermore, the percentage of Ig+ cells reactive with two of these monoclonals, tentatively shown to react with two different types of catfish light chains, was found to be nearly additive when the two antibodies were mixed. Thus it seems that fish lymphocytes, like their mammalian counterparts, have two different populations of lymphocytes; one which contains abundant surface Ig and one which does not. Whether these two types of cells represent the fish equivalents of B and T cells remains to be determined.

Animals↗