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G W Warr

Publications and source records attributed to G W Warr.

At least 37 records · Page 2Linked to original sources
PubMed1997

The adaptive immune system of fish.

Fish above the level of the agnatha display typical vertebrate adaptive immune responses characterized by immunoglobulins, T-cell receptors, cytokines, and major histocompatibility complex molecules. However, while the immune system of fish is quite different in its efficiency and complexity from that of higher vertebrates, our knowledge is sufficiently limited that we do not yet have a full understanding of the reasons for this difference. Filling the substantial gaps in our knowledge of the adaptive immune responses of fish will improve our understanding of their response to vaccines, and aid in the rational design of vaccines and protocols for vaccination.

Adaptation, Physiological↗
PubMed1995

Purification of duck immunoglobulins: an evaluation of protein A and protein G affinity chromatography.

Duck serum proteins binding to protein A Sepharose CL-4B and protein G Sepharose 4 Fast Flow and eluted at pH 2.8 or 11.5 were characterized by sodium dodecyl sulphate polyacrylamide gel electrophoresis, radial/immunodiffusion against defined anti-immunoglobulin (Ig) reagents, and by the reactivity in immunoelectrophoresis of antisera raised in rabbits inoculated with the eluates. The results indicated that IgY (previous nomenclature 7.8S IgG) and IgY (delta Fc) (previously 5.7S IgG) bound to protein A efficiently and to protein G weakly, while IgM bound to protein A and protein G weakly. Some binding of non-Ig proteins also occurred. Attempts to separate the non-Ig proteins from the Igs by elution at different pHs (5.0, 4.0, 3.0 and 2.5) were unsuccessful, but it was found that precipitation of Igs in day-old duck serum with Na2SO4, followed by chromatography on protein A Sepharose, yielded relatively pure IgY. The efficient binding of the duck IgYs to protein A resembles high affinity binding of mammalian Igs but cannot be attributed to the Fc, as it is in mammals, since the IgY (delta Fc) does not have an Fc region. Instead, binding probably occurs through unique histidine residues occurring predominantly in the CH1 domain.

Amino Acid Sequence↗
PubMed1995

IgY: clues to the origins of modern antibodies.

IgY is the functional equivalent of IgG in birds, reptiles and amphibia, but many aspects of its biology are poorly understood. Recent studies have increased awareness of the genetics and functions of this molecule, and have revealed its position as the ancestor of the uniquely mammalian antibodies IgG and IgE. Here, Greg Warr, Kathy Magor and David Higgins review current knowledge of IgY structure, function and expression in the context of the evolutionary role of this primitive immunoglobulin.

Amphibians↗
PubMed1994

One gene encodes the heavy chains for three different forms of IgY in the duck.

IgY, the major Ab of the duck (Anas platyrhynchos), exists in two secreted forms and a transmembrane (TM) form. To investigate the genetic relationships of the multiple IgY we cloned the gene encoding the IgY (upsilon) heavy chains. The heavy chain of the smaller (5.7S) IgY, which lacks the third and fourth constant domains, results from the use of a unique terminal exon found in the intron between the second and third C region exons. Alternate pre-mRNA processing pathways also produce a full-length upsilon-chain and a TM form, each having four C region domains. Although the number of secretory exons and the inferred positions of intramolecular disulfide bonds indicate structural similarity between IgY and IgE, the TM exons of duck IgY share high sequence identity and a similar pattern of RNA processing with those of IgG. These results suggest that IgG and IgE may have diverged from an ancestral molecule resembling IgY.

Alternative Splicing↗
PubMed1994

An Ig heavy chain enhancer of the channel catfish Ictalurus punctatus: evolutionary conservation of function but not structure.

The teleost fishes are among the earliest evolutionary lineages to have an Ig heavy chain (IgH) locus whose organization approximates that of mammals. To understand transcriptional control of the IgH locus in a teleost fish and to gain insight into the evolution of the control elements, the enhancer activity in the IgH locus of the channel catfish, Ictalurus punctatus, was investigated. Segments of the locus extending from upstream of the proximal JH gene to 2.5 kb downstream of the second transmembrane (TM2) exon of the mu gene were tested in transient transfection expression assays in murine myeloma and T cell lines, and in catfish B lymphoblastoid, monocyte-like, and putative T cell lines. In marked contrast to mammals, no enhancer activity was observed in the catfish JH to C mu intron, but strong enhancer activity (approaching that of the murine IgH intronic enhancer) was identified in a 1.8-kb segment that included the TM2 exon. This catfish enhancer was active in a B lineage-specific manner in both catfish and murine cells. It was not localized in a small core region, but appeared to contain multiple, dispersed cooperative elements rich in octamer- and mu E5-related motifs. Although the catfish IgH enhancer shares functional characteristics with the mammalian IgH intronic and 3' enhancers, its unusual organization does not permit any obvious inferences concerning evolutionary relationships between the catfish enhancer and any one of the murine IgH enhancers.

Animals↗
PubMed1994

Development and characterization of channel catfish long term B cell lines.

The establishment of channel catfish long term cloned B cell lines, the first such cell lines from ectothermic vertebrates, is described. These diploid cell lines were developed by in vitro LPS stimulation of B cells from normal channel catfish peripheral blood in the absence of overt attempts to transform or immortalize the cells. The resultant cell lines were cloned and maintained continuously in vitro for more than 12 mo without restimulation, feeder cells, or exogenous factors. Southern blot analyses of the parental cell lines revealed multiple mu-chain gene rearrangements, suggesting a polyclonal origin for the cell lines. Additional evidence for polyclonal development was provided by the demonstration that the parental cell lines transcribed mRNA for all of the six known channel catfish VH gene families. The characterization of several clonal cell lines revealed mRNA expression for both the secreted and membrane forms of the catfish mu-chain; however, the cloned cell lines each expressed only a single VH gene and analysis of the Ig H chain locus was consistent with allelic exclusion having occurred in these cells. Flow cytometry demonstrated that the cloned and uncloned cell lines produced both cytoplasmic and cell surface IgM. This IgM contained only one of the two L chain isotypes of the channel catfish, suggesting preferential L chain usage. Although these cells did not appear morphologically to be plasma cells, they secreted moderate levels of IgM in culture. These cell lines have considerable potential for addressing questions concerning the evolution of B cell function.

Animals↗
PubMed1993

Expression of a mouse-channel catfish chimeric IgM molecule in a mouse myeloma cell.

Fusion genes encoding a murine VH domain and the constant region domains of the mu chain from the channel catfish, Ictalurus punctatus, were stably expressed in the lambda light chain producing mouse myeloma cell line J558L. Although the pathways of pre-mRNA processing for expression of membrane (micron and secreted (microsecond) forms of the mu chain differ between mammals and teleosts, mRNAs encoding both catfish micron and microsecond were correctly expressed in the mouse myeloma cells. The mouse-channel catfish chimeric mu chain polypeptide was able to associate covalently with the mouse lambda light chain and assemble, intracellularly, into polymers of covalent structure (microL)2-8 which resembled those seen with native catfish IgM. In contrast to native catfish IgM, the mouse-catfish chimeric IgM showed the property of binding strongly to protein A of Staphylococcus aureus. The mouse-channel catfish chimeric IgM was core-glycosylated, but did not contain terminal sialic acid. Secretion rates for the chimeric IgM were low, and the possibility could not be excluded that extracellular chimeric IgM was released from dead or dying cells. The reason(s) for the intracellular retention of the chimeric IgM (probably in the endoplasmic reticulum) are not known, but those mechanisms involving retention via cysteine residues were excluded.

Animals↗
PubMed1992

Structural relationship between the two IgY of the duck, Anas platyrhynchos: molecular genetic evidence.

cDNA clones encoding the H chains of the 7.8S and 5.7S IgY of the White Pekin duck have been isolated and sequenced. The H chain of the 7.8S IgY possesses four C region domains and thus resembles the H chain of chicken IgY with which it shows, in the C region, 54% inferred amino acid sequence identity, and complete conservation of the C region cysteine and tryptophan residues. The H chain of the 5.7S IgY possesses only two C region domains, that are virtually identical to CH1 and CH2 of the 7.8S IgY H chain. Although Southern blot genomic analysis did not resolve whether the two transcripts encoding the H chains of the 7.8S and 5.7S IgY are derived from one or two H chain-encoding genes, the CH 1, 2, 3, and 4 exons are apparently colinear, and no evidence was found for a separate locus in which CH1 and 2 exons were present and CH3 and 4 exons were lacking. The VH domain-encoding sequences of the cDNA for the two IgY H chains showed high similarity in the inferred VH gene (93% nucleotide and 91% inferred amino acid identity) and in the inferred JH segment (89% nucleotide and 93% inferred amino acid identity) but low similarity in the D region (26% nucleotide and 7% inferred amino acid identity). Genomic Southern blot hybridization analysis showed multiple VH-hybridizing sequences represented on up to 20 restriction fragments.

Amino Acid Sequence↗
PubMed1992

Clonal proliferation of murine lymphohemopoietic progenitors in culture.

We have used a two-step clonal culture system to unequivocally demonstrate that individual primitive lymphohemopoietic progenitor cells have the capacity for differentiation along either the myeloid or the B-lymphoid lineage. Highly enriched murine marrow cells were plated individually in culture by micromanipulation in the presence of pokeweed mitogen-stimulated spleen cell conditioned medium, erythropoietin, steel factor (SF), and interleukin (IL) 7. Forty-five percent of the single cells formed primary colonies expressing multiple hemopoietic lineages. When aliquots from individual colonies were replated in secondary methyl cellulose culture containing SF and IL-7, 41% of the primary colonies gave rise to lymphocyte colonies. Cells of the lymphocyte colonies were blast-like and B220+, sIg-, Mac-1-, Gr-1-, Ly-1-, L3T4-, Ly-2-, and CD3-. Thirty to 70% of the cells were Thy-1+. mu-chain mRNA was detected in most of the cells by in situ hybridization with an antisense RNA probe. When lymphocyte colonies derived from a single cell were pooled and individually injected into scid mice, donor-type IgM was measurable in the serum of mice and spleens contained donor-type B cells. We then carried out initial screening of growth factors to identify growth factors that might replace pokeweed mitogen-stimulated spleen cell conditioned medium in the primary culture. Combinations of two factors that included SF plus IL-6, IL-11, or granulocyte colony-stimulating factor were all effective in the primary culture in the maintenance of the B-lymphoid potential. Interestingly, IL-3 could neither replace nor act synergistically with SF to support the lymphoid potential of the primary cultures. Our observations demonstrate that many primitive progenitors previously believed to be myeloid-committed also possess B-lymphoid potential. This culture system should prove valuable for elucidation of the mechanisms regulating early stages of lymphohemopoiesis.

Animals↗
PubMed1992

Alternate splicing pathways of the immunoglobulin heavy chain transcript of a teleost fish, Ictalurus punctatus.

Previously we sequenced a partial cDNA clone encoding the 3' region of the message for the membrane receptor form of the heavy (mu) chain of the channel catfish which indicated that the first transmembrane (TM1) exon is spliced directly to the C mu 3 exon and not into a cryptic site within the CH4 exon, as occurs in other vertebrates. Studies utilizing polymerase chain reaction analysis of mRNA and further analysis of cDNA clones now confirm that the only detectable splicing pattern used in micron production by the channel catfish utilizes this C mu 3----TM1 pathway of pre-mRNA splicing.

Animals↗
PubMed1992

Cellular pathway(s) of antigen processing and presentation in fish APC: endosomal involvement and cell-free antigen presentation.

Studies were conducted to address further the role(s) of antigen processing and presentation in the induction of immune responses in a phylogenetically lower vertebrate, specifically a teleost, the channel catfish. In particular, studies were aimed at determining the subcellular compartments involved in antigen degradation by channel catfish antigen-presenting cells (APC) as well as ascertaining the reexpression of immunogenic peptides on the surfaces of APC. The results showed that exogenous protein antigens were actively endocytosed by APC as detected by flow cytometry. Use of radiolabeled antigen and subcellular fractionation protocols also showed that antigen localized in endosomes/lysosomes. Furthermore, there was an apparent redistribution of antigen between these organelles and the plasma membrane during the course of antigen pulsing. Functional assays for the induction of in vitro antigen-specific proliferation of immune catfish peripheral blood leukocytes (PBL) showed that membrane preparations from antigen-pulsed autologous APC were highly stimulatory. The magnitude of responses elicited with such membrane preparations was very similar to that of PBL cultures stimulated with native antigen-pulsed and fixed intact APC or prefixed intact APC incubated with a peptide fragment of the nominal antigen. Current data further corroborate our previous findings that steps akin to antigen processing and presentation are clearly important in the induction of immune responses in lower vertebrates like fish, in a manner similar to that seen in mammalian systems. Consequently, it would appear that many immune functions among the diverse taxa of vertebrates are remarkably conserved.

Animals↗
PubMed1990

The immunoglobulin M heavy chain constant region gene of the channel catfish, Ictalurus punctatus: an unusual mRNA splice pattern produces the membrane form of the molecule.

The immunoglobulin (IgM) heavy chain constant region gene of the channel catfish, Ictalurus punctatus, has been cloned and characterized. The gene contains four constant region domain-encoding exons (CH1 to CH4) expressed in the secreted form of the immunoglobulin, and two exons encoding the transmembrane (TM) domain utilized in the lymphocyte membrane receptor form of the immunoglobulin. The sequence of a cDNA clone encoding the 3' region of the message for the membrane receptor form of the mu chain indicates that the TM1 exon is spliced directly to the CH3 exon, and not into a site within the CH4 exon, as occurs in the mammals, a shark and an amphibian. This unusual pattern of splicing, which produces a membrane heavy chain that is characteristically smaller than the secreted heavy chain, may be common to all teleost fish.

Amino Acid Sequence↗
PubMed1990

A 12 kDa protein in chicken serum antigenically cross-reactive with, but unrelated to, beta 2-microglobulin.

A protein, isolated from chicken serum, showed the following beta 2-microglobulin-like properties; an apparent molecular size of 12000 Mr, predominantly beta-sheet secondary structure, and antigenic cross-reaction with human beta 2-microglobulin. However, amino acid composition analysis and partial amino acid sequence analysis showed that this molecule was not only not a beta 2-microglobulin, but was unrelated to any other known class of protein.

Amino Acid Sequence↗
PubMed1988

Immunoglobulin heavy chain variable region gene evolution: structure and family relationships of two genes and a pseudogene in a teleost fish.

Nucleotide sequences for two immunoglobulin heavy chain variable region (VH) genes and one pseudogene in the goldfish (Carassius auratus) and the family relationships and distribution of these genes in individual fish are presented. Comparison of the nucleotide and inferred amino acid sequences of goldfish and other vertebrate VH genes indicates that goldfish VH genes show the major VH gene regulatory and structural features (5'-putative promoter region, split hydrophobic leader, three framework and two complementarity-determining regions, and 3'-recombination signals for VH to diversity region joining) and that goldfish VH genes are not more closely related to one another than they are to VH genes of evolutionarily distant vertebrates such as the mammals. Goldfish VH genes appear to exist in distinct families, and individual goldfish can carry from none to apparently greater than 15 genes of a given family. These results suggest that whereas the basic structure of VH genes has been conserved in evolution, there may be substantial variation in the nature and population distribution of VH gene families in the vertebrates.

Amino Acid Sequence↗
PubMed1986

Putative immunoglobulin VH genes of the goldfish, Carassius auratus, detected by heterologous cross-hybridization with a murine VH probe.

By using a defined cDNA probe for the VH region of a murine phosphocholine-binding myeloma protein (S107) we have defined a family of distinct cross-hybridizing DNA sequences in genomic DNA of the goldfish. The estimated number of the goldfish putative VH family detectable by the S107 probe is about 36. By using two putative goldfish VH probes to analyze, by hybridization, the relationships among seven of the goldfish genomic clones, we have determined that the putative goldfish VH genes recognized by the S107 probe comprise at least several distinct families that are not closely related.

Animals↗
PubMed1985

Anomalous behavior of goldfish IgM heavy chain in sodium dodecylsulfate polyacrylamide gel electrophoresis.

By sodium dodecylsulfate polyacrylamide gel electrophoresis, the heavy chain of the serum immunoglobulin (IgM) of the goldfish (Carassius auratus) differs not only from other studied vertebrate serum IgM heavy chains, but also from other vertebrate lymphocyte membrane IgM heavy chains including those from the goldfish itself. This difference, an increase in apparent Mr of approximately 5000, was investigated by assessing in comparison with the IgM heavy chain of human and rainbow trout (Salmo gairdneri) the following properties: (1) molecular size by gel filtration in denaturing buffers; (2) carbohydrate content, by direct analysis; (3) intrinsic net charge, by isoelectric focusing; (4) net hydrophobicity, deduced from amino acid analysis; and (5) sodium dodecylsulfate binding by direct measurement. Results indicate that goldfish IgM heavy chain is indistinguishable from other IgM heavy chains in terms of (a) its gel-filtration behavior in denaturing conditions, (b) its carbohydrate content (which is similar to trout IgM heavy chain) and (c) its intrinsic net charge and hydrophobicity. However, goldfish IgM does differ from the other proteins studied in its detergent-binding ability and it is this behavior that is concluded to be the cause of its unusual mobility in sodium dodecylsulfate polyacrylamide gel electrophoresis.

Amino Acids↗