Variability of the immunoglobulin light chain in the Siberian sturgeon, Acipenser baeri

All sturgeon VL segments isolated in this study belong to a single family, VLI, which can be divided into two subfamilies. Of the 79 cDNA clones isolated, 76 belong to the larger subfamily, VLIa, and only 3 clones constitute the smaller subfamily, VLIb. To evaluate variability, the Shannon entropy was estimated for each individual amino acid position, and to facilitate comparisons of variability between species the mean entropy of the CDR regions was calculated. In such a comparison, the sturgeon was found to have CDR1 and CDR3 variability approaching those found in mouse and clawed frog, but showed very low variability for CDR2. Amino acid position 50 does however display variability in the range of mouse and clawed frog. It is further confirmed that the sturgeon has numerous J segments, but that the junctional diversity does not contribute greatly to the diversity of the light chain. Comparisons of cDNA clones and a genomic VL segment indicate that the VL undergoes changes, particularly in the CDR regions, in a manner that can be explained by somatic hypermutation and/or gene conversion.     

Cloning of a CXCR4 homolog in chondrostean fish and characterization of the CXCR4-specific structural features

The chemokine receptor CXCR4 and its ligand SDF-1 are essential components of hematopoiesis, organogenesis and immunomodulation in mammalian species. We cloned a cDNA encoding CXCR4 homolog of sterlet (Acipenser ruthenus), a representative of chondrostean fishes. The deduced amino acid sequence of sterlet CXCR4 is almost equally distant from mammalian and teleost CXCR4 (66–68% identical residues). Percent identity with the other chemokine receptors varies in the 30–35% range. The CXCR4 sequences from the three phylogenetically diverged lineages were compared with the sequences of the other chemokine receptors to determine the CXCR4-specific structural elements that were conserved during vertebrate evolution. The characteristic residues and/or motifs are located predominantly in the intracellular and extracellular regions and in the third, fourth and fifth transmembrane domains. The data presented may be helpful for structure-function analysis of the CXCR4 ligand binding and signal transduction.     

Characterization of a myosin-like antigen from Onchocerca volvulus

Recombinant cDNAs expressing an immunodominant antigen (Onchoag-1) of Onchocerca volvulus were identified by immunoscreening a cDNA expression library. The Onchoag-1 cDNAs are derived from an 8-kb mRNA that codes for a protein with an apparent molecular mass of 200 kDa. Indirect immunofluorescence using antisera against a recombinant fusion protein showed that Onchoag-1 is located in the muscle tissues of adult O. volvulus. The 2-kb sequence of one of the cDNAs contains a single open translation reading frame that encodes a protein with sequence similarities to Caenorhabditis elegans myosin heavy chain. Analysis of the 3′ region of Onchoag-1 chromosomal gene reveals that it is frequently interrupted by short introns that follow the GT/AG rule at their splice sites. Studies on this myofibrillar antigen should contribute to our understanding of muscle function in O. volvulus as well as provide useful insight to the genesis of the immunopathological damage that is often associated with allergic reactions (the Mazzotti reactions) in onchocerciasis patients, following the administration of a chemotherapeutic agent such as diethylcarbamazine.     

Cloning and structural analysis of IgM (μ chain) and the heavy chain V region repertoire in the marsupial Monodelphis domestica

To address the question of the Ig isotype repertoire of non placental mammals, we have examined the Ig expression in the marsupial Monodelphis domestica (grey short tailed opossum). Screening of an opossum spleen cDNA library has previously led to the isolation of full length clones for opossum IgG (γ chain), IgE ( chain) and IgA (α chain). We now present the isolation of several cDNA clones encoding the entire constant regions of the opossum IgM (μ chain). A comparative analysis of the amino acid sequences for IgM from various animal species showed that opossum IgM, within the various animals studied, is the most divergent member of its Ig class. However, it still conforms to the general structure of IgM in other vertebrates. Four Ig classes have now been identified in opossum and only one isotype is apparently present within each Ig class, IgM, IgG, IgA and IgE. Opossum has previously been shown to have a limited VH region diversity, with only two V gene families. Both of these belong to the group III of mammalian VH sequences. This limitation in variability is to some extent compensated for by a large variation in D, P and N regions, both in size and in sequence. However, evidence for the expression of only two functional J segments has so far been detected, which indicates a rather limited diversity also of the J segments in the opossum.     

Characterization of a snake venom neurotoxin which blocks nicotinic transmission in the avian ciliary ganglion

Bungarus multicinctus venom was fractionated by ion exchange chromatography and the various fractions were assayed for their ability to block synaptic transmission through the chick ciliary ganglion. alpha-Bungarotoxin purified from this venom failed to block transmission at 50 micrograms/ml. A second neurotoxin, which we designate Toxin F, blocked transmission at 1-3 micrograms/ml and also blocked ganglionic depolarizations induced by carbachol. Toxin F was clearly distinguishable from alpha-bungarotoxin on the basis of molecular weight (estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis) and isoelectric point. Binding assays revealed that 125I-labeled toxin F bound to two sites in the ciliary ganglion: one site that was shared by alpha-bungarotoxin and toxin F and another site that was recognized solely by toxin F. Carbachol and d-tubocurarine displaced only that [125I]toxin F bound to the shared site and had no effect on [125I]toxin F bound to the site recognized by toxin F alone. The results suggest that toxin F blocks synaptic transmission in the chick ciliary ganglion by a postsynaptic mechanism. Further study is required to determine whether this effect of toxin F is mediated through a direct interaction with ganglionic nicotinic receptors.     

Synaptology of the olfactory bulb of an elasmobranch fish, Sphyrna tiburo

The ultrastructure of the elasmobranch olfactory bulb was examined in order to determine the synaptology of the olfactory circuitry in the bonnethead shark, Sphyrna tiburo. The compartmentalization of the bulb, together with the lack of mitral cell basal dendrites, suggests a different way of performing lateral communication between mitral cells of the olfactory bulb. The results show that granule cells assume an important role by directly interlinking mitral cells. A corollary of this is the segregation of the input onto the mitral cell dendritic arborization: afferent fibers synapse onto the intraglomerular mitral terminals, whereas most local circuit interactions utilize extraglomerular synapses located on the shafts and the somas of the mitral dendrites. Therefore, the elasmobranch synaptic pattern is different from that of higher vertebrates; This might represent the use of a different neural route to achieve the same processing task.     

Characterization of a naturally occurring protease inhibitor in the hemolymph of the scorpion, Heterometrus bengalensis

A protease inhibitor has been purified by ultracentrifugation, affinity chromatography on trypsin-sepharose 4B, and chromatofocusing on PBE-94 from hemolymph of the scorpion Heterometrus bengalensis. Homogeneity of the protease inhibitor was demonstrated by high performance liquid chromatography (HPLC). The protease inhibitor is a monomeric glycoprotein with a molecular weight of 120,000 dalton, which is stable between pH 4 and pH 8. The molecule inhibits serine proteases like trypsin and α-chymotrypsin and shows a noncompetitive mode of inhibition towards trypsin, with a Ki value of 6.1 × 10⁻⁶ mM. Amino acid analysis shows a preponderance of aspartic acid, glutamic acid, serine, and glycine. The protease inhibitor is efficient in inhibiting phenoloxidase activity in both the hemolymph and the isolated phenoloxidase. Melanin synthesis by phenoloxidase may be influenced by this protease inhibitor.     

Reduced postischemic expression of a glial glutamate transporter,GLT1, in the rat hippocampus

Perturbations of the synaptic handling of glutamate have been implicated in the pathogenesis of brain damage after transient ischemia. Notably, the ischemic episode is associated with an increased extracellular level of glutamate and an impaired metabolism of this amino acid in glial cells. Glutamate uptake is reduced during ischemia due to breakdown of the electrochemical ion gradients across neuronal and glial membranes. We have investigated, in the rat hippocampus, whether an ischemic event additionally causes a reduced expression of the glial glutamate transporter GLT1 (Pines et al. 1992) in the postischemic phase. Quantitative immunoblotting, using antibodies recognizing GLT1, revealed a 20% decrease in the hippocampal contents of the transporter protein, 6 h after an ischemic period lasting 20 min induced by four vessel occlusion. In situ hybridization histochemistry with ³⁵S labelled oligonucleotide probes or digoxigenin labelled riboprobes directed to GLT1 mRNA showed a decreased signal in the hippocampus, particularly in CA1. This reduction was more pronounced at 3 h than at 24 h after the ischemic event. We conclude that the levels of GLT1 mRNA and protein show a modest decrease in the postischemic phase. This could contribute to the delayed neuronal death typically seen in the hippocampal formation after transient ischemia.     

Characterization of a 30-kDa Borrelia burgdorferi substrate-binding protein homologue

A Borrelia burgdorferi chromosomal gene encodes a 30-kDa antigen (P30) that has considerable homology with periplasmic substrate-binding proteins of Gram-negative bacteria, and is recognized by antibodies in sera from a subset of patients with Lyme disease and from B. burgdorferi-infected mice. The p30 gene is 801 nucleotides in length and P30 contains 267 amino acids, with predicted molecular mass of 30 kDa. The P30 amino acid region 36–258 has homology to conserved domains of the oligopeptide permease A of Gram-negative bacteria. Immunofluorescence studies using murine anti-P30 serum suggest that P30 is on the outer surface of B. burgdorferi. P30 expression could be detected in representatives of all 3 subspecies of B. burgdorferi sensu lato, but not in all of the tested strains. Antibodies to P30 were detected in sera of 18 out of 82 patients (22%) with Lyme disease, including individuals with early- or late-stage infection. Although antibodies to P30 are present in the sera of C3H/HeN mice infected with B. burgdorferi for at least 90 days, immunization with recombinant P30 does not protect mice from infection. We conclude that P30 is a putative substrate-binding protein of B. burgdorferi and is immunologically recognized in human and murine Lyme borreliosis.     

Characterization of β β β-ADRENERGIC receptors in fish and amphibian lymphoid organs

Cells from goldfish and amphibian lymphoid organs, mainly leukocytes, express high affinity β-adrenergic receptors specific for β-adrenergic ligands (agonists: adrenaline, noradrenaline, terbutaline, and fenoterol; antagonists: CGP-12177, dihydroalprenolol, propranolol, atenolol, and butoxamine). The rank order of ligand potency does not allow their being classified into any known mammalian subtype. Among features that distinguish them from mammalian β¹ and β²-adrenoceptors is much lower affinity for (-)-CGP-12177, obtained in both saturation and kinetic experiments (about 25 nM for goldfish head kidney cells). The density of receptors on goldfish and anuran cells is organ-dependent and comparable to that estimated on mammalian leukocytes. The extraordinarily high receptor density on salamander splenic cells (about 183,000) correlates with the large size of urodele cells. The competition experiments on goldfish cells with propranolol and CGP-12177 suggest the existence of yet another binding site, which may be either another β-AR subtype, or a serotonergic receptor.     

Sleep in a schooling fish, Tilapia mossambica

There are only a few infra-mammalian studies on sleep. For this reason, behavioural sleep in the fish T. mossambica was investigated and the increased response threshold to electrical and feeding stimuli measured. The results of these studies afford little evidence for REM-like sleep; however, the possibility of this kind of sleep is not precluded. Behavioural sleep is established for this species of fish.     

Characterization of lineage restricted forms of a Xenopus CD45 homologue

The leukocyte common antigen, also known as CD45, is a structurally heterogenous molecule ranging in molecular weight from 180 to 220 kDa. CD45 belongs to a family of high molecular weight, cell surface glycoproteins expressed on all hematopoietic lineages with the exception of mature erythrocytes. In higher vertebrates, the highly conserved cytoplasmic domain of CD45 exhibits protein tyrosine phosphatase activity and has been implicated in lymphocyte activation through dephosphorylation of critical tyrosine residues on substrates associated with signal transduction pathways. The monoclonal antibody CL21 recognizes a high molecular weight determinant expressed on the surface of Xenopus leukocytes which was postulated to be a CD45 homologue. In order to determine if lymphocyte subpopulations expressed different molecular weight variants, splenic B cells were identified and isolated on the basis of surface IgM and the CL21 determinant expressed by these cells was compared to the determinant expressed by thymocytes. Immunoprecipitation revealed that IgM+B cells expressed a 220 kDa molecular weight variant whereas thymocytes and IgM-cells expressed a 180 kDa variant. Bone marrow myeloid cells, isolated on the basis of light scatter properties, expressed a determinant which ranged from 150 to 160 kDa. Dephosphorylation experiments utilizing p-nitrophenyl phosphate, ³²P-labeIed Raytide [tyr(P)], or Kemptide [ser(P)] as substrates demonstrated that immunoprecipitated CL21 antigen exhibited tyrosine specific phosphatase activity which was inhibited by sodium orthovanadate. Thus, data based on the presence of enzymatic activity and lineage restricted molecular weight variants support the hypothesis that the CL21 determinant is the amphibian homologue of mammalian CD45, and suggest that both structural and functional elements of CD45 have been conserved during vertebrate evolution.     

Characterization of a divergent glycosomal microbody phosphoglycerate kinase from Trypanosoma brucei

There are 3 loci in the phosphoglycerate kinase (PGK) gene complex of Trypanosoma brucei. The PGK-A gene product, which we term 56PGK, is targeted to glycosomal microbodies and is highly homologous to the parasite's 2 known PGKs (one cytoplasmic and one glycosomal). However, 56PGK contains an 80 amino acid insertion as well as numerous substitutions compared to the other PGKs. The complementation and kinetic analyses described here demonstrate that 56PGK is an authentic phosphoglycerate kinase - the largest yet described. When expressed in Escherichia coli, 56PGK complements the pgk⁻ phenotype. 56PGK was expressed as a fusion protein and purified to near homogeneity. The Michaelis constants are similar to those of other PGKs, being 0.12 and 2.4 mM for Mg-ATP and 3-phosphoglycerate, respectively. As with other T. brucei PGKs, ATP but not GTP or ITP can serve as a phosphate donor during catalysis. No evidence was obtained for phosphate transfer to atypical substrates. 56PGK shows sulfate inhibition at all concentrations tested, rather than the sulfate activation observed with yeast PGK.     

Detection of IgM to hepatitis B core antigen in a reductant containing, chemiluminescence assay

The Abbott PRISM® hepatitis B core (HBc) antigen assay is an automatic in vitro competitive chemiluminescence immunoassay for the detection of total antibody to HBc (anti-HBc) antigen in human serum or plasma. The assay utilizes cysteine solution as a reducing reagent in order to maximize specificity. To help understand the effect of cysteine on detection of anti-HBc antigen, we separated and purified anti-HBc IgM and IgG from human plasma using size exclusion, protein A/G, and affinity chromatography techniques. We showed that cysteine affected the reactivity of anti-HBc IgM with recombinant HBc (rHBc) antigen but not the reactivity of anti-HBc IgG. Anti-HBc IgM treated with cysteine yielded byproducts which were reactive in the PRISM HBcore assay. Reduction-sensitive factor (RSF) — IgM fraction from serum known to be non-specific for anti-HBc activity, similarly treated with cysteine, was no longer reactive in the PRISM HBcore assay. We showed that cysteine treatment is effective against non-specific IgM in human blood. Also, the inclusion of cysteine in the PRISM HBcore assay does not compromise the detection of HBc specific antibodies.     

The winter ulcer bacterium Moritella viscosa demonstrates adhesion and cytotoxicity in a fish cell model

Moritella viscosa is considered the main aetiological agent of ‘winter ulcer’ disease in farmed salmonid fish. To further understand the pathogenesis of this disease, M. viscosa interaction with fish cells was studied using a Chinook salmon embryo cell line (CHSE-214). As winter ulcer appears exclusively at temperatures below 7–8 °C, we attempted to identify if this connection is explained by temperature regulated bacterial virulence. Therefore, infection studies were performed at a temperature range from 4 to 15 °C. At all temperatures, M. viscosa caused CHSE cells to retract and round up, lose their attachment abilities and finally disintegrate. The bacterium adhered to CHSE cells and caused changes to the cytoskeleton, however, it did not invade the cells. Increased adherence was demonstrated at 4 °C compared to adherence at higher temperatures. Extracellular proteins exerted rapid pore formation and lysis of CHSE cells at a temperature range from 4 to 22 °C. Furthermore, only small differences were found comparing extracellular proteomes of M. viscosa from 4 and 15 °C. We propose that the pathogenic mechanisms exerted by M. viscosa on CHSE cells are disruption of the cytoskeleton which affects cell rigidity and structure, followed by pore formation and lysis caused by secreted products from the bacterium. These processes can also occur at temperatures above those experienced from winter ulcer outbreaks. However, the adhesion mechanisms appear to be temperature regulated and may contribute to temperature dependent disease outbreaks.