Identification and characterization of clonal NK-like cells from channel catfish (Ictalurus punctatus)

TcR alpha, beta, and gamma chain negative cytotoxic NK-like cells were cloned from alloantigen-stimulated PBL obtained from nai;ve channel catfish. Stimulation with allogeneic cells and growth promoting factors are required for their continued in vitro proliferation and cytotoxic activity. These granular cells kill not only the stimulating allogeneic cells, but also unrelated allogeneic targets by a perforin/granzyme-mediated apoptosis pathway. In addition, they are negative for markers that define neutrophils, monocytes/macrophages, and non-specific cytotoxic cells. Although these NK-like clones kill a number of different allogeneic targets, they display interclonal variation in cytotoxicity toward a panel of allogeneic targets, i.e. some clones have no apparent target specificity, while others display a target preference. In addition, flow cytometric analyses revealed that expression of a putative FcmuR, an LFA-1-like molecule, and a putative thymocyte/T cell antigen varies among the different clones, with no clear correlation between surface antigen expression and cytotoxic activity. Although not all clones express a putative FcmuR, it was noted that they all expressed an ITAM containing FcepsilonR gamma chain homolog. This finding suggests that the catfish FcepsilonR gamma chain may potentially be used as an accessory molecule for not only FcmuRs, but also for other unknown activation receptors. These results support the hypothesis that catfish NK-like cells are heterogeneous in terms of target specificities and cell surface phenotype.     

Motoneuronal innervation and mechanical properties of extraocular muscles in the catfish, (Ictalurus punctatus)

Mechanical characteristics and electrical activity were studied in the extraocular muscles of the catfish, Ictalurus punctatus. The contractile properties were determined by stimulation of the individual muscle nerve branches to lateral and medial rectii and the superior and inferior oblique muscles. The speed of contraction was higher than in most other fish muscle, with a twitch contraction time of about 12 ms and a tetanus fusion frequency of 150–170 Hz in all four eye muscles. The fatigue resistance was also high. These properties were the same in fully innervated and partially innervated muscle, largely irrespective of what part of the muscle that was activated. Although different fibre types are known to exist in fish extraocular muscle, it was not possible to obtain functional separation of the mechanical force profile even in the lateral rectus with two distinct motoneuronal innervations. We suggest that polyneuronal innervation of the muscle fibres produces the mechanical responses. Since EMG activity during spontaneous eye movements was similar in the global and the orbital parts of the muscle, all types of fibres in fish extraocular muscle are probably recruited for all types of eye movements.     

Preparation of primary cultures of adult and juvenile catfish (Ictalurus punctatus) hepatocytes

Summary We describe a modified perfusion and collagenase dissociation in a trypsinizing flask for hepatocyte isolation from adult or juvenile catfish liver. When cells isolated in this manner are allowed to attach to plastic culture dishes in serum-free medium, good viabilities and prolonged function can be obtained.     

MHC class II A genes in the channel catfish (Ictalurus punctatus)

In order to characterize the Major histocompatibility complex (MHC) class II A genes of the channel catfish (Ictalurus punctatus) a cDNA library was screened and PCR was performed. Four different full-length cDNA sequences for MHC class II A genes were obtained from a clonal B cell line derived from an outbred fish. Two different genomic sequences and corresponding cDNAs were obtained from a presumably homozygous gynogenetic catfish. The A genes have five exons and four phase one introns. The first exon encodes the 5' untranslated region (UTR) and leader peptide; the second and third exons encode the alpha1 and alpha2 domains, respectively. The connecting peptide, transmembrane and cytoplasmic domains, as well as part of the 3' UTR, are encoded by the fourth exon and the rest of the 3' UTR is encoded by the fifth exon. Southern blot analyses using an exon three probe revealed two to four hybridizing fragments with considerable restriction fragment length polymorphisms evident among randomly selected outbred channel catfish. These findings are consistent with the presence of at least two functional polymorphic MHC class II A gene loci. An unusual aspect of the channel catfish MHC class II alpha chain is its lack of N-linked glycosylation sites.     

Granulomatous inflammation and monstrous giant cells in response to intraperitoneal hormone implants in channel catfish (Ictalurus punctatus)

Plastic implants (2.7 mm maximum dimension) of an ethyl vinyl acetate copolymer (EVAc) matrix, containing inulin, bovine serum albumin (BSA) and luteinizing hormone releasing hormone (LHRH), were covered with impervious EVAc and then surgically placed into the peritoneal cavity of 1-year-old channel catfish, Ictalurus punctatus. In fish kept in cold water (13 degrees C), 10 per cent of the implants per month were encapsulated by granulation tissue. In fish kept in warm water (27 degrees C), 20 per cent of the implants per month were encapsulated, with a total of 86 per cent encapsulated at 5 months. In addition to fibroblasts and capillaries, the granulation tissue included macrophages, neutrophils, lymphocytes, plasma cells, multinucleated giant cells and a matrix of collagen fibres. The density of the fibrous capsule increased with time. In a separate investigation, it was found that the thickness of the capsule was directly proportional to the degree of exposure of the EVAc matrix to the fish (exposure influenced by the rate of dissolution of the capsule content). Monstrous giant cells with up to 600 nuclei per 5 microns thick section were seen in capsules around implants. On intraperitoneally implanted cover glasses, whole giant cells contained up to 6000 nuclei and were interconnected by cytoplasmic bridges. Signs of neoplasia, implant expulsion or massive adhesions were not seen.     

Voltage-clamp analysis of currents produced by glutamate and some glutamate analogues on horizontal cells isolated from the catfish retina

Horizontal cells isolated from the catfish retina were exposed to radiolabeled glutamate, glycine, gamma-aminobutyric acid (GABA), and sucrose to determine if the enzymatic dissociation procedure altered the high-affinity uptake mechanism for GABA and generally reduced membrane selectivity. As in the intact retina, isolated cells could transport GABA but not the other substances. The horizontal cells were voltage clamped using a single low-resistance patch-type electrode. The acidic amino acid L-glutamate, and its analogues kainate and quisqualate, were applied to the cell by pressure ejection from a nearby pipette. All three agonists produced inward currents that reversed near O mV. Quisqualate produced a current with a similar time course as glutamate, but the time course of the response to kainate was faster. The agonists N-methyl-D-aspartate and L-aspartate had little effect on the membrane conductance. The current-to-voltage (I-V) relationship for all three agonists was nonlinear when the membrane potential was hyperpolarized. The nonlinearity was, at least in part, a result of the decreased response to the three agonists. Removal of Mg did not alter this nonlinear relationship. When the inward potassium rectifier was blocked with 100 microM Ba, the response to glutamate was increased compared with the control experiment before block by barium; however, the I-V relationship was still highly nonlinear. Thus glutamate block of the inward potassium current cannot account entirely for the nonlinear I-V. The increase in membrane permeability to specific ions in the presence of an agonist was determined by ion substitution experiments and measuring the shift in the reversal potential. The three agonists appear to increase the membrane permeability to cations but not to anions. The amino acid antagonists cis-2,3-piperidine dicarboxylic acid (PDA) and D-glutamyl glycine (DGG) were bath applied to test their ability to block the depolarizing effects of glutamate. DGG had no measureable effect at 100 microM concentration, whereas PDA reversibly reduced the glutamate response at 1 mM concentration although block was incomplete. Isolated horizontal cells responded to bath-applied glutamate in concentrations of 10-500 microM. In concentrations of glutamate greater than 50 microM, when the membrane potential was held at the resting potential, the inward current reached a maximum followed by a decrease to a steady-state level. This apparent time-dependent desensitization at high agonist concentrations was at least partially removed when Mg was removed from the bathing solution.(ABSTRACT TRUNCATED AT 400 WORDS)     

Antimicrobial peptides derived from hemoglobin are expressed in epithelium of channel catfish (Ictalurus punctatus, Rafinesque)

The beta-chain of the respiratory protein hemoglobin (Hbbeta), has recently been identified in novel sites, including mammalian macrophages and alveolar epithelium, as well as in gill microsomes of fish. However, the functional significance of extra-erythrocytically expressed hemoglobin has been unclear. Here we show inducible expression and upregulation of antimicrobial peptides (AMPs) homologous to Hbbeta in the gill epithelium of channel catfish (Ictalurus punctatus) in response to parasitic (Ichthyophthirius multifiliis, ich) infection. One peptide (HbbetaP-1), while having activity against some fish bacterial pathogens (e.g., Aeromonas hydrophila), had especially potent antiparasitic activity that was specifically lethal (lytic) to the feeding (trophont) stage of ich and also appeared to accelerate the differentiation of trophonts. However, it had no apparent effect on either the disseminative (theront) or reproductive (tomont) stages, nor was it lytic to channel catfish erythrocytes. Fish experimentally challenged with ich confirmed that the HbbetaP-1 sequence was both transcribed and translated in skin and gill epithelium, the target tissues for ich. The Hb AMP concentration expressed in vivo appeared to be well within the antiparasitic concentrations measured in vitro. Our findings suggest that hemoglobin-derived AMPs might play a significant role in the non-specific immune response.     

Molecular identification and expression analysis of tumor necrosis factor in channel catfish (Ictalurus punctatus)

A tumor necrosis factor (TNF) -like gene, encoding a propeptide of 230 amino acids and a mature (soluble) peptide of 162 amino acids, was identified in channel catfish (Ictalurus punctatus). While the catfish protein shared features in common with both mammalian TNF and TNFβ homologs, overall sequence identity/similarity was slightly higher vs. TNF genes when mature TNF sequences were compared. Phylogenetic analysis placed catfish and other fish TNF sequences within their own cluster apart from mammalian TNF and β genes, and supported the suggestion that TNF and β genes separated after the divergence of mammals and teleosts. In contrast to trout and carp, but similar to flounder, catfish TNF was present as a single copy gene. Expression studies demonstrated that catfish TNF mRNA was present in all tested tissues (i.e. liver, spleen, head kidney, mesonephros, gill, thymus, and PBLs) from an unstimulated fish. Moreover, catfish TNF was constitutively expressed in actively proliferating, but otherwise unstimulated, macrophage (42TA) and T cell (G14D; TS32.17) lines, but not in B cell (1G8 or 3B11) or fibroblast lines. TNF expression was upregulated in PBLs, and in G14D and 42TA cells, but not in 3B11 cells, by PMA/calcium ionophore treatment. These results demonstrate that a catfish homolog of TNF has been identified, and indicate that catfish TNF is expressed in catfish in a manner similar to that seen in mammals.     

Proton modulation of ion channels in isolated horizontal cells of the goldfish retina

Transient changes in extracellular pH (pH_o) occur in the retina and may have profound effects on neurotransmission and visual processing due to the pH sensitivity of ion channels. The present study characterized the effects of acidification on the activity of membrane ion channels in isolated horizontal cells (HCs) of the goldfish retina using whole-cell patch-clamp recording. Currents recorded from HCs were characterized by prominent inward rectification at potentials negative to −80 mV, a negative slope conductance between −70 and −40 mV, a sustained inward current, and outward rectification positive to 40 mV. Inward currents were identified as those of inward rectifier K⁺ (Kir) channels and Ca²⁺ channels by their sensitivity to 10 m m Cs⁺ or 20 μ m Cd²⁺, respectively. Both of these currents were reduced when pH_o decreased from 7.8 to 6.8. Glutamate (1 m m )-activated currents were also identified, as were hemichannel currents that were enhanced by removal of extracellular Ca²⁺ and application of 1 m m quinidine. Both glutamate-activated and hemichannel currents were suppressed by a similar reduction of pH_o. When all of these H⁺-inhibited currents were blocked, a small, sustained inward current at −60 mV increased following a decrease in pH_o from 7.8 to 6.8. In addition, slope conductance between −70 and −20 mV increased during this acidification. Suppression of this H⁺-activated current by removal of extracellular Na⁺, and an extrapolated E _rev near E _Na, indicated that this current was carried predominantly by Na⁺ ions.     

Membrane currents of horizontal cells isolated from turtle retina.

1. Membrane currents of horizontal cells isolated from the retina of the turtle, Pseudemys, were characterized by the whole-cell patch-clamp technique. 2. Four membrane currents were identified: an anomalous rectifier blocked by barium, a transient A-current, a sustained L-type calcium current enhanced by Bay K 8644, and a fast, tetrodotoxin-sensitive sodium current. Each of these four currents was found in both horizontal cell somata and axon terminals. 3. The current-voltage relations of axon terminals and somata were similar, but, in the normal operating range of the cell (-30 to -50 mV), the mean slope resistance of the axon terminal was higher (1.38 G omega) than that of the soma (0.26 G omega). 4. Exposure to either glutamate, kainate, or quisqualate induced a sustained inward current in horizontal cell axon terminals. The reversal potential for this current was -3 mV when tested with voltage steps and +9.1 mV when measured by a voltage ramp. The same horizontal cells were insensitive to N-methyl-D-aspartate. 5. A continuum model was developed to compute the degree of signal transfer between a horizontal cell body and its axon terminal. The model consisted of a network of electrically coupled somata that communicates with a network of electrically coupled axon terminals through the connecting axons. The specific membrane resistances used for the model derived from the patch-clamp measures. 6. We computed the voltage change elicited in either the layer of somata or of axon terminals by a static light stimulus of arbitrary dimensions. The amplitude of a spot response as a function of its radius was given by the weighted sum of two Bessel functions with different space constants. 7. The computed responses of the cell body were dominated by the Bessel function with the smaller space constant, whereas those of the axon terminal depended primarily on the Bessel function with the larger space constant. 8. The model predicts that, in contrast to the findings in teleost retina, there is little signal transfer between the somata and axon terminals of horizontal cell in the turtle retina.     

A culture system for mitogen-induced proliferation of channel catfish (Ictalurus punctatus) peripheral blood lymphocytes

Summary Techniques are described for the isolation and culture of channel catfish peripheral blood leukocytes in the presence of lymphocyte mitogens. The mitogens used are lipopolysaccharide, concanavalin A, and a mixture of a phorbol ester and the calcium ionophore A23187. Each of these mitogens cause channel catfish lymphocytes to proliferate vigorously in vitro at an optimal culture temperature of 27° C. Essential components of the tissue culture medium employed are 10% human plasma and 5% catfish sera, which act synergistically in supporting the in vitro proliferation of channel catfish lymphocytes.     

Bacterial sialic acid modulates activation of the alternative complement pathway of channel catfish (Ictalurus punctatus)

The alternative complement pathway (ACP) provides the non-immune channel catfish (Ictalurus punctatus) with protection against many Gram-negative bacteria. Very little serum bactericidal activity (0-13%) was found against 8 fish pathogens, but a strong bactericidal response (100%) was found against 7 non-pathogens. MgEGTA chelation of catfish serum did not essentially change the bactericidal results. Catfish serum heated at 56 degrees C and serum adsorbed with zymosan had no bactericidal activity. This demonstrated that the ACP was responsible for the bactericidal response. The molecular nature of the microbial surface determines whether or not the ACP will be activated. A relative lack of surface sialic acid has been found to be important for binding complement Factor B of the ACP by susceptible microbial surfaces. This study therefore examined the 15 Gram-negative bacterial fish pathogens and non-pathogens by determining their sialic acid content and their ability to elicit a bactericidal response by the catfish ACP. It was found that there was very little bactericidal activity against the fish pathogens that contained sialic acid but a very strong bactericidal response (100%) against the non-pathogens that lacked sialic acid (p = .0043). A relative lack of sialic acid or no sialic acid therefore correlated with a strong bactericidal response by the catfish ACP. Neuraminidase treatment of the bacterial fish pathogens to remove sialic acid greatly increased the bactericidal response against them by the catfish ACP when compared with untreated bacteria (p = .0431).     

Ionic currents of cultured horizontal cells isolated from white perch retina

Horizontal cells from the retinas of white perch were isolated and maintained in cell culture for 3 days to 3 wk. Four morphologically distinct types of horizontal cells could be identified in culture and were labeled types H1, H2, H3, and H4. Whole-cell patch-clamp techniques were used to study the ionic currents present in the four cell types. In all cells, depolarizing commands above threshold elicited a fast-inward current followed by an outward current. The fast-inward current was abolished by tetrodotoxin (TTX) or 0 Na+ Ringer's, indicating the current was carried by Na+. In H1, H2, and H3 cells, the outward current, carried by K+, consisted of two components: a transient current (IA), blockable with 4-aminopyridine (4-AP), tetraethylammonium (TEA), or intracellular cesium and a sustained current that could be blocked with TEA. The H4 cell had only the sustained current. An inward rectifying K+ current (anomalous rectifier) was observed in the four cell types. The current was sensitive to the extracellular K+ concentration. Its activation showed two components: an instantaneous component and a slower component. The slow component becomes faster with greater hyperpolarizations. The four cell types possessed a small, sustained Ca2+ current that, under normal conditions, was masked by the inward Na+ current and outward K+ currents.     

Identification,annotation and expression analysis of 29 Rho GTPase genes from channel catfish (Ictalurus punctatus) after bacterial infections

The Rho family GTPases are a group of small monomeric G proteins, which are molecular switches in signaling pathways. They have been known to regulate a diverse range of cellular processes including actin cytoskeleton rearrangement and microtubule dynamics. In particular, their participations in immune responses are also significant. However, little information of the Rho GTPases is available in teleost including channel catfish, an economically important species and one of the best teleost models forimmunological research. In this study, Rho GTPase genes were identified from channel catfish and well annotated by phylogenetic and syntenic analyses. Their expression profiles were determined in channel catfish healthy tissues and infected tissues. Altogether seven Rho GTPase genes were significantly regulated after bacterial infection, with six genes in the gill after Flavobacterium columnare challenge and two genes in the intestine in response to Edwardsiella ictaluri. All the differentially expressed genes were up-regulated soon after bacterial infection. Different expression patterns between the two experiments were observed, which may be attributed to tissue-specific regulation or pathogen-specific regulation. These results suggested that Rho GTPases play important roles in immune responses to bacterial pathogens, setting a foundation for future investigation on Rho GTPases.