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PubMed2005

Constitutive expression of tumor necrosis factor-alpha in cytotoxic cells of teleosts and its role in regulation of cell-mediated cytotoxicity.

Cytotoxic T lymphocytes (CTL) and natural killer (NK) cells are the main killer cell populations of the immune system. The mechanisms by which these cells recognize target cells vary considerably, while the effector molecules used to facilitate target cell death are highly conserved. The main pathways utilized by killer cells consist of granule exocytosis and those mediated by members of the TNF superfamily. Nonspecific cytotoxic cells (NCC) are the first identified cytotoxic cell population in teleosts. We have previously demonstrated the expression of granzymes and Fas ligand in these cells. This is the first report of the expression of tumor necrosis factor-alpha in these killer cells. A cDNA coding for TNF was cloned and sequenced from NCC purified from Nile tilapia (Oreochromis niloticus). Factors regulating the transcriptional modulation of TNF in these cells were identified by RT-PCR analysis. The mature form of tilapia TNF was expressed as a recombinant protein and biological activities were analyzed. Using a cross-reacting anti-TNF polyclonal antibody, analysis of TNF expression suggested that tilapia NCC constitutively express the membrane-bound as well as secreted forms of TNF. Recombinant tilapia TNF effectively induced cytotoxicity in the mammalian cell line WEHI, although to a lesser extent compared to the murine TNF. Treatment with recombinant TNF protected NCC from activation-induced cell death. Recombinant tilapia TNF was also effective in upregulation of granzyme transcription in tilapia NCC. These data suggest that teleost TNF may play a role in diverse effector functions of cytotoxic cells from ectotherms, similar to the biological functions described for mammalian TNF.

Animals↗
PubMed2005

Nonspecific cytotoxic cells of teleosts are armed with multiple granzymes and other components of the granule exocytosis pathway.

Granzymes are members of the serine protease family and major components of cytotoxic granules of professional killer cells. Multiple granzymes have been identified from human and rodents with different substrate specificities. Although the significance of granzymes A and B in cell-mediated cytotoxicity has been extensively investigated, recent reports suggest that other granzymes may have either equal or greater importance in mediating cell death. Studies on the evolution of these closely related proteases were hindered by the lack of sequence and biochemical information of granzymes from "lower vertebrates." Here we report the generation of a catalytically active recombinant granzyme identified in the cytotoxic cells of an ectothermic vertebrate. Fully active, soluble recombinant catfish granzyme-1 (CFGR-1) was generated using a yeast-based expression system. In vitro enzyme kinetic assays using various thiobenzyl ester substrates verified its tryptase activity in full agreement with previous observations by sequence comparison and molecular modeling. The tryptase activity that was secreted from catfish NCC during an in vitro cytotoxicity assay strongly correlated with the cytotoxicity induced by these cells. Evidence for additional granzymes with different substrate specificities in NCC was obtained by analysis of the protease activity of supernatants collected from in vitro cytotoxicity assays. Searches of the catfish EST database further confirmed the presence of teleost granzymes with different substrate specificities. Granzyme activity measurements suggested a predominance of chymase and tryptase activities in NCC. Further proof that the granule exocytosis pathway is one of the cytotoxic mechanisms in NCC was provided by the expression of granule components perforin, granulysin and serglycin detected by RT-PCR analysis. These results demonstrate the evidence for a parallel evolution of effector molecules of cell-mediated cytotoxicity in teleosts.

Amino Acid Sequence↗
PubMed2006

Cardiac expression and distribution of nitric oxide synthases in the ventricle of the cold-adapted Antarctic teleosts, the hemoglobinless Chionodraco hamatus and the red-blooded Trematomus bernacchii.

The presence of nitric oxide synthase (NOS) was investigated in the ventricle of two Antarctic teleosts, the hemoglobinless icefish Chionodraco hamatus and its red-blooded counterpart, Trematomus bernacchii. Under unstimulated conditions, in both teleosts, NADPH-diaphorase localised NOS activity in the endocardial-endothelial cells (EEc) and in the myocardiocytes. Application of anti-mammalian endothelial and inducible NOS (eNOS and iNOS, respectively) primary antibodies for immunofluorescence revealed a comparable tissue-specific basal expression of the two isoforms in the two species. eNOS strongly localised at the level of the EEc and, in T. bernacchii, of the vascular endothelium (VE). The enzyme is also localised, albeit to less extent, within the myocardiocytes, and in the epicardium. In contrast, iNOS immunostaining only labels the cytoplasm of the ventricular myocytes. Western blotting analysis identified two peptides with molecular masses of about 135 and 130kDa, similar to those of the mammalian eNOS and iNOS. To verify whether this NOS system is susceptible to septic stimulation, C. hamatus and T. bernacchii were exposed to bacterial lipopolysaccharides (LPS). The treatment did not modify the distribution pattern of the two isoenzymes while it increased the amount of NADPH-diaphorase-dependent reaction product and the expression of both eNOS and iNOS. These results indicate a high phylogenetic conservation of the intracardiac NOS system, emphasizing its importance in the control of the vertebrate heart and its relevance as a general mechanism of defense against pathogens.

Adaptation, Biological↗
PubMed2004

Characterisation and distribution of calcitonin gene-related peptide in a primitive teleost, the eel, Anguilla anguilla and comparison with calcitonin.

Radioimmunoassay (RIA), radioreceptor assay and chromatography were used to study the occurrence of calcitonin gene-related peptide (CGRP) in a primitive teleost, the eel, Anguilla anguilla. Immunologically and biologically active CGRP-like molecules were found in brain, heart, kidney, liver, spleen and ultimobranchial body with the higher concentrations in brain, spleen and heart. Gel exclusion chromatography of heart and spleen extracts followed by SDS-PAGE showed that the eel CGRP-like molecules presented a molecular weight between 3.30 and 3.95 kDa similar to that of human CGRP. The wide distribution of CGRP reflects its multiple role as brain neuromediator and peripheral paracrine effector as described in mammals. In comparison, the distribution of calcitonin (CT) was much more restricted, immunologically and biologically active CT-like molecules being localised in the ultimobranchial bodies (UBB) that is the site of CT synthesis in non-mammalian vertebrates. In plasma, CGRP-like concentrations were 10 to 100 higher than those of CT. These high concentrations in a primitive teleost strengthen the possible endocrine role of CGRP in early vertebrates and emphasise the important role of this hormone in evolution.

Anguilla↗
PubMed2006

Genes coding for intermediate filament proteins closely related to the hagfish "thread keratins (TK)" alpha and gamma also exist in lamprey, teleosts and amphibians.

The "thread keratins (TK)" alpha and gamma so far have been considered highly specialized intermediate filament (IF) proteins restricted to hagfish. From lamprey, we now have sequenced five novel IF proteins closely related to TKalpha and TKgamma, respectively. Moreover, we have detected corresponding sequences in EST and genomic databases of teleosts and amphibians. The structure of the TKalpha genes and the positions of their deduced amino acid sequences in a phylogenetic tree clearly support their classification as type II keratins. The genes encoding TKgamma show a structure typical for type III IF proteins, whereas their positions in phylogenetic trees favor a close relationship to the type I keratins. Considering that most keratin-like sequences detected in the lancelet also exhibit a gene structure typical for type III IF proteins, it seems likely that the keratin gene(s) originated from an ancient type III IF protein gene. According to EST analyses, the expression of the thread keratins in teleost fish and amphibians may be particularly restricted to larval stages, which, in conjunction with the observed absence of TKalpha and TKgamma genes in any of the available Amniota databases, indicates a thread keratin function closely related to larval development in an aquatic environment.

Amino Acid Sequence↗
PubMed2004

Evolutionary aspects of GnRHs, GnRH neuronal systems and GnRH receptors in teleost fish.

Gonadotrophin-releasing hormone (GnRH) was originally believed to be released by a unique set of hypophysiotrophic neurons to stimulate the release of gonadotrophins from the pituitary, therefore acting as a major initiator of the hormonal cascade controlling the reproductive axis. However, it now appears that each vertebrate species expresses two or three GnRH forms in multiple tissues and that GnRHs exert pleiotropic actions via several classes of receptors. This new vision of the GnRH systems arose progressively from numerous comparative studies in all vertebrate classes, but fish in general, and teleosts in particular, have often plaid a leading part in changing established concepts. To date fish still appear as attractive models to decipher the evolutionary mechanisms that led to the diversification of GnRH functions. Not only do teleosts exhibit the highest variety of GnRH variants, but recent data and whole genome analyses indicate that they may also possess multiple GnRH receptors. This paper intends to summarize the current situation with special emphasis on interspecies comparisons which provide insights into the possible evolutionary mechanisms leading to the diversification of GnRH functions.

Animals↗
PubMed2004

Hormone release is tied to changes in cell size in the osmoreceptive prolactin cell of a euryhaline teleost fish, the tilapia, Oreochromis mossambicus.

Prolactin (PRL) cells from a teleost fish, the tilapia, Oreochromis mossambicus, facilitate the direct study of osmoreception. The release of two prolactins, PRL(188) and PRL(177), which act in freshwater osmoregulation in teleost fish, rises in vitro within 5 min after extracellular osmolality falls. An increase in cell size accompanied this rise. Cell size and PRL release also increased, albeit more slowly, following the partial replacement of medium NaCl (55 mOsmolal) with an equivalent concentration of urea, a membrane-permeant molecule. Similar replacement using mannitol, which is membrane-impermeant, elicits no response. These findings suggest that osmoreception is linked to changes in cell volume rather than to extracellular osmolality per se.

Animals↗
PubMed2005

Relationships between aromatase and estrogen receptors in the brain of teleost fish.

Teleost fish are known for exhibiting a high aromatase activity mainly due to the expression of the cyp19b gene, encoding aromatase B (AroB). Recent studies based on both in situ hybridization and immunohistochemistry have demonstrated in three different species that this activity is restricted to radial glial cells. In agreement with measurements of aromatase activity, such aromatase-expressing cells are more abundant in the telencephalon, preoptic area, and mediobasal hypothalamus, although positive cells are also found in the midbrain and hindbrain. Comparative distribution of AroB and estrogen receptor (ERalpha, ERbeta1, and ERbeta2) expression indicates that the preoptic region and hypothalamus are major target for locally produced estradiol (E2) which is likely involved in controlling expression of genes implicated in neuroendocrine regulations. However, AroB and ER have never been reported to be co-expressed in the same cells which is intriguing given that, at least in some species, AroB is strongly up-regulated by E2 itself in agreement with the presence of an estrogen-responsive element (ERE) in the proximal promoter of the cyp19b gene. In vivo data in zebrafish have shown that E2 up-regulates AroB only in radial glial cells. This is in agreement with in vitro transfection experiments indicating that this ERE is functional, but not sufficient, as the E2 regulation of AroB only occurs in glial cell contexts, suggesting a cooperation between ER and so far unidentified glial-specific factors. These data also suggest that radial glial cells may express low amounts of ER that escaped detection until now. The expression of AroB in radial cells, well known for their roles in neurogenesis and now considered as progenitor cells, suggests that local E2 production within these cells could influence the well-documented capacity of the brain of teleosts to grow during adulthood.

Animals↗
PubMed2005

Cranial movements during suction feeding in teleost fishes: Are they modified to enhance suction production?

Suction is produced during prey capture by most teleost fishes. Here, we ask two questions about the functional basis of suction feeding. First, is there variation in the kinematic pattern produced by different species while suction feeding? Second, do species termed 'suction specialists' demonstrate similar modifications to their feeding behavior? We used 10 kinematic variables in a principal component analysis to identify axes of variation among 14 suction feeding teleost species (representing nine families and five orders within the Euteleostei) that demonstrate different feeding habits and habitats. MANOVA and Tukey post hoc tests were used to assess differences among species. Most species clustered together on the principal component axes, suggesting a generalized mechanism that facilitates unidirectional flow. Typically, only one species stood out as 'extreme' on each functional axis, and a species that stood out on one axis did not stand out on others. Only one species, the flatfish Pleuronichthys verticalis, an obligate benthic feeder, demonstrated modifications consistent with enhanced suction production. This species displayed a suite of changes that should enhance suction production, including large hyoid depression, large cranial rotation, and small gape. We suggest that suction performance may be greatest in such obligate benthic feeders because cranial morphology is highly modified and prey are captured from the substrate.

Analysis of Variance↗
PubMed2006

Fine structure of the organ of attachment of the teleost, Garra gotyla gotyla (Ham).

We describe the morphology of the attachment organ (AO) of the teleost, Garra gotyla gotyla (Cyprinidae). It is located ventrally around the mouth opening and used by the species for attachment to submerged rocks in sub-Himalayan streams and rivers where it lives. The AO consists of three crescentic parts and a central callus part. Scanning electron microscopy (SEM) shows the former to possess numerous tubercles, each of which bears about 23-27 curved spines. Light microscopy shows the epidermis of the tuberculated parts to possess one type of cell arranged into 7-8 rows. Transmission electron microscopy (TEM) reveals these cells to contain abundant tonofilaments (hence called the filament cells). The epidermis of the callus part possesses the filament cells and additionally mucous cells, which are absent in the tuberculated parts. The superficial epidermis is apparently keratinized (thickness: 5-8 microm), and a part of the cells of the outer row is modified into spines. These cells show a thick plasma membrane envelope and possess mucous granules (diameter: 0.1-0.3 microm) and bundles of tonofilaments. The cells of the inner two to four rows possess similar organelles and additionally, prominent Golgi bodies and rough endoplasmic reticulum. Immunohistochemically, the cells of the outer row and the spines stain positively for cytokeratin. The cells of the innermost rows (five to eight) possess few tonofilaments and no mucous granules. It is evident that the filament cells of the mid- to upper epidermis are specialized for the production of mucous granules and tonofilaments, which is unique for the teleost epidermis concerned. It appears that the tuberculated parts with spines assist in anchorage and interlocking with the substratum, while the central callus part probably utilizes both suction and frictional mechanisms, and mucous secretion protects the spines from damage during anchorage and abrasion.

Animals↗
PubMed2002

Intestinal bicarbonate secretion by marine teleost fish--why and how?

Intestinal fluids of most marine teleosts are alkaline (pH 8.4-9.0) and contain high levels of HCO(3)(-) equivalents (40-130 mM) which are excreted at a significant rate (>100 microEq kg(-1) h(-1)). Recent research reveals the following about this substantial HCO(3)(-) secretion: (1) It is not involved in acid-base regulation or neutralisation of stomach acid, but increases in parallel with drinking rate at elevated ambient salinities suggesting a role in osmoregulation; (2) In species examined so far, all sections of the intestine can secrete bicarbonate; (3) The secretion is dependent on mucosal Cl(-), sensitive to mucosal DIDS, and immuno-histochemistry indicates involvement of an apical Cl(-)/HCO(3)(-) exchanger. In addition, hydration of CO(2) via carbonic anhydrase in combination with proton extrusion appears to be essential for bicarbonate secretion. The mode of proton extrusion is currently unknown but potential mechanisms are discussed. One consequence of the luminal alkalinity and high bicarbonate concentrations is precipitation of calcium and magnesium as carbonate complexes. This precipitation is hypothesised to reduce the osmolality of intestinal fluids and thus play a potential role in water absorption and osmoregulation. The present studies on European flounder reveal that elevated luminal calcium (but not magnesium) concentrations stimulate intestinal bicarbonate secretion both acutely and chronically, in vitro and in vivo. At the whole animal level, the result of this elevated bicarbonate secretion was increased calcium precipitation with an associated reduction in the osmolality of rectal fluids and plasma. These observations suggest direct functional links between intestinal bicarbonate secretion, divalent cation precipitation and osmoregulation in marine teleost fish.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed2003

Trigeminal somatosensory innervation of the head of a teleost fish with particular reference to nociception.

Trigeminal somatosensory receptors have not been characterised in teleost fish and studies in elasmobranchs have failed to identify nociceptors. The present study examined the trigeminal nerve of a teleost fish, the rainbow trout (Oncorhynchus mykiss) to determine what types of somatosensory receptors were present on the head of the trout specifically searching for nociceptors. Single unit recordings were made from receptive fields on the head of the fish innervated by the trigeminal nerve. Each receptive field was tested for sensitivity to mechanical, thermal and chemical stimulation. Five different receptor types were found: fast adapting receptors responding to mechanical stimulation; slowly adapting receptors responding to mechanical stimuli; polymodal nociceptors responding to mechanical, noxious thermal and chemical stimulation; mechanothermal nociceptors responding to mechanical stimulation and noxious heat; and mechanochemical receptors responsive to mechanical and chemical stimulation. Mechanical thresholds, receptive field diameter, conduction velocities and thermal thresholds of the receptors were determined and there was no significant difference between the receptor types in terms of these properties. Three shapes of action potential (AP) were recorded from these receptors: type 1 with no inflexion; type 2 with an inflexion on depolarisation; and type 3 with an inflexion on repolarisation. Conduction velocity, amplitude and duration of the APs, afterhypolarisation amplitude and duration, as well as the maximum rate of depolarisation were measured for each action potential type. No major differences were found when making comparisons within receptor type and between receptor types. The fish nociceptors had similar physiological properties to nociceptors found in higher vertebrates.

Action Potentials↗
PubMed2001

Identification of rod- and cone-specific phosducins in teleost retinas.

Phosducin (PD) is a regulatory protein of vertebrate phototransduction cascades. In mammalian retina, it has been thought that only one kind of PD commonly exists in both rods and cones. However, we have found two kinds of PD (OIPD-R and OIPD-C) in the retina of a teleost, medaka (Oryzias latipes). In situ hybridization and immunohistochemical analysis demonstrated that OIPD-R and -C are selectively expressed in rods and cones, respectively. The antiserum against medaka PDs recognized two kinds of proteins in bluegill (Lepomis macrochirus) retina. These results suggest that rod- and cone-specific PDs exist in teleost retinas, probably creating differences in light adaptation between rods and cones.

Amino Acid Sequence↗
PubMed2001

A novel second myostatin gene is present in teleost fish.

We report on the isolation and characterisation of the complete cDNA sequence encoding a novel bone morphogenetic protein-like protein (sbMSTN-b) in the teleost fish Sparus aurata. The encoded protein is 68% identical to S. aurata MSTN at the amino acid level, and homologues were also found in Umbrina cirrosa and Tetraodon nigroviridis. Phylogenetic analysis suggests that the MSTN-b gene may be present in most, perhaps all, teleost fish species. RT-PCR on different tissues/stages indicates that MSTN-b is expressed almost exclusively in the central nervous system, starting from late larval stages. Quantitative analyses indicate an increase of sbMSTN-b expression in the brain associated with metamorphosis, at the same time as completion of nervous system differentiation.

Amino Acid Sequence↗
PubMed2002

Progestin membrane receptors involved in the meiotic maturation of teleost oocytes: a review with some new findings.

The endocrine control of oocyte maturation in fish has proven to be a valuable model for investigating rapid, nongenomic steroid actions at the cell surface. Considerable progress has been made over the last decade in identifying and characterizing progestin membrane receptors mediating these actions in fish, in understanding the hormonal regulation and physiological roles of these receptors in oocyte maturation, in elucidating the signal transduction pathways they activate, and in determining their nature. Recent advances on these topics are briefly reviewed. New data demonstrating the involvement of pertussis toxin-sensitive inhibitory G-proteins in induction of oocyte maturation by the maturation-inducing steroid (MIS) in teleosts is also presented. In addition, the cloning strategy to isolate the MIS receptor gene from spotted seatrout ovaries and the characteristics of a novel gene and protein discovered by this approach are discussed. Current evidence suggests this G-protein-coupled receptor-like protein is the long sought after MIS receptor mediating meiotic maturation of teleost oocytes.

Animals↗
PubMed2003

Ovarian follicle ultrastructure in the teleost Synbranchus marmoratus (Bloch, 1795), with special reference to the vitelline envelope development.

Synbranchus marmoratus is a protogynous diandric teleost fish widely distributed throughout South America. The aim of this work was to study the ultrastructure of the vitelline envelope and the relationship among oocyte and their follicular cells during oogenesis. During perinucleolar stage, the oocyte and the follicular cells form microvillar processes that project into the perivitelline space. The oocyte secretes a dense and amorphous material, which appears as the first evidence of the vitelline envelope (VE) development. The VE passes from a double to a multilayered structure during oocyte growth. In mature oocytes, the VE reach a mean thickness of 11 microm, having up to 30 layers. Oocyte microvilli are thinner than the follicular ones and were seen in contact with the follicular plasmalema, however we could not find any contact between the follicular microvilli and the oolemma. Before ovulation, microvillar processes retract and the pore canals seem to collapse. An outer electron dense layer occludes the superficial pore and forms a continuous layer. No jelly or adhesive coatings were seen at least in ovulated eggs sampled from ovarian lumen. Follicular cell and oocyte cytological characteristics do not differ from those described in other teleosts species.

Animals↗
PubMed1998

Morphological basis of the interactions between endocrine cell types in the pancreatic islets of the teleost, Blennius gattoruggine.

The endocrine pancreas of the teleost fish Blennius gattoruggine was studied by immunochemistry using both light and electron microscopy. Generally, one large Brockmann body, along with intermediate and small islets, was found. Cells immunoreactive (IR) to anti-insulin (B), anti-glucagon (A) anti-somatostatin (D) anti-pancreatic polypeptide and anti-PYY sera were detected with B cells located at the center of the islet and the other cell types forming a peripheral mantle. The B-cell cytoplasm showed rows of microtubules close to the secretory granules and perpendicular to the plasmalemma. The ultrathin section images revealed exocytotic and endocytotic features, and the presence of intercellular gap junctions between the plasmalemma of contiguous cells, suggesting intercellular routes of communication, e.g. via autocrine and/or paracrine mechanism. These features were observed in all of the cell types, and were abundant in D cells. D cells were particularly numerous in the islets and were disposed close to A and B cells, as observed in other teleost species. The most peripheral B cells, in closer contact with D cells than the central ones, appeared strongly immunolabeled, perhaps owing to the inhibitory action of somatostatin. Some D cells exhibited a long protrusion directed towards the center of the islet. In view of their cytological characteristics and their secretion, D cells might have an important role in the modulation of A and B-cell secretion in an endocrine and/or paracrine fashion.

Animals↗
PubMed1980

An immune histochemical and enzymic characterization of the muscle fibres in myotomal muscle of the teleost Brachydanio rerio, Hamilton-Buchanan.

The myotomal muscles of teleosts have a complex internal structure. For a better understanding of the functional anatomy of these muscles we analysed the myofibrillar properties of the myotomal muscle fibres in a small teleost: Brachydanio rerio, the zebrafish. By immune histochemical techniques we discerned 4 types of muscle fibres: red lateral fibres, red muscle rim (RMR) fibres, intermediate fibres and white fibres, each with a distinct type of heavy chain myosin. In addition we discerned 2 populations of muscle fibres in which the myofibrils contain mixtures of white and intermediate heavy chain myosin. The immune histochemical classification correlates well with an enzyme histochemical classification of the muscle fibres. In order to find out whether the immune and enzyme histochemical differences correlate with differences in dynamic properties of the muscle fibres, we analysed the ATP-ase activity of glycerinated muscle fibres. 3 of the 5 muscle fibre types which are discriminated by immune histochemical methods could thus be analysed. Differences in histochemical reactions coincide with differences in ATP-ase activities of the glycerinated muscle fibres. 2 classes of muscle fibres were homogeneous as to the ATP-ase activity of their muscle fibres. The 3rd class, the white fibres, could be divided in 2 populations with slightly different ATP-ase activities. The results indicate that the myotomal muscle fibre population is much more heterogeneous than has been considered thusfar. This heterogeneity may allow an almost continuous range of contraction speeds in the myotomal muscle.

Adenosine Triphosphatases↗