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PubMed2006

Proteome analysis identifies novel protein candidates involved in regeneration of the cerebellum of teleost fish.

In contrast to mammals, adult teleost fish exhibit an enormous potential to regenerate neuronal tissue after injuries to the CNS. By combining a well-defined cerebellar lesion paradigm with differential proteome analysis at a post-lesion survival time of 3 days, we screened for protein candidates involved in repair of the fish brain. Out of nearly 900 protein spots detected on 2-D gels, spot intensity was significantly increased at least twofold in 30 spots and decreased to at least half the intensity of control tissue in 23 spots. The proteins associated with 24 of the spots were identified by PMF and MS/MS fragmentation. The cellular localization and the spatiotemporal patterns of two of these proteins, beta-actin and beta-tubulin, were further characterized through immunohistochemistry. Comparison of the observed changes in protein abundance with the previously characterized events underlying regeneration of the cerebellum suggests that the proteins identified are especially involved in cellular proliferation and survival, as well as axonal sprouting.

Animals↗
PubMed2003

Acute CO2 tolerance during the early developmental stages of four marine teleosts.

Ocean sequestration of CO2 is proposed as a possible measure to mitigate climate changes caused by increasing atmospheric concentrations of the gas, but its impact on the marine ecosystem is unknown. We investigated the acute lethal effect of CO2 during the early developmental stages of four marine teleosts: red sea bream (Pagrus major), Japanese whiting (Sillago japonica), Japanese flounder (Paralichthys olivaceus), and eastern little tuna (Euthynnus affinis). The percentages of larvae that hatched and survived were not affected by exposure to water with a PCO2 of 1.0 kPa (= 7.5 mmHg) within 24 h. Median lethal PCO2 values for a 360-min exposure were 1.4 kPa (cleavage), 5.1 kPa (embryo), 7.3 kPa (preflexion), 4.2 kPa (flexion), 4.6 kPa (postflexion), and 2.5 kPa (juvenile) for red sea bream; 2.4 kPa (cleavage), 4.9 kPa (embryo), 5.9 kPa (preflexion), 6.1 kPa (flexion), 4.1 kPa (postflexion), and 2.7 kPa (juvenile) for Japanese whiting; 2.8 kPa (cleavage) and > 7.0 kPa (young) for Japanese flounder; and 11.8 kPa (cleavage) for eastern little tuna. Red sea bream and Japanese whiting of all ontogenetic stages had similar susceptibilities to CO2: the most susceptible stages were cleavage and juvenile, whereas the most tolerant stages were preflexion and flexion.

Animals↗
PubMed1999

Functional diversity of vertebrate ARNT proteins: identification of ARNT2 as the predominant form of ARNT in the marine teleost, Fundulus heteroclitus.

The aryl hydrocarbon receptor nuclear translocator (ARNT) is a member of the bHLH/PAS protein superfamily. ARNT dimerizes with several PAS superfamily members, including the ligand-activated aryl hydrocarbon receptor (AHR), forming a complex that alters transcription by binding specific elements within the promoters of target genes. Two genes encode different forms of the protein in rodents: ARNT1, which is widely expressed, and ARNT2, which is limited to the brain and kidneys of adults and specific neural and branchial tissues of embryos. In an effort to characterize aryl hydrocarbon signaling mechanisms in Fundulus heteroclitus, a marine teleost that can develop heritable xenobiotic resistance, we have isolated a liver cDNA encoding an ARNT homolog. The protein exhibits AHR-dependent DNA binding capability typical of other vertebrate ARNTs. Unexpectedly, phylogenetic analysis reveals that the cDNA encodes an ARNT2. This is the only detectable ARNT sequence in Fundulus liver, gill, ovary, and brain, suggesting that ARNT2 is the predominant form of ARNT in this species. Also surprising is the relative lack of sequence identity with another fish ARNT protein, rainbow trout ARNTb, which we show forms a distinct branch outside the ARNT1 and ARNT2 clades in phylogenetic analyses. Functional diversity of ARNT proteins in fish may have important implications for the assessment of aryl hydrocarbon effects on natural populations. The increasing use of fish models in developmental and toxicological studies underscores the importance of identifying taxon-specific roles of ARNT proteins and their potential dimeric partners in the PAS superfamily.

Amino Acid Sequence↗
PubMed2000

Identification and characterization of a cDNA encoding cytochrome P450 3A from the fresh water teleost medaka (Oryzias latipes).

A new member of the CYP3A gene family has been cloned from the teleost fish medaka (Oryzias latipes) by reverse-transcriptase polymerase chain reaction (RT-PCR). Degenerate primers homologous to highly conserved regions of known CYP3A sequences were used for initial RT-PCRs. Individual PCR products were cloned, sequenced, and identified as those belonging to the cytochrome P450 superfamily based on amino acid sequence similarity and the presence of the highly conserved heme-binding region. PCR products were subsequently used as probes to screen a complementary DNA library. A full-length cDNA clone was identified containing a 1758-base-pair (bp) insert with an open reading frame encoding a single peptide of 500 amino acids. Comparisons of the deduced amino acid sequence to other known cytochrome P450 sequences indicate that this gene product is most similar to the CYP3A gene family and has been designated as CYP3A38 by the cytochrome P450 nomenclature committee. Northern blot analysis identified two abundant CYP3A related transcripts in liver of both male and female adults and demonstrated quantitative differences in abundance according to gender. Similarly, Western blot analysis demonstrated the presence of two abundant cytochrome P450 related proteins in liver of both male and female adults. These results suggests that O. latipes contains multiple forms of CYP3A. Heterologous expression of CYP3A38 cDNA in HEK 293 cells produced a single protein that was reactive with anti-scup P450A (CYP3A) polyclonal antibody. Microsomes of HEK 293 cells expressing recombinant CYP3A38 protein actively catalyzed the hydroxylation of testosterone.

Amino Acid Sequence↗
PubMed1997

Isolation of a cytochrome P450 3A cDNA sequence (CYP3A30) from the marine teleost Fundulus heteroclitus and phylogenetic analyses of CYP3A genes.

A reverse transcriptase polymerase chain reaction (RT-PCR) protocol, using degenerate PCR-primers specific to highly conserved regions of mammalian CYP3A genes, was employed to amplify a 400 base pair cDNA fragment from Fundulus heteroclitus liver RNA. The 124 amino acid sequence deduced from this cDNA sequence was aligned with corresponding sequences from representative members from the CYP1, 2, 3, and 4 gene families retrieved from the GenBank database. Phylogenetic trees were constructed using distance-matrix and maximum parsimony methods. The F. heteroclitus sequence and all mammalian CYP3A sequences cluster together when compared to sequences of members of CYP gene families 1, 2, and 4. This fish sequence was 57 to 70% identical to the corresponding region of mammalian CYP3A genes. These data indicate that the sequence obtained from F. heteroclitus represents a teleost fish CYP3A gene and it has been designated CYP3A30.

Amino Acid Sequence↗
PubMed1995

New rods move before differentiating in adult teleost retina.

To understand the cellular context of neuronal differentiation in the vertebrate retina, we analyzed the behavior of a class of progenitor cells in the outer nuclear layer of the teleost retina which divide throughout the animal's life and generate only rod photoreceptors. We present evidence that these progenitors reside adjacent to the outer limiting membrane of the retina during all phases of cell cycle. After final cell division, postmitotic cells move inward, toward the vitreal boundary of the outer nuclear layer, before they give rise to new rods. This movement is remarkable because it occurs in the mature, functioning retina. We hypothesize that only one of the two progenitor daughter cells moves while the other remains adjacent to the outer limiting membrane to divide again.

Animals↗
PubMed1999

Changes in liver enzyme activity in the teleost Sparus aurata in response to cadmium intoxication.

Enzyme activity modulation by cadmium in the liver of the teleost fish Sparus aurata was investigated in vivo following 3 and 6 days of CdCl2 administration (2.5 mg/kg body wt). The specific activities of the mitochondrial enzymes NAD-isocitrate dehydrogenase, succinate dehydrogenase, and malate dehydrogenase were stimulated by approximately 20% after 3 days administration and were further increased (by about 40%) after 6 days treatment. In comparison with these enzymes, the activities of glutamate-oxaloacetate transaminase (GOT) and glutamate-pyruvate transaminase (GPT) in mitochondria were less stimulated after the two indicated intervals of treatment. Cadmium significantly reduced the activities of liver cytoplasmic GOT and GPT while a simultaneous increase occurred in the serum activities of these same enzymes. The activity of liver NADPH-cytochrome P450 reductase was stimulated by 25 and 40% after 3 and 6 days cadmium intoxication, respectively. Lastly, the antioxidant enzymes glutathione peroxidase and glutathione reductase in liver and catalase in both liver and blood were strongly reduced after 3 and 6 days cadmium administration. These data suggest that cadmium in fish hepatocytes alters cell membrane structure and concomitantly induces some perturbation in the integrity of the mitochondrial membrane.

Alanine Transaminase↗
PubMed1993

Studies on the hypocalcemic actions of salmon calcitonin and ultimobranchial gland extracts in the freshwater teleost Cyprinus carpio.

A single injection of synthetic salmon calcitonin (sCT) or homologous ultimobranchial gland (UBG) extract caused a significant reduction in total and ultrafiltrable plasma Ca2+ in a freshwater teleost, Cyprinus carpio. A time-bound analysis on the effect of sCT or UBG extract caused a short-duration hypocalcemia and the response was more pronounced in fish kept in low-Ca2+ water. Sexually immature fish showed a greater response than did mature fish. In all the studies the protein-bound Ca2+ fraction remained unaffected. The observation thus indicate a potent hypocalcemic role of calcitonin or UBG in freshwater common carp under laboratory conditions.

Animals↗
PubMed1995

Evidence that the inhibitory effects of stress on reproduction in teleost fish are not mediated by the action of cortisol on ovarian steroidogenesis.

Ovarian follicles of goldfish, common carp, and the sparid Pagrus auratus (New Zealand snapper) were incubated in vitro to assess the effects of cortisol (F) on ovarian steroidogenesis. Unstimulated goldfish follicles produced little testosterone (T) or 17 beta-estradiol (E2), whereas both carp and snapper follicles spontaneously produced E2 and to a lesser extent T. Goldfish follicles produced increased amounts of E2 in response to treatment with human chorionic gonadotropin (hCG), whereas carp and snapper follicles did not. However, stimulation of carp follicles with maturational carp gonadotropin (cGTH-II) resulted in dose-dependent increases in production of E2. Treatment of follicles of all three species with T resulted in E2 production to levels as high as or higher than those following treatment with hCG or cGtH-II. Cotreatment of follicles with T and hCG or cGTH-II did not result in higher E2 production than treatment with T alone. Goldfish follicles treated with 25-hydroxycholesterol showed increases in E2 production that were similar to those occurring following treatment with T. Treatment of follicles with F at a range of doses up to 1 microgram.ml-1 had no inhibitory effect on T or E2 production in any treatment combination in any of the species examined. In several cases, follicles incubated with T and/or hCG produced more E2 in the presence of F than they did without F. The results suggest either that the observed inhibitory effects of stress in a range of teleost species are not mediated by F or that they arise higher in the endocrine pathway than at the level of ovarian steroidogenesis.

Animals↗
PubMed1996

Immunochemical detection of parathyroid hormone-related protein in the saccus vasculosus of a teleost fish.

Using antisera to regions of human parathyroid hormone-related protein (PTHrP) the saccus vasculosus (SV) of the sea bream (Sparus aurata) has been shown to contain immunoreactive PTHrP. By immunohistochemistry (IHC) the epithelial coronet cells in fixed and wax-embedded SV tissue reacted with antisera to the prepro region of human PTHrP (-13 to +2), the N-terminus PTHrP (1-16), and the midmolecule PTHrP (50-69). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of saccus extracts and incubation media contained two major proteins of 14.3 and 15 kDa. By Western blotting these two proteins both reacted with the three antisera used for IHC, suggesting that they are immunochemically similar to human PTHrP (1-84). Ultrastructurally the coronet cells of Sparus saccus vasculosus resembled coronet cells described for other teleosts, with an abundant smooth endoplasmic reticulum (SER) which was more highly organized in the coronets. IHC at EM level showed reaction mainly with the membranes of the SER. These results suggest that S. aurata saccus vasculosus may produce a PTHrP-like molecule similar to human PTHrP.

Animals↗
PubMed1997

Changes in plasma arginine vasotocin (AVT) concentration and dorsal aortic blood pressure following AVT injection in the teleost Platichthys flesus.

Dorsal aortic blood pressure and plasma arginine vasotocin (AVT) concentrations have been assessed in free swimming, chronically cannulated flounder following AVT injection. Intraarterial AVT at doses of 4.76 x 10(-12) mol.kg-1 and greater caused a biphasic change in blood pressure (an initial fall followed by a sustained pressor response). Doses above 4.76 x 10(-12) mol.kg-1 were associated with plasma AVT concentrations (20 min after injection) 2-3 orders of magnitude greater than the physiological range and must be considered pharmacological. Injection of the lowest pressor AVT dose (4.76 x 10(-12) mol.kg-1), 20 min after injection, increased plasma AVT concentrations to 23.4 +/- 6.1 fmol x ml-1. This increase is close to plasma AVT concentrations recently reported in untreated fish; however, in the initial period after injection plasma levels were calculated to be considerably higher than the physiological range. These results confirm that AVT is pressor in flounder but suggest that the pressor response may occur only at circulating AVT levels above the normal physiological range. The biphasic response to AVT and the differing responses to mammalian V1 and V2 type receptor agonists in the current work suggests that AVT may contribute to regional blood flow distribution in teleosts rather than blood pressure regulation.

Animals↗
PubMed1997

Osmoregulatory actions of growth hormone and prolactin in an advanced teleost.

To date, growth hormone (GH) is known to contribute to seawater adaptation only in salmonid fishes (primitive Euteleostei). Accordingly, the effects of homologous GH and two forms of homologous prolactin (PRL177 and PRL188) on hypoosmoregulatory ability and gill Na+,K(+)-ATPase activity in a more advanced euryhaline cichlid fish, the tilapia (Oreochromis mossambicus), were examined. Following adaptation of hypophysectomized fish to 25% seawater for 3 weeks, fish were given four injections of hormone or vehicle. They were then exposed to 100% seawater for 12 hr and examined for changes in plasma osmolality. Tilapia GH (0.02 and 0.2 microgram/g) significantly improved the ability of tilapia to decrease plasma osmolality following transfer to full-strength seawater, in a dose-related manner. Growth hormone treatment also significantly stimulated gill Na+,K(+)-ATPase activity (0.5 microgram/g). Both tilapia PRLs (PRL177 and PRL188) increased plasma osmolality in 100% seawater and reduced gill Na+,K(+)-ATPase activity, the effects induced by PRL188 being more significant than those by PRL177. Thus, GH may be involved in seawater adaptation of tilapia, a species belonging to the most advanced teleost super-order (Acanthopterygii), whereas both PRLs in tilapia are not involved in seawater adaptation.

Adaptation, Physiological↗
PubMed1998

FMRFamide-like immunoreactivity in the olfactory system responds to morphine treatment in the teleost Clarias batrachus: involvement of opiate receptors.

In view of the close relationship between the FMRF-related peptides and the central opiate-sensitive system, we investigated the effects of morphine, alone and in combination with naloxone, on the FMRFamide-like immunoreactivity in the olfactory system of the teleost, Clarias batrachus. In the olfactory system of normal and untreated fish, FMRFamide-like immunoreactivity was confined to the ganglion cells and fibers of the terminal nerve; the cells in the olfactory epithelium per se or the olfactory nerve were not immunoreactive. Intensely immunoreactive cells appeared in the olfactory epithelium following 2 h of intracranial morphine administration. FMRFamide-like immunoreactivity also appeared in the olfactory nerve fibers as they ran caudally and arborized in the glomerular layer of the bulb. However, immunoreactivity in the ganglion cells of the terminal nerve and the ensuing fibers was abolished, suggesting the transport/release of the immunoreactive material. Pretreatment with naloxone, a potent opiate receptor antagonist, reversed the effects of morphine, suggesting the involvement of opiate receptors in the regulation of the ganglion cells of the terminal nerve. The results provide initial immunocytochemical evidence in favor of a relationship between the opiates and FMRFamide-containing systems within the framework of the olfactory system.

Amino Acid Sequence↗
PubMed1999

Effects of prolactin and growth hormone on strategies of hypoosmotic adaptation in a marine teleost, Sparus sarba.

Silver seabream (Sparus sarba) held in seawater (33 per thousand) or acclimated to a hypoosmotic environment of 6 per thousand were given intraperitoneal injections of saline (0.8% NaCl), recombinant bream growth hormone (rbGH, 1 microg/g), or ovine prolactin (oPRL, 6microg/g) for 7 consecutive days. Serum Na+ levels were unaffected by hypoosmotic acclimation and rbGH and oPRL treatment. Treatment of seawater fish with oPRL resulted in hyperchloremia. In 6 per thousand, saline-treated fish exhibited elevated branchial chloride cell (CC) numbers and exposure indices, all of which were markedly reduced by oPRL. CC numbers and morphometrics were unaffected by oPRL in seawater fish. In contrast, rbGH treatment of seawater fish resulted in elevated CC numbers, apical area, and fractional area and, in 6 per thousand fish, elevated CC fractional area and exposure numbers. Branchial Na+-K+-ATPase activity reduced in saline-treated fish adapted to 6% but was unaffected by rbGH regardless of salinity. oPRL reduced activity in both seawater and 6 per thousand-adapted fish. Neither hypoosmotic adaptation nor oPRL had any effect on renal Na+-K+-ATPase activity whereas rbGH reduced activity in both 33 and 6 per thousand. Saline-treated fish adapted to 6 per thousand exhibited reduced Na+-K+-ATPase activity in most regions of the intestine. Treatment with rbGH did not change intestinal Na+-K+-ATPase activity of seawater fish but elevated activity in the anterior regions (esophagus and stomach) of 6 per thousand-adapted fish. Treatment with oPRL elevated Na+-K+-ATPase activity throughout the gastrointestinal tract of seawater fish and in the anterior reaches of 6 per thousand-adapted fish. The data indicated that the as yet uncharacterized osmoregulatory roles of PRL and GH in seabream may warrant further attention as the present study connoted differing responses to that of other teleosts studied.

Adaptation, Physiological↗
PubMed1999

Beta-endorphin-like immunoreactivity in the forebrain and pituitary of the teleost Clarias batrachus (Linn.).

The organization of beta-endorphin-like immunoreactivity in the olfactory system, forebrain, and pituitary of the teleost Clarias batrachus was investigated. Immunoreactivity was prominently seen in the sensory neurons and basal cells in the olfactory epithelium and in some cells in the periphery and center (granule cells) of the olfactory bulb. Immunoreactive fibers in the olfactory nerve enter the olfactory nerve layer of the olfactory bulb and branch profusely to form tufts organized as spherical neuropils in the glomerular layer. While fascicles of immunoreactive fibers were seen in the medial olfactory tracts, the lateral olfactory tracts showed individual immunoreactive fibers. Immunoreactive fibers in the medial olfactory tract extend into the telencephalon and form terminal fields in discrete telencephalic and preoptic areas; some immunoreactive fibers decussate in the anterior commissure, while others pass into the thalamus. While neurons of the nucleus lateralis tuberis revealed weak immunoreactivity, densely staining somata were seen at discrete sites along the wall of the third ventricle. Although a large population of immunoreactive cells was seen in the pars intermedia of the pituitary gland, few were seen in the rostral pars distalis and proximal pars distalis; immunoreactive fibers were seen throughout the pituitary gland.

Animals↗
PubMed1999

Teleost-type angiotensin is present in Australian lungfish, Neoceratodus forsteri.

Angiotensin I (ANG I) was produced from the incubation of lungfish plasma with homologous kidney extracts. The purified peptide was found to have the sequence of H-Asn-Arg-Val-Tyr-Val-His-Pro-Phe-Thr-Leu-OH, which is homologous for the first eight residues with all teleost angiotensins so far sequenced, although lungfish generally possess tetrapod-type hormones. The lungfish decapeptide (ANG I) induced dose-dependent increases in arterial pressure in the rat. The lungfish octapeptide (ANG II) released aldosterone from kidney-adrenal tissue in vitro in a dose-dependent manner and induced dose-dependent increases in arterial pressure of the lungfish. Substitution of asparagine with aspartic acid in the first position (tetrapod-type ANG II) did not alter the blood pressure response significantly, but a second substitution of the valine in the (5)-position with isoleucine (ANG II form found in human and rat) abolished the rise in arterial pressure in lungfish over the same dose range.

Angiotensin I↗
PubMed1999

Characterization of teleost insulin receptor family members.

Insulin from mammals and fish has been used to determine insulin-binding affinities and receptor numbers with remarkable similarities between these two vertebrates, suggesting functional conservation. Yet, the nature and structure of teleost insulin receptors are not known. Therefore, the cloning and mRNA characterization of rainbow trout insulin receptors were undertaken. Three insulin receptor cDNAs were isolated by screening a cDNA library, confirmed as separate genes by genomic Southern hybridization, and designated as rainbow trout insulin receptor a (rtIR a), rainbow trout insulin receptor b (rtIR b), and rainbow trout insulin receptor c (rtIR c). A high degree of amino acid identity was observed between rainbow trout insulin receptors (rtIRs) and their human homolog, confirming the structural similarities between mammalian and fish insulin receptors. Reverse transcription-polymerase chain reaction from total RNA using either oligo(dT) or random hexamer primers resulted in a diminished ability to detect rtIR a and rtIR b mRNA when oligo(dT) was used, suggesting developmental and tissue-specific polyadenylation. The highest steady-state levels of rtIR mRNAs were consistently detected in juvenile and adult pyloric caeca (which also contained adipose and pancreatic tissue), while the lowest levels were consistently found in muscle. A high level of rtIR b and rtIR c mRNA was also found in ovary, while a high level of rtIR a was found in adult brain. Significant differences were also found between steady-state rtIR mRNA levels in corresponding juvenile and adult tissues. These results suggest a complex expression pattern of insulin receptor mRNAs in partial tetraploid fish.

Aging↗
PubMed2000

Circulating C-21 steroids in relation to reproductive condition of a viviparous marine teleost, Sebastes rastrelliger (grass rockfish).

Plasma concentrations of free, glucuronidated, and sulfated steroids were measured in grass rockfish (Sebastes rastrelliger) at identified stages of ovarian development and pregnancy using radioimmunoassays validated for the detection of individual steroids or compounds with a particular configuration. Changes in reproductive status were most clearly reflected in concentrations of free C-21 steroids. Previtellogenic, vitellogenic, and postspawn fish exhibited uniformly low concentrations of circulating C-21 steroids while pregnant fish showed a pronounced and significant increase in a series of free 17,20beta-dihydroxylated steroids together with 17,20alpha-P. Among individual steroids, the compound exhibiting the greatest fluctuation in relation to reproductive condition was 17,20beta-P-5beta, which during pregnancy showed a 22-fold increase from basal concentrations. Smaller relative increases in association with pregnancy were also seen in 17, 20beta-P, 17,20beta,21-P, and 17,20alpha-P (3.5-, 3.5-, and 5.5-fold increases, respectively). Fish in the final stages of pregnancy or which had recently spawned exhibited uniformly low concentrations of the C-21 steroids, indicating a drop in circulating amounts of these compounds around the time of parturition. The hormone profiles established during the annual reproductive cycle of the grass rockfish suggest that C-21 steroids may contribute to the endocrine mechanisms which regulate viviparity in this highly fecund marine teleost. The C-21 steroids characterized in this study may provide appropriate reference compounds in the future evaluation of this concept.

Aging↗