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Age dependence of O6-methylguanine-DNA methyltransferase activity and its depletion after carcinogen treatment in the teleost medaka (Oryzias latipes).

O6-Methylguanine-DNA methyltransferase (O6-MT) is considered to play an important role in the repair of DNA lesions induced by alkylating carcinogens in a wide range of animals. The activity of O6-MT was compared in liver extracts from the teleost medaka (Oryzias latipes) at various ages (3-5 years old) reared under natural conditions. O6-MT activity decreased significantly with advancing age. When medaka were exposed continuously to the alkylating agent methylazoxymethanol (MAM) acetate at levels of 0.1, 0.15 and 0.3 ppm in water, O6-MT activity was markedly reduced from days 1 to 7, with a slight increase thereafter. Furthermore, when fish were exposed to MAM acetate at levels of 1-2 ppm for 1 h and then maintained in normal tap water, O6-MT activity remained suppressed for 2 weeks, followed by a partial recovery.

Aging↗

Teleost HoxD and HoxA genes: comparison with tetrapods and functional evolution of the HOXD complex.

In tetrapods, Hox genes are essential for the proper organization and development of axial structures. Experiments involving Hox gene inactivations have revealed their particularly important functions in the establishment of morphological transitions within metameric series such as the vertebral column. Teleost fish show a much simpler range of axial (trunk or appendicular) morphologies, which prompted us to investigate the nature of the Hox system in these lower vertebrates. Here, we show that fish have a family of Hox genes, very similar in both number and general organization, to that of tetrapods. Expression studies, carried out with HoxD and HoxA genes, showed that all vertebrates use the same general scheme, involving the colinear activation of gene expression in both space and time. Comparisons between tetrapods and fish allowed us to propose a model which accounts for the primary function of this gene family. In this model, a few ancestral Hox genes were involved in the determination of polarity in the digestive tract and were further recruited in more elaborate axial structures.

Amino Acid Sequence↗

A comparative assessment of the adrenotoxic effects of cadmium in two teleost species, rainbow trout, Oncorhynchus mykiss, and yellow perch, Perca flavescens.

Rainbow trout (Oncorhynchus mykiss) and yellow perch (Perca flavescens) have a different sensitivity to cadmium (Cd) in vivo (trout < LC50 < perch). Metals and particularly Cd impair cortisol secretion by adrenocortical cells in both species. The purpose of the present study was to assess in vitro the effect of Cd on cortisol secretion by adrenocortical cells of trout and perch, to compare the sensitivity of adrenal steroidogenesis in these two teleosts. Adrenocortical cells were exposed to Cd for 60 min, then stimulated with ACTH, dbcAMP or with pregnenolone, a cortisol precursor. Cd inhibited ACTH-stimulated cortisol secretion in a dose-dependent manner in both fish species, however, the EC50s (concentration resulting in 50% inhibition of cortisol secretion) was significantly lower in trout (EC50 = 0.09 mM) than perch (EC50 = 0.26 mM). To test the specificity of Cd to act as an endocrine disrupter, the LC50 (concentration that kills 50% of the cells) was also evaluated to determine the LC50/EC50 ratio ( LC50/EC50(O.mykiss) = 175.6 > LC50/EC50(P.flavescens) = 37.7). Adrenocortical cells of trout were more sensitive than those of perch and Cd had a higher endocrine-disrupting potential and specificity in trout than in perch. However, in both species, Cd had the same effect on ACTH, dbcAMP and pregnenolone-stimulated cortisol secretion, with pregnenolone maintaining cortisol secretion until cell viability was impaired. These results confirm that for both species, Cd interferes in the signalling pathway of cortisol synthesis in a step prior to the pregnenolone formation. Data provided by the present study revealed important differences in vulnerability of adrenal steroidogenesis between rainbow trout and yellow perch.

Adrenal Glands↗

The Chinese rare minnow (Gobiocypris rarus) as an in vivo model for endocrine disruption in freshwater teleosts: a full life-cycle test with diethylstilbestrol.

Chinese rare minnow (Gobiocypris rarus), a freshwater teleost, was exposed to diethylstilbestrol (DES) at 0.05, 0.5, 1 and 5 microg/L from fertilized eggs for up to mature period under flow-through condition. Several endpoints that related to development, reproductive fitness and transgenerational effects were evaluated. It was found that body length and body weight were significantly reduced and vitellogenin (Vtg) levels were significantly increased for fish exposed to DES. Histological examination showed that the sex ratios of F0 fish skewed to female and about 2% of the fish exposed to 0.05 microg/L DES developed testes-ova. The reproductive success, as determined from data on egg production, was reduced in female fish exposed to 0.05, 0.5, 1 and 5 microg/L DES. The lowest-observed-effect concentrations (LOEC) for changes of sex ratios, reproductive success and histology alteration of F0 are 0.05 microg/L. In the offspring, transgenerational effects on egg hatching rate, egg fertilization and Vtg levels of juvenile individuals were not observed. However, survival of F1 generation fry significantly declined. The analysis of sex steroid levels revealed a significant decrease of testosterone (T) in the whole body homogenates (WBH) of male progeny and somewhat elevation of estradiol (E2) in the WBH of female offspring. These findings indicate that exposure to DES causes a variety of developmental, reproductive and transgenerational effects.

Animals↗

Intestinal bicarbonate secretion in marine teleost fish-source of bicarbonate, pH sensitivity, and consequences for whole animal acid-base and calcium homeostasis.

Whole animal studies using seawater European flounder (Platichthys flesus) revealed that increasing intestinal [Ca(2+)] to 20 mM stimulated net HCO(3)(-) base secretion by 57%, but this was effectively balanced by an increase in net acid secretion, likely from the gills, to maintain whole animal acid-base status. Higher Ca(2+) concentrations (40 and 70 mM) in ambient seawater resulted in reduced plasma total CO(2). This indicates (1) imperfect acid-base compensation, and (2) that endogenous metabolic CO(2) is insufficient to fuel intestinal HCO(3)(-) secretion, under hyper-stimulated conditions. Bicarbonate secretion plays an important role in preventing calcium absorption by precipitating a large fraction of the imbibed calcium as CaCO(3). Indeed, under high Ca(2+) conditions (20 mM), up to 75% of the intestinal Ca(2+) is precipitated as CaCO(3) and then excreted. This is undoubtedly important in protecting the marine teleost kidney from the need for excessive calcium excretion and risk of renal stone formation. Using an in vitro pH-stat technique with the isolated intestinal epithelium, the replacement of serosal CO(2) with a HEPES buffered saline had no effect on HCO(3)(-) secretion, indicating that the endogenous supply of HCO(3)(-) from CO(2) hydration within epithelial cells is adequate for driving baseline secretion rates. Further, in vitro data demonstrated a stimulatory effect of low pH on intestinal HCO(3)(-) secretion. Thus, both luminal Ca(2+) and H(+) can regulate HCO(3)(-) secretion but the precise mechanisms and their potential interaction are currently unresolved.

Acid-Base Equilibrium↗

ProCRH in the teleost Ameiurus nebulosus: gene cloning and role in LPS-induced stress response.

The procorticotrophin-releasing hormone (proCRH) gene from the teleost Ameiurus nebulosus was cloned by direct and inverse PCR-based technologies and characterized. The structure of the proCRH gene shows the presence of four exons and three introns giving a total length of 1416bp. Sequence similarity with the corresponding proCRH coding sequences in Tilapia mossambica and Homo sapiens is 97.7 and 78%, respectively. Western blot experiments performed with an anti-human CRH (1-41) antibody revealed the presence of an immunoreactive molecule with an approximate MW of 18kDa, a value comparable to that of the putative catfish proCRH peptide. These data suggest that proCRH could be active in A. nebulosus without any cleavage. ProCRH immunoreactive molecules were found in the central nervous system (CNS) and were immunocytochemically detected in the head kidney and in the pancreatic gland. Western blot and immunocytochemical experiments showed an increase in proCRH expression in the CNS after 15min but not after 120min exposure to LPS. In contrast, the increased immunopositivity was detectable in the pancreas only after 120min of treatment, but in the head kidney throughout the entire period of exposure. Our findings indicate that the CNS responds to the altered conditions for a shorter period of time than the peripheral organs, suggesting a hierarchical and time-regulated stress response. However, an independent response in the peripheral organs cannot be excluded in this scenario.

Amino Acid Sequence↗

Identification of a novel adrenomedullin gene family in teleost fish.

Adrenomedullin (AM) is a multifunctional peptide known to form a hormone family with calcitonin gene-related peptide (CGRP) and amylin. We have cloned five distinct AM cDNAs from the pufferfish, Takifugu rubripes, and named them TrAM-1, -2, -3, -4, and -5. Judging from the deduced precursor sequences and processing pattern of the C-terminal mature peptides, TrAMs may be divided into at least two groups; AM-2 and -3, and AM-1, -4, and possibly -5. Phylogenetic analysis of the mature peptides, exon-intron structure of their genes, and tissue distribution of their mRNA also support this classification. TrAM-1 and -4 were ubiquitously expressed in various tissues including the kidney and interrenal (adrenal homolog) as in the case of mammalian AM, while TrAM-2 and -3 were expressed most abundantly in the brain followed by the vascular tissues. Synteny of the genes around AM gene showed that TrAM-1 is the ortholog of mammalian AM. The presence of a PAMP-like sequence in the prosegment of TrAM-1 also supports this notion. Multiple AMs were also detected in another pufferfish, Tetraodon nigroviridis, and in zebrafish, Danio rerio. The present study shows for the first time the presence of a novel AM family in teleost fish that is independent from CGRP and amylin, which further suggests the possible existence of multiple AMs in mammals.

Adrenomedullin↗

Phosphorylation of teleost phosducins and its effect on the affinity to G-protein beta gamma subunits.

Phosducin (PD) is a regulatory protein involved in the phototransduction cascade of vertebrate photoreceptor cells. We have previously demonstrated that there are rod- and cone-specific PDs (OlPD-R and OlPD-C) in the retina of the teleost fish, medaka (Oryzias latipes) [FEBS Lett. 502 (2001) 117]. A 6x His affinity precipitation assay revealed that phosphorylation by either protein kinase A (PKA) or Ca(2+)/calmodulin-dependent kinase II (CaMKII) reduced the affinity of recombinant medaka PDs to endogenous medaka G-protein beta gamma subunits (Gbetagamma). These results suggest that the affinity of medaka PDs to Gbetagamma is regulated by cAMP and Ca(2+) concentrations as also found for mammalian PDs. However, we found a specific difference in the phosphorylation patterns between recombinant OlPD-R and OlPD-C, which resulted in different affinities to Gbetagamma. These differences may affect the light/dark-adaptation between medaka rods and cones.

Animals↗

Co-localization of neuronal nitric oxide synthase with arginine-vasotocin in the preoptic-hypothalamo-hypophyseal system of the teleost Oreochromis niloticus.

This study provides evidence that, in the preoptic-hypothalamo-hypophysial system of the teleost Oreochromis niloticus, several sub-populations of arginine-vasotocin (AVT)-producing neurons and neurosecretory fibers terminals express neuronal nitric oxide synthase (nNOS)-like molecules. The co-localization between nNOS and AVT was demonstrated by means of double immunofluorescence staining with the confocal microscope. This study is the first to provide evidence that nNOS may be co-localized with AVT in neurons of a non-mammalian vertebrate.

Animals↗

The anterograde and retrograde axonal transport of biotinylated dextran amine and biocytin in the nervous system of teleosts.

Biotinylated dextran amine (BDA) and biocytin are well transported both retrogradely and anterogradely. Both tracers have stable molecular structure for long-term storage and examination, and their visualizations can be realized by simple histochemical reactions. Therefore, the BDA and biocytin are widely used in neuroanatomical studies as the tract-tracing markers. The results obtained by BDA and biocytin applications to various areas of the nervous system in teleosts were qualitatively identical, and the retrogradely and anterogradely labeled structures could be clearly identified with reference to the counter-staining. Iontophoretic injections or crystal insertions resulted in filling of cell bodies, dendrites and terminals in the core of injection side, revealing morphological details of the local and distant somata, dendritic arborizations and axonal terminals. However, biocytin exhibited superior to BDA in anterograde transport, and could label very thin axons, axonal collaterals and terminal ramifications. In contrast, retrograde transport of BDA was superior to that of biocytin, and resulted in more complete dendritic filling of retrograde labeled neurons including dendritic arborizations and spines.

Animals↗

Rhythmic expression, light entrainment and alpha-MSH modulation of rhodopsin mRNA in a teleost pigment cell line.

To investigate whether teleost fish GEM-81 erythrophoroma cells were photosensitive, the cells were submitted to constant darkness (DD), 14 h of light and 10 h of darkness (14L:10D), and 10 h of light and 14 h of darkness (10L:14L). The doubling times (hours) were: DD 35.33+/-0.05; 14L:10D 67.85+/-0.04; and 10L:14D 49.60+/-0.08. In order to verify whether proliferation was dependent on light phase length, GEM-81 cells were submitted to 7L: 5D. The proliferation curves and doubling times were similar in 14L:10D and 7L:5D (respectively 69.44+/-0.03 and 67.85+/-0.04), suggesting that the cell cycle was regulated by the length of the light phase within 24 h, or by the light/dark ratio. We have also demonstrated the expression of Carassius retinal rhodopsin mRNA in GEM-81 cells, which cycles in a circadian rhythm, entrained by light. In addition, we showed that alpha-melanocyte stimulating hormone (alpha-MSH, 10(-10) to 10(-8) M), a conspicuous hormone that exerts mitogenic and melanogenic activity in most vertebrates, decreased rhodopsin mRNA in the first 3 days; after 4 days the inhibition was reversed, and after 5 days an increase in rhodopsin mRNA level was elicited. This is the first report of rhythmic expression of extra-ocular rhodopsin and its modulation by light and hormones.

Animals↗

Intestinal anion exchange in teleost water balance.

Simultaneous measurements of all major electrolytes including HCO3(-) and H+ as well as water demonstrated that fluids absorbed by the anterior intestine of the marine gulf toadfish under in vivo-like conditions on an overall net basis are hypertonic at 380 mOsm and acidic ([H+] = 27 mM). This unusual composition of fluids absorbed across the intestinal epithelium is due to the unusual intestinal fluid chemistry resulting from seawater ingestion and selective ion and water absorption along the gastro-intestinal tract. Measurement under near symmetrical conditions with high NaCl concentrations and low MgSO4 concentrations revealed absorption of iso-osmotic and much less acidic fluids by the intestinal epithelium, a situation resembling that of other water absorbing leaky vertebrate epithelia. Reduced luminal NaCl concentrations seen in vivo results in lower absolute water absorption rates but higher Cl-/HCO3(-) exchange rates which are associated with higher net H+ absorption rates. It appears that apical anion exchange is important for net Cl- uptake by the marine teleost intestine especially when luminal NaCl concentrations are low and/or when MgSO4 concentrations are high. Observations indicate that fluid absorption from solutions of low NaCl but high MgSO4 concentrations is energetically more demanding than absorption from NaCl rich solutions at the level of the intestinal epithelium. Furthermore, the high luminal MgSO4 concentration which is an unavoidable consequence of seawater ingestion projects a demand for renal and branchial compensation for intestinal MgSO4 uptake and absorption of hypertonic and acidic fluid by the intestine.

Animals↗

Existence of an extra-retinal and extra-pineal photoreceptive organ that regulates photoperiodism in gonadal development of an Osmerid teleost, ayu (Plecoglossus altivelis).

Ayu (Plecoglossus altivelis) is an Osmerid teleost whose gonadal development exhibits clear photoperiodism: it is stimulated and prevented under short and long photoperiod, respectively. However, the photoreceptor organ involved in this process remains to be identified. In the present study, we examined whether gonads of ophthalmectomized (Ex) and pinealectomized (Px) ayu respond to short photoperiod to test whether photoreceptors other than lateral eyes and pineal complex are involved in the photoperiodic response of gonadal development. Gonadosomatic index (GSI) and plasma levels of sex steroids (testosterone and 11-ketotestosterone for males and testosterone and estradiol 17-beta for females) were significantly increased in the Ex+Px ayu kept under short photoperiod in both males and females as compared with the initial control. On the other hand, there were no significant increases in GSI and sex steroids in the Ex+Px ayu kept under long photoperiod. Histological observation of gonads in the Ex+Px ayu revealed that oocytes undergoing final maturation in females and proliferation of germ cells in males were observed under short photoperiod but not under long photoperiod. These results indicate that extra-retinal and extra-pineal photoreceptive organ regulates photoperiodic gonadal development in this species.

Animals↗

Activities of antioxidant enzymes and cytochrome c oxidase in liver of Arctic and temperate teleosts.

Enhanced antioxidant status in polar fishes may occur due to high dissolved oxygen levels and membranes rich in peroxidation-sensitive polyunsaturated fatty acids. To evaluate the importance of antioxidant enzymes in polar fishes, activities of catalase (CAT), superoxide dismutase (SOD), and glutathione reductase (GR), as well as the aerobic enzyme cytochrome c oxidase (CCO), were measured at 6 degrees C and 1 degrees C in livers of confamilial Arctic and temperate teleosts: the Arctic fourhorn sculpin Triglopsis quadricornis (Cottidae) and saddled eelpout Lycodes mucosus (Zoarcidae) vs. the temperate longhorn sculpin (Myoxocephalus octodecimspinosus) (Cottidae) and ocean pout (Zoarces americanus) (Zoarcidae), respectively. At both assay temperatures, CAT activities were substantially lower in both Arctic species, SOD was similar in the cottids but lower in the Arctic zoarcid, and GR was similar in temperate and Arctic fishes. Activities at respective habitat temperatures were always significantly lower in the Arctic fishes. The lower antioxidant enzyme activities in the Arctic fishes cannot be attributed to lower aerobic status because CCO activity was similar or higher in the Arctic fishes; significant negative relationships were found between CCO and CAT and GR (but not SOD) when all species were combined, indicating that a higher apparent aerobic status does not necessarily coincide with higher antioxidant enzyme activities. Antioxidant enzyme activities may not be enhanced as part of cold adaptation in Arctic fishes, at least in the liver.

Acclimatization↗

Characterization and comparison of fatty acyl Delta6 desaturase cDNAs from freshwater and marine teleost fish species.

Fish are the most important dietary source of the n-3 highly unsaturated fatty acids (HUFA), eicosapentaenoic (EPA) and docosahexaenoic acid (DHA), that have particularly important roles in human nutrition reflecting their roles in critical physiological processes. The objective of the study described here was to clone, functionally characterize and compare expressed fatty acid desaturase genes involved in the production of EPA and DHA in freshwater and marine teleost fish species. Putative fatty acid desaturase cDNAs were isolated and cloned from common carp (Cyprinus carpio) and turbot (Psetta maximus). The enzymic activities of the products of these cDNAs, together with those of cDNAs previously cloned from rainbow trout (Oncorhynchus mykiss) and gilthead sea bream (Sparus aurata), were determined by heterologous expression in the yeast Saccharomyces cerevisiae. The carp and turbot desaturase cDNAs included open reading frames (ORFs) of 1335 and 1338 base pairs, respectively, specifying proteins of 444 and 445 amino acids. The protein sequences possessed all the characteristic features of microsomal fatty acid desaturases, including three histidine boxes, two transmembrane regions, and N-terminal cytochrome b(5) domains containing the haem-binding motif, HPGG. Functional expression showed all four fish cDNAs encode basically unifunctional Delta6 fatty acid desaturase enzymes responsible for the first and rate-limiting step in the biosynthesis of HUFA from 18:3n-3 and 18:2n-6. All the fish desaturases were more active towards the n-3 substrate with 59.5%, 31.5%, 23.1% and 7.0% of 18:3n-3 being converted to 18:4n-3 in the case of turbot, trout, sea bream and carp, respectively. The enzymes also showed very low, probably physiologically insignificant, levels of Delta5 desaturase activity, but none of the products showed Delta4 desaturase activity. The cloning and characterization of desaturases from these fish is an important advance, as they are species in which there is a relative wealth of data on the nutritional regulation of fatty acid desaturation and HUFA synthesis, and between which substantive differences occur.

Amino Acid Sequence↗

Cu,Zn superoxide dismutase from Trematomus bernacchii: functional conservation and erratic molecular evolution in Antarctic teleosts.

In the present study, we describe the purification and molecular characterization of Cu,Zn superoxide dismutase (SOD) from Trematomus bernacchii, a teleost widely distributed in many areas of Antarctica, that plays a pivotal role in the Antarctic food chain. The amino acid and cDNA sequences have been obtained using both biochemical and molecular biology approaches and are compared with Cu,Zn SODs from other fishes. Assessment of the primary sequences highlights that the catalytically important residues are fully conserved in Cu,Zn SOD from T. bernacchii. Phylogenetic analyses performed on Cu,Zn SOD amino acid sequences permit speculation regarding the evolution of this protein. In particular, the data confirms the erratic differentiation of these proteins and concurs with the theory of the "unclock-like" behaviour of Cu,Zn SOD evolution.

Amino Acid Sequence↗

Hematological responses of the Neotropical teleost matrinxã (Brycon cephalus) to environmental nitrite.

Environmental increase in nitrite impairs the function of several aquatic species, including fishes. Nitrite reacts with hemoglobin yielding the non-functional methemoglobin (metHb), and many physiological disturbances can arise. The physiological mechanisms to cope with nitrite are still unclear in fish. Hematological parameters, the role of NADH-methemoglobin reductase system and the electrolytic balance were studied in the freshwater teleost Brycon cephalus (matrinxã) exposed to 0.2, 0.4 and 0.6 mg/L of nitrite N-NO(2) for 24 and 96 h. Hematocrit, total hemoglobin and the red blood cell (RBC) number decreased. Methemoglobin content increased from 1% to 69% for 24 h of exposure and drastically from 5-6% to 90% for 96 h. The activity of NADH-methemoglobin reductase system displayed a tendency of increase in response to nitrite concentration or time of exposure. In the plasma, nitrite was accumulated to values 30-fold higher than the environmental concentration. The plasma K(+) concentration increased only in fish exposed to NO(2) for 24 h. No changes in plasma protein and Na(+) were observed during nitrite exposure but Cl-presented a punctual increase at 0.2 mg/L N-NO(2)-96 h. The hematological data suggest that nitrite caused functional and hemolytic anemia. Furthermore, the electrolytic balance was relatively undisturbed, and the nitrite clearance in matrinxã is likely depending on other factors than NADH-methemoglobin reductase system.

Animals↗