Evidence for enkephalin-like antigenic determinants in the gonads of the icefish Chionodraco hamatus

A possible involvement of enkephalin-like substances in the control of gonadal activities has been suggested in vertebrates. Recently, proopiomelanocortin-related peptides have been detected in the gonads of amphibians and teleosts living in temperate waters. The aim of this study was to look for enkephalin-like peptides in the ovary and in the testis of the icefish Chionodraco hamatus. Our results confirmed the presence of enkephalinergic systems in the gonads of Antarctic notothenioid fishes.A preliminary account of this work was presented to the XII International Congress of Comparative Endocrinology, 16–21 May 1993, Toronto, Canada (Pestarino et al. 1983b)     

Structure and membrane interactions of chionodracine,a piscidin-like antimicrobial peptide from the icefish Chionodraco hamatus

Chionodracine (Cnd) is a 22-residue peptide of the piscidin family expressed in the gills of the Chionodraco hamatus as protection from bacterial infections. Here, we report the effects of synthetic Cnd on both Psychrobacter sp. TAD1 and Escherichia coli bacteria, as well as membrane models. We found that Cnd perforates the inner and outer membranes of Psychrobacter sp. TAD1, making discrete pores that cause the cellular content to leak out. Membrane disruption studies using intrinsic and extrinsic fluorescence spectroscopy revealed that Cnd behaves similarly to other piscidins, with comparable membrane partition coefficients. Membrane accessibility assays and structural studies using NMR in detergent micelles show that Cnd adopts a canonical topology of antimicrobial helical peptides, with the hydrophobic face toward the lipid environment and the hydrophilic face toward the bulk solvent. The analysis of Cnd free energy of binding to vesicles with different lipid contents indicates a preference for charged phospholipids and a more marked binding to native E. coli extracts. Taken with previous studies on piscidin-like peptides, we conclude that Cnd first adsorbs to the membrane, and then forms pores together with membrane fragmentation. Since Cnd has only marginal hemolytic activity, it constitutes a good template for developing new antimicrobial agents.     

Apparent deficiency of metallothionein in the liver of the Antarctic icefish Chionodraco hamatus. Identification and isolation of a zinc-containing protein unlike metallothionein.

1. A zinc-binding protein has been isolated and purified from the liver of the icefish Chionodraco hamatus. 2. The icefish Zn-protein has characteristics distinct from those of metallothionein. 3. The amino acid composition shows a low content of cysteine and a high content of glutamate and aspartate. 4. No metallothionein has been detected in the extracts from icefish liver.     

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.     

Characterization of a Tc1-like transposon in the Antarctic ice-fish,Chionodraco hamatus

We report the presence of Tc1 transposon-like sequences in the Antarctic ice-fish Chionodraco hamatus, belonging to the Notothenioidei. The complete DNA sequence of these transposon-like elements is reduced in length compared to other Tc1 transposons, but it appears to share significant structural similarities with them. It contains a degenerate open reading frame, whose inferred 264 amino acid sequence shares sequence similarity with the 'aspartic acid, aspartic acid (35) glutamic acid' family of transposases, particularly those from Caenorhabditis species (sp.) and Drosophila sp. Southern blot analysis and polymerase chain reaction amplification indicate that Tc1 transposon-like sequences are present in other notothenioid species, though their amount can vary in the different lineages.     

Obestatin regulates cardiovascular function and promotes cardioprotection through the nitric oxide pathway

Patients with ischaemic heart disease or chronic heart failure show altered levels of obestatin, suggesting a role for this peptide in human heart function. We have previously demonstrated that GH secretagogues and the ghrelin gene‐derived peptides, including obestatin, exert cardiovascular effects by modulating cardiac inotropism and vascular tone, and reducing cell death and contractile dysfunction in hearts subjected to ischaemia/reperfusion (I/R), through the Akt/nitric oxide (NO) pathway. However, the mechanisms underlying the cardiac actions of obestatin remain largely unknown. Thus, we suggested that obestatin‐induced activation of PI3K/Akt/NO and PKG signalling is implicated in protection of the myocardium when challenged by adrenergic, endothelinergic or I/R stress. We show that obestatin exerts an inhibitory tone on the performance of rat papillary muscle in both basal conditions and under β‐adrenergic overstimulation, through endothelial‐dependent NO/cGMP/PKG signalling. This pathway was also involved in the vasodilator effect of the peptide, used both alone and under stress induced by endothelin‐1. Moreover, when infused during early reperfusion, obestatin reduced infarct size in isolated I/R rat hearts, through an NO/PKG pathway, comprising ROS/PKC signalling, and converging on mitochondrial ATP‐sensitive potassium [mitoK(ATP)] channels. Overall, our results suggest that obestatin regulates cardiovascular function in stress conditions and induces cardioprotection by mechanisms dependent on activation of an NO/soluble guanylate cyclase (sGC)/PKG pathway. In fact, obestatin counteracts exaggerated β‐adrenergic and endothelin‐1 activity, relevant factors in heart failure, suggesting multiple positive effects of the peptide, including the lowering of cardiac afterload, thus representing a potential candidate in pharmacological post‐conditioning.     

Specific immunity proteomic profile of the skin mucus of Antarctic fish Chionodraco hamatus and Notothenia coriiceps

The white-blooded Antarctic icefish is the only known vertebrate lacking oxygen-transporting haemoglobins. Fish skin mucus, as the first line of defence against pathogens, can reflect fish welfare. In this study, we analysed the skin mucus proteome profiles of the two Antarctic fish species, the white-blooded Antarctic icefish, Chionodraco hamatus, and the red-blooded Antarctic fish, Notothenia coriiceps, unfolding the different proteins by liquid chromatography coupled with tandem mass spectrometry isobaric tags for relative and absolute quantitation (iTRAQ) technology. Of the 4444 totally identified proteins, 227 differentially expressed proteins (DEPs) were found in the comparison between C. hamatus and N. coriiceps, of which 121 were upregulated and 106 were downregulated in the icefish. In the Kyoto Encyclopedia of Genes and Genomes pathway annotation, we found two pathways “Legionellosis” and “Complement and coagulation cascades” were significantly enriched, among of which innate immune candidate proteins such as C3, CASP1, ASC, F3 and C9 were significantly upregulated, suggesting their important roles in C. hamatus immune system. Additionally, the DEP protein–protein interaction network analysis and “Response to stress” GO category provided candidate biomarkers for deep understanding of the distinct immune response of the two Antarctic fish underlying the cold adaptation.     

Population genetic structure and gene flow patterns between populations of the Antarctic icefish Chionodraco rastrospinosus

Aim A lack of genetic structure is predicted for Antarctic fish due to the duration of pelagic larval stages and the strength of the currents in the Southern Ocean, particularly the Antarctic Circumpolar Current. In this study we explored the population structure of the ocellated icefish, Chionodraco rastrospinosus, by means of analysing a total of 394 individuals collected at four geographical areas off the Antarctic Peninsula in the period 1996–2006. Location Elephant Island, southern South Shetlands, Joinville Island and South Orkneys in the Southern Ocean. Methods The spatio‐temporal genetic structure of Chionodraco rastrospinosus was explored using seven microsatellite loci. Existence and direction of gene flow across sampling locations were investigated using the isolation‐by‐migration procedure. Results Microsatellite data showed a lack of genetic structuring in the area studied, with no differences found at both the geographical or temporal level, and an eastward unidirectional gene flow among sites. This suggested a lack of genetic barriers for this species, attributable to larval dispersal following the Antarctic Circumpolar Current, which fits well with the predicted pattern for Antarctic fish. Re‐examination of genetic data of the closely related icefish Chaenocephalus aceratus, with similar larval duration but displaying genetically structured populations, indicated a weak but significant bidirectional gene flow. Main conclusions Our results point to a relationship that is more complex than expected between potential for dispersal and realized gene flow in the marine environment. In addition to ocean circulation and larval dispersal, other major life‐history traits might be driving connectivity, particularly larval retention.     

Adverse effects of pneumoperitoneum on renal function: involvement of the endothelin and nitric oxide systems

Increased intra-abdominal pressure (IAP) during laparoscopy adversely affects kidney function. The mechanism underlying this phenomenon is largely unknown. This study was designed to investigate the involvement of endothelin (ET)-1 and nitric oxide (NO) systems in IAP-induced renal dysfunction. Rats were subjected to IAP of 14 mmHg for 1 h, followed by a deflation for 60 min (recovery). Four additional groups were pretreated with 1) ABT-627, an ET(A) antagonist; 2) A-192621, an ET(B) antagonist; 3) nitroglycerine; and 4) N(G)-nitro-L-arginine methyl ester, a NO synthase inhibitor, before IAP. Urine flow rate (V), absolute Na+ excretion (U(Na)V), glomerular filtration rate (GFR), and renal plasma flow (RPF) were determined. Significant reductions in kidney function and hemodynamics were observed when IAP was applied. V decreased from 8.1 +/- 1.0 to 5.8 +/- 0.5 microl/min, U(Na)V from 1.08 +/- 0.31 to 0.43 +/- 0.10 microeq/min, GFR from 1.84 +/- 0.12 to 1.05 +/- 0.06 ml/min (-46.9 +/- 2.7% from baseline), and RPF from 8.62 +/- 0.87 to 3.82 +/- 0.16 ml/min (-54 +/- 3.5% from baseline). When the animals were pretreated with either ABT-627 or A-192621, given alone or combined, the adverse effects of IAP on GFR, RPF, V, and U(Na)V were significantly augmented. When the animals were pretreated with nitroglycerine, the adverse effects of pneumoperitoneum on GFR and RPF were substantially improved. In contrast, pretreatment with N(G)-nitro-L-arginine methyl ester remarkably aggravated pneumoperitoneum-induced renal dysfunction. In conclusion, decreased renal excretory function and hypofiltration are induced by increased IAP. These effects are related to impairment of renal hemodynamics and could be partially ameliorated by pretreatment with nitroglycerine and aggravated by NO and ET blockade.     

Satellite DNAs,Heterochromatin and sex chromosomes in Chionodraco hamatus (Channichthyidae,Perciformes)

We studied the genome of an antarctic ice fish, Chionodraco hamatus, in order to detect highly repetitive DNAs that may play a role in heterochromatinization processes and sex chromosome differentiation. We used two different experimental approaches. Hybridization of a Bkm probe to genomic DNA showed slight differences between the two sexes. Using restriction enzymes, a Bgl II satellite (pIF) was isolated. In situ hybridization revealed a preferential localization of pIF on the centromeres and the telomeres of most chromosomes, as well as an interstitial band on the long arms of the neo-Y sex chromosome, where probably the hypothetical fusion took place. Dot-blot experiments showed that pIF is still present in species belonging to different families of the same suborder. Though preliminary, our results suggest a conservative nature of this DNA which might have played a definite functional role in the genome of these polar fishes.     

Interaction of nitric oxide and serotonin in aggressive behavior

Nitric oxide (NO) modulates many behavioral and neuroendocrine responses. Genetic or pharmacological inhibition of the synthetic enzyme that produces NO in neurons evokes elevated and sustained aggression in male mice. Recently, the excessive aggressive and impulsive traits of neuronal NO synthase knockout (nNOS-/-) mice were shown to be caused by reductions in serotonin (5-HT) turnover and deficient 5-HT1A and 5-HT1B receptor function in brain regions regulating emotion. The consistently high levels of aggression observed in nNOS-/- mice could be reversed by 5-HT precursors and by treatment with specific 5-HT1A and 5-HT1B receptor agonists. The expression of the aggressive phenotype of nNOS-/- knockout mice requires isolated housing prior to testing. The effects of social factors such as housing condition and maternal care can affect 5-HT and aggression, but the interaction among extrinsic factors, 5-HT, NO, and aggression remains unspecified. Taken together, NO appears to play an important role in normal brain 5-HT function and may have significant implications for the treatment of psychiatric disorders characterized by aggressive and impulsive behaviors.     

Ultrastructure and proteins of the egg chorion of the antarctic fish Chionodraco hamatus (Teleostei, Notothenioidei)

The chorion morphology and protein content of unfertilised eggs of the antarctic icefish Chionodraco hamatus were characterised. Scanning and transmission electron microscopy showed that the chorion of this antarctic species is organised differently from that of non-polar species, with several concentric layers varying in thickness. Purified chorions were dissolved in 8-M urea buffer, and polypeptides were revealed by one- and two-dimensional gel electrophoresis. Glycoproteins were detected using affinity blotting with concanavalin-A. The principal glycoproteins had a molecular weight of 49 and 93 kDa. Purification of the main polypeptides between 40 and 92 kDa was achieved by preparative electrophoresis followed by electroelution of excised bands. The main biochemical composition of C. hamatus chorion was similar to that of other teleost species investigated.     

The nitric oxide pathway in the cardiovascular system

The present review analyzes the role nitric oxide (NO) plays in the homeostasis of the cardiovascular system. By regulating vascular smooth muscle cell and myocyte contractility, myocardial oxygen consumption and renal tubular transport, this simple molecule plays a central role in the control of vascular tone, cardiac contractility and short and long term regulation of arterial pressure. Fifteen years ago, all we knew about NO is that it had very similar properties as those of endothelium-derived relaxing factor and that its action was probably mediated by cGMP. An enormous amount of knowledge has since been amassed on the biochemical pathways that NO follows from the moment it is synthesized from L-arginine until the physiological or pathological actions take place in the effector cells. This review intends to organize this knowledge in a fashion that is easy to understand. We will dissect the NO pathway in different steps, focusing on the physiological and pathophysiological actions of the isoenzymes which synthesize NO, the molecules involved in this synthesis such as caveolins, protein kinases and cofactors, the situations in which endogenous inhibitors of NO synthase are formed from L-arginine instead of NO, the way in which NO exerts its physiological actions through cGMP-dependent protein kinases and finally, the pathological routes NO may follow when the oxidative status of the cell is high.     

Ion transport systems in the kidney and urinary bladder of two Antarctic teleosts, Chionodraco hamatus and Trematomus bernacchii

Na⁺-K⁺-Cl^- cotransport and Na⁺/K⁺ATPase were studied by immunohistochemistry in the kidney and urinary bladder of Trematomus bernacchii and Chionodraco hamatus. The activity was correlated to the density of mitochondria. The first segment of the renal proximal tubule was more active than the second one. In T. bernacchii and the temperate marine teleost Pagellus bogaraveo, the immunoreactivity for the antibody to cotransporters and to the !-subunit of the sodium pump was stronger than in the icefish. This difference indicates in the kidney of the icefish, a weaker secretory activity, a consequent lower osmolarity in the lumen and lower water loss, which correlates well with the need for a greater blood volume in the icefish. The epithelium of the urinary bladder in T. bernacchii, where intense immunostaining was observed, was composed of columnar cells. In C. hamatus the columnar cells, where the immunostaining was weaker, lined only a portion of the urinary bladder, the other region being composed of cuboidal cells.     

Lymphomyeloid organs of the Antarctic fish Trematomus nicolai and Chionodraco hamatus (Teleostei: Notothenioidea): a comparative histological study

Lymphomyeloid organs of two common species of Antarctic fish, Trematomus nicolai and Chionodraco hamatus, were studied with the aim of analysing some morphological aspects of these organs in relation to adaptation to low environmental temperature. The thymuses of T. nicolai and C. hamatus were flattened, incompletely lobated, with numerous Hassall-like bodies, which were mainly located in the central part of the organ in C. hamatus. In T. nicolai, thymocytes, erythroid and reticular epithelial cells filled the organ. In C. hamatus, the thymocytes intermingled with reticular epithelial cells were often close to groups of melano-macrophages. In both species, the thymus did not show distinct compartmentalisation; however, the thymocytes had significantly different sizes in the outer and inner portions of the thymus. The head kidney of both species was completely filled by haematopoietic tissue, highly vascularised and mainly lymphopoietic in T. nicolai, while both erythropoietic and lymphopoietic in C. hamatus. The spleen appeared mainly erythropoietic in T. nicolai and mainly lymphopoietic in C. hamatus. Solitary melano-macrophages in T. nicolai were close to numerous small vascular ellipsoids where erythroid and lymphoid cells were intermingled without the formation of red and white pulp areas. In C. hamatus, large lymphoid areas were organised around the capillaries. The possible adaptation of lymphoid organs to the low temperature of polar water is discussed. Accepted: 8 November 1999     

Dynamics of nitric oxide release in the cardiovascular system

The endothelium plays a critical role in maintaining vascular tone by releasing nitric oxide (NO). Endothelium derived NO diffuses to smooth muscles, triggering their relaxation. The dynamic of NO production is a determining factor in signal transduction. The present studies were designed to elucidate dynamics of NO release from normal and dysfunctional endothelium. The nanosensors (diameter 100-300 nm) exhibiting a response time better than 100 micros and detection limit of 1.0 x 10(-9) mol L(-1) were used for in vitro monitoring of NO release from single endothelial cells from the iliac artery of normotensive (WKY) rats, hypertensive (SHR) rats, and normal and cholesterolemic rabbits. Also, the dynamics and distribution of NO in left ventricular wall of rabbit heart were measured. The rate of NO release was much higher (1200 +/- 50 nmol L(-1) s(-1)) for WKY than for SHR (460 +/- 10 nmol L(-1) s(-1)). Also, the peak NO concentration was about three times higher for WKY than SHR. Similar decrease in the dynamics of NO release was observed for cholesterolemic rabbits. The dynamics of NO release changed dramatically along the wall of rabbit aorta, being highest (0.86 +/- 0.12 micromol L(-1)) for the ascending aorta, and lowest for the iliac aorta (0.48 +/- 0.15 micromol L(-1)). The distribution of NO in the left ventricular wall of rabbit heart was not uniform and varied from 1.23 +/- 0.20 micromol L(-1) (center) to 0.90 +/- 0.15 micromol L(-1) (apex). Both, the maximal concentration and the dynamics of NO release can be useful diagnostic tools in estimating the level of endothelial dysfunction and cardiovascular system efficiency.