Stability and conformational dynamics of metallothioneins from the antarctic fish Notothenia coriiceps and mouse

The structural properties and the conformational dynamics of antarctic fish Notothenia coriiceps and mouse metallothioneins were studied by Fourier-transform infrared and fluorescence spectroscopy. Infrared data revealed that the secondary structure of the two metallothioneins is similar to that of other metallothioneins, most of which lack periodical secondary structure elements such as alpha-helices and beta-sheets. However, the infrared spectra of the N. coriiceps metallothionein indicated the presence of a band, which for its typical position in the spectrum and for its sensitivity to temperature was assigned to alpha-helices whose content resulted in 5% of the total secondary structure of the protein. The short alpha-helix found in N. coriiceps metallothionein showed an onset of denaturation at 30 degrees C and a T(m) at 48 degrees C. The data suggest that in N. coriiceps metallothionein a particular cysteine is involved in the alpha-helix and in the metal-thiolate complex. Moreover, infrared spectra revealed that both proteins investigated possess a structure largely accessible to the solvent. The time-resolved fluorescence data show that N. coriiceps metallothionein possesses a more flexible structure than mouse metallothionein. The spectroscopic data are discussed in terms of the biological function of the metallothioneins.     

Hemoglobin from the antarctic fish Notothenia coriiceps neglecta. Amino acid sequence of the beta chain

1. Notothenia coriiceps neglecta is a cold-adapted notothenioid teleost, widely distributed in the Antarctic waters. 2. In comparison with fishes from temperate waters, the blood of this teleost contains a reduced number of erythrocytes and concentration of hemoglobin; the erythrocytes contain two hemoglobins, Hb1 and Hb2, respectively accounting for approximately 90, and 5% of the total. 3. The two components differ by the alpha chain; the amino acid sequence of the beta chain in common to the two hemoglobins has been established, thus completing the elucidation of the primary structure of the major component Hb 1.     

Multiple molecular forms of gonadotropin-releasing hormone in teleost fish brain

Gonadotropin-releasing hormone (GnRH) immunoreactive peptides in extracts of hake (Merluccius capensis) and tilapia (Tilapia sparrmanii) brain were investigated by high performance liquid chromatography (HPLC) and radioimmunoassay with region-specific antisera. In hake brain, content and concentration of GnRH was higher in the pituitary gland than in the hypothalamic lobes or extrahypothalamic brain. Hake pituitary gland GnRH was purified by six consecutive HPLC systems. The major GnRH molecular form co-eluted with salmon brain GnRH (Trp7, Leu8-GnRH) in four different HPLC systems which were specifically designed to separate the four natural vertebrate GnRHs (mammalian, salmon, chicken I and II). The immunoreactive peak in the final purification step had a retention time identical to that of Trp7, Leu8-GnRH and an UV absorbance (280 nm) peak appropriate for two tryptophan residues in the peptide, as in Trp7, Leu8-GnRH. Six additional less hydrophobic forms of GnRH were detected. Tilapia brain extract contained two major GnRH molecular forms which had identical retention times to chicken GnRH I (Gln8-GnRH) and Trp7, Leu8-GnRH in an HPLC system which separates the natural vertebrate GnRHs. The immunological properties of these two immunoreactive peaks, determined by relative interaction with four region-specific GnRH antisera raised against vertebrate GnRHs, were identical to those of Gln8-GnRH and Trp7, Leu8-GnRH. Additional GnRH molecular forms were also detected. In summary, these findings indicate that a major GnRH molecule in hake pituitary gland is Trp7, Leu8-GnRH, while tilapia brain contains both Trp7, Leu8-GnRH and Gln8-GnRH. Additional GnRH molecular forms were detected in both species.(ABSTRACT TRUNCATED AT 250 WORDS)     

Distribution of gonadotropin-releasing hormone immunoreactive systems in the brain of the Senegalese sole, Solea senegalensis

The present paper reports the immunohistochemical distribution of the gonadotropin-releasing hormone (GnRH) structures in the brain of the Senegalese sole, Solea senegalensis. In this study, we have used two antibodies against the salmon GnRH and chicken GnRH-II forms and the streptavidin–biotin-peroxidase complex method. Immunoreactive cell bodies are observed at the junction between the olfactory bulbs and the telencephalon (terminal nerve ganglion cells), in the ventral telencephalon, in the preoptic parvocellular nucleus, and in the synencephalic nucleus of the medial longitudinal fasciculus. GnRH-immunoreactive fibres were found extensively throughout the brain, located in the telencephalon, preoptic area, hypothalamus, hypophysis, optic tectum, midbrain and rhombencephalon. The antisera used in this study against the two GnRH forms exhibited cross-reactivity on the same cell masses and did not allow cell populations expressing different GnRH forms to be discriminated clearly. However, anti-salmon GnRH immunostained the GnRH cells and fibres of the forebrain much more intensely, whereas the anti-chicken GnRH antiserum shows a higher immunoreactivity on synencephalic cells of the medial longitudinal fasciculus.     

Localization of immunoreactive lamprey gonadotropin-releasing hormone in the rat brain

A highly specific antiserum against lamprey gonadotropin-releasing hormone (GnRH) was used to localize 1-GnRH in areas of the rat brain associated with reproductive function. Immunoreactive 1-GnRH-like neurons were observed in the ventromedial preoptic area (POA), the region of the diagonal band of Broca and the organum vasculosum lamina terminalis, with fiber projections to the rostral wall of the third ventricle and the organum vasculosum lamina terminalis. Another population of 1-GnRH-like neurons was localized in the dorsomedial and lateral POA, with nerve fibers projecting caudally and ventrally to terminate in the external layer of the median eminence. Other fibers apparently projected caudally and circumventrically to terminate around the cerebral aqueduct in the mid-brain central gray. By using a highly specific antiserum directed against mammalian luteinizing hormone-releasing hormone (m-LHRH), the localization of the LHRH neuronal system was compared to that of the 1-GnRH system. There were no LHRH neurons in the dorsomedial or the lateral region of the POA that contained the 1-GnRH neurons. As expected, there was a large population of LHRH neurons in the ventromedial POA associated with the diagonal band of Broca and organum vasculosum lamina terminalis. In both of these regions, there were many more LHRH neurons than 1-GnRH neurons and the LHRH neurons extended more dorsally and laterally than the 1-GnRH neurons. The LHRH neurons seemed to project to the median eminence in the same areas as those that were innervated by the 1-GnRH neurons. Absorption studies indicated that 1-GnRH cell bodies were eliminated by adding 1 microg of either 1-GnRH-I or 1-GnRH-III, but not m-LHRH to the antiserum before use. Fibers were largely eliminated by the addition of 1 microg 1-GnRH-III to the antiserum. No chicken GnRH-II neurons or nerve fibers could be visualized by immunostaining. Because the antiserum recognized GnRH-I and GnRH-III equally, we have visualized an 1-GnRH system in rat brain. The results are consistent with the presence of either one or both of these peptides within the rat hypothalamus. Because 1-GnRH-I has only weak nonselective gonadotropin-releasing activity, whereas 1-GnRH-III is a highly selective releaser of follicle-stimulating hormone, and because 1-GnRH neurons are located in areas known to control follicle-stimulating hormone release selectively, our results support the hypothesis that 1-GnRH-III, or a closely related peptide, may be mammalian follicle-stimulating hormone-releasing factor.     

Solution structure of MT_nc,a novel metallothionein from the Antarctic fish Notothenia coriiceps

The structure of [113Cd(7)]-metallothionein (MT_nc) of the Antarctic fish Notothenia coriiceps, the first three-dimensional structure of a fish metallothionein, was determined by homonuclear 1H NMR experiments and heteronuclear [1H, 113Cd]-correlation spectroscopy. MT_nc is composed of an N-terminal beta domain with 9 cysteines and 3 metal ions and a carboxy-terminal alpha-domain with 11 cysteines and 4 metal ions. The position of the ninth Cys of the alpha domain of MT_nc is different from the corresponding Cys of mammalian MTs. As a result, the last CXCC motif in the mammalian MT sequence becomes CXXXCC in the fish MT. This difference leads to a structural change of the alpha domain and, in turn, to a different charge distribution with respect to that observed in mammalian metallothioneins.     

Metal (Fe, Zn, Mn and Cu) levels in the Antarctic fish Notothenia coriiceps

During the 1994/1995 Antarctic summer 20 Antarctic cod (Notothenia coriiceps) of both sexes were collected at King George Island, South Shetland Islands, Antarctica, to determine the levels of Fe, Zn, Mn and Cu in muscle, liver, gonad and kidney. Metal levels were assessed using the inductively coupled plasma-atomic emission spectrometry (ICP-AES) technique. The levels of Fe, Zn, Mn and Cu observed in both sexes of N. coriiceps were significantly lower in muscle tissue than in liver, kidney and gonad; the livers of males and gonads of females had the highest levels. The differences between the sexes were significant for Zn and Mn in gonads and for Cu in liver. Despite the modest size-range of the specimens, the Cu levels in liver, muscle and kidney were significantly inversely correlated with female mass (P < 0.05) as was gonad Cu level and male body length. Fe mainly presented the highest concentrations and Cu and Mn the lowest ones. Mean muscle metal levels are similar to those of hake, cod, herring and mackerel; however, the level of Fe was slightly higher. Accumulation may occur for Fe, Zn and Mn in liver, kidney and gonad, which have higher levels than those of most prey. Received: 21 October 1997 / Accepted: 1 February 1998     

Fat absorptive processes in the intestine of the Antarctic fish Notothenia coriiceps (Richardson, 1844)

Absorption of lipids by the enterocytes of Notothenia coriiceps, an omnivorous Antarctic and subAntarctic fish, was studied by light and electron microscopy. The lipids are absorbed by the anterior and middle intestine segments. They appear as fat droplets that measure from 0.5 to 7 μm of diameter and which accumulate in the apical cytoplasm within the first 24 h and seem to be the main fat storage form in the enterocytes. Fat is also observed as lipid particles with 60–300 nm inside the rough endoplasmic reticulum and cytoplasmic smooth vesicles. The epithelial intercellular space and the mucosal inner lamina contain lipid particles, which probably are the fat transport form. Our observations show that an intense lipid absorptive process takes place in N. coriiceps digestive system, due to the great extension of the intestine involved and due to the great lipid accumulation found in the epithelial compartment.     

Characterization of the cytoplasmic chaperonin containing TCP-1 from the Antarctic fish Notothenia coriiceps

The cytoplasmic chaperonin containing TCP-1 (CCT) plays a critically important role in the folding and biogenesis of many cytoskeletal proteins, including tubulin and actin. For marine ectotherms, the chronically cold Southern Ocean (−2 to +2°C) poses energetic challenges to protein folding, both at the level of substrate proteins and with respect to the chaperonin/chaperone folding system. Here we report the partial functional and structural characterization of CCT from an Antarctic notothenioid fish, Notothenia coriiceps. We find that the mechanism of folding by the Antarctic fish CCT differed from that of mammalian CCT: (1) the former complex was able to bind denatured β-tubulin but (2) when reconstituted with rabbit Cofactor A, failed to release the protein to yield the tubulin/cofactor intermediate. Moreover, the amino acid sequences of the N. coriiceps CCT β and θ chains contained residue substitutions in the equatorial, apical, and intermediate domains that would be expected to increase the flexibility of the subunits, thus facilitating function of the chaperonin in an energy poor environment. Our work contributes to the growing realization that protein function in cold-adapted organisms reflects a delicate balance between the necessity of structural flexibility for catalytic activity and the concomitant hazard of cold-induced denaturation.     

Effect of starvation and experimental feeding on the proximate composition and caloric content of an antarctic teleost,Notothenia coriiceps neglecta

• 1.1. Starving Notothenia coriiceps nn/lecta at 1°C for 20 days resulted in a loss of 4.22 gcal/kcal per day.
• 2.2. During starvation energy was obtained from lipid and carbohydrate stores of the liver and red muscle.
• 3.3. Feeding N. coriiceps neglecta low lipid, high protein shrimp meat at 18.9 gcal/kcal per day at 1°C for 20 days resulted in a gain of 8.5 gcal/kcal per day.
• 4.4. The level of carbohydrate in the liver and red muscle increased five times.
• 5.5. Gross growth efficiency (K₁) equalled 0.52.
• 6.6. Net growth efficiency (K₂) equalled 0.67.

     

Hemoglobin from the Antarctic fish Notothenia coriiceps neglecta. 1. Purification and characterisation

Antarctic fishes live at a constant temperature of -1.8 degrees C, in an oxygen-rich environment. In comparison with fishes that live in temperate or tropical waters, their blood contains less erythrocytes and hemoglobin. A study was initiated on the structure and function of Antarctic fish hemoglobin. The erythrocytes of the Antarctic benthic teleost Notothenia coriiceps neglecta, of the family Nototheniidae, have been shown to contain two hemoglobins, accounting for about 90% and 5% of the total content. These hemoglobins have been isolated, and obtained in crystalline form. They are tetramers and contain two pairs of globin chains. The globin chains of each hemoglobin have been purified and characterised. The two hemoglobins appear to have one of the two globin chains in common. The Root and Bohr effects have been investigated in erythrocytes, 'stripped' hemolysates and pure hemoglobins, indicating that the functional properties are finely regulated by pH and allosteric effectors.     

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.     

Karyotypes of two Antarctic fishes,Notothenia gibberifrons andNotothenia coriiceps neglecta

The chromosomes of two species of Antarctic fishes,Notothenia (Gobionotothen) gibberifrons andNotothenia (Notothenia) coriiceps neglecta, were prepared by the air-drying method at the Polish Antarctic Station “Henryk Arctowski” during the austral summer 1984–1985. ForN. (G.) gibberifrons the diploid number is2n = 46 consisting of 2 metacentric (m) pairs, 1 submetacentric (sm) pair and 20 telocentric (t) or subtelocentric (st) pairs. ForN. (N.) coriiceps neglecta the diploid number is 2n = 22 consisting of 9 m pairs, 1 sm pair and 1st pair. Some aspects of karyological evolution of these fishes are discussed.     

Neuronal systems immunoreactive with antiserum to lamprey gonadotropin-releasing hormone in the brain of Petromyzon marinus

Summary The wall structure of arteriovenous anastomoses in the rabbit ear was investigated. (1) Clusters of epithelioid smooth muscle cells form 3–4 longitudinally oriented plicae. The channel shows a single, irregularly outlined lumen, and its wall is very thin between adjacent plicae. (2) Endothelial cells covering the plicae protrude into the lumen, thus suggesting active contraction or shortening of the plicae. (3) The tunica adventitia is composed of 4–6 sheaths of flat fibroblasts, which may serve as a barrier to prevent loss of neurotransmitters. Processes of some of the fibroblasts also extend into the tunica media. (4) The tunica media is composed of an outer circular layer of typical smooth muscle cells, and an inner longitudinally running plica of ramified smooth muscle cells. Wide intercellular spaces between these ramified cells are filled with collagen fibrils, microfibrils, amorphous intercellular substances, and fibroblasts. Fibroblasts form close membrane contacts with each other, and with the smooth muscle cells. (5) Fibroblasts and other connective tissue components may function as an elastic support during active motility of the anastomotic channel.     

Comparison of fatty acid composition in nine organs of the sympatric Antarctic teleost fish species Notothenia coriiceps and Notothenia rossii (Perciformes: Nototheniidae)

Fatty acid (FA) composition of nine organs from two closely related Antarctic fish species, Notothenia coriiceps and Notothenia rossii, was determined through gas chromatography with flame ionization detection. A data set for each species was obtained using major FA profiles from specimens caught in the sea waters of Admiralty Bay during the summer season. The FA profiles for both species are overall similar, but organ peculiarities have been found, which could reflect metabolic specificities and feeding habits between species. With the exception of liver, the most abundant FA in organs was the n?3 polyunsaturated FA. The total n?6 polyunsaturated FAs were minor components in all evaluated organs. Palmitic acid was identified as the major saturated FA, whereas oleic acid was the most represented of the monounsaturated FA in almost all assessed organs of both species. The n? 3/n? 6 ratios of all organs were higher than 3.5. Differences in individual FA and FA metabolic profiles of some organs observed between N. coriiceps and N. rossii suggest specific requirements in the mobilization, transport, incorporation, and/or catabolism of lipids that were reinforced by differences on some FA ratios expressing the activity coefficient of enzymes implicated on the FA pathway flux.     

Posttranslational modification of brain tubulins from the Antarctic fish Notothenia coriiceps: reduced C-terminal glutamylation correlates with efficient microtubule assembly at low temperature

We have shown previously that the tubulins of Antarctic fish assemble into microtubules efficiently at low temperatures (-2 to +2 degrees C) due to adaptations intrinsic to the tubulin subunits. To determine whether changes in posttranslational glutamylation of the fish tubulins may contribute to cold adaptation of microtubule assembly, we have characterized C-terminal peptides from alpha- and beta-tubulin chains from brains of adult specimens of the Antarctic rockcod Notothenia coriiceps by MALDI-TOF mass spectrometry and by Edman degradation amino acid sequencing. Of the four fish beta-tubulin isotypes, nonglutamylated isoforms were more abundant than glutamylated isoforms. In addition, maximal glutamyl side-chain length was shorter than that observed for mammalian brain beta tubulins. For the nine fish alpha-tubulin isotypes, nonglutamylated isoforms were also generally more abundant than glutamylated isoforms. When glutamylated, however, the maximal side-chain lengths of the fish alpha tubulins were generally longer than those of adult rat brain alpha chains. Thus, Antarctic fish adult brain tubulins are glutamylated differently than mammalian brain tubulins, resulting in a more heterogeneous population of alpha isoforms and a reduction in the number of beta isoforms. By contrast, neonatal rat brain tubulin possesses low levels of glutamylation that are similar to that of the adult fish brain tubulins. We suggest that unique residue substitutions in the primary structures of Antarctic fish tubulin isotypes and quantitative changes in isoform glutamylation act synergistically to adapt microtubule assembly to low temperatures.