Molecular cloning and biochemical characterization of medaka (Oryzias latipes) lysosomal neu4 sialidase

Glycoconjugates are known to be involved in many physiological events in vertebrates. Sialidase is one of the glycosidases, which removes sialic acid from glycoconjugates. In mammals, the properties and physiological functions of sialidases have been investigated, while there is little understanding of fish sialidase. Here, to investigate the significance of fish neu4 sialidase, neu4 gene was cloned from medaka brain mRNA and identified. Sialidase-specific motifs (GPG, YRVP and Asp-Box) were well conserved in the medaka neu4 polypeptide. Optimal pH of medaka neu4 sialidase was 4.6, but its activity was sustained even at neutral and weak alkaline pH. The neu4 considerably cleaved sialic acid from 4-methylumbelliferyl-N-acetyl-α-d-neuraminic acid and sialyllactose, but not from ganglioside and fetuin, which are good substrates for human NEU4. neu4 activity was mostly detected in mitochondria/lysosome fraction after biochemical fractionation, and indirect immunofluorescence assays revealed neu4 localization in lysosome in neu4 overexpressed cells. Next, developmental change in medaka neu4 and other sialidase mRNA levels were estimated by real-time PCR. Each sialidases showed different expression patterns in embryonic development: neu4 was up-regulated at late developmental stage in embryo, and neu3a mRNA level was quite high in 0.5 dpf. On the other hand, neu3b expression was drastically increased after hatching, suggesting that each sialidase may play a different role in embryonic development.     

Cloning,molecular characterization and expression analysis of insulin-like growth factor binding protein-2 (IGFBP-2) cDNA in goldfish,Carassius auratus

In the present study, a full-length cDNA encoding the insulin-like growth factor binding protein-2 (IGFBP-2) was cloned from the liver of goldfish (Carassius auratus) by rapid amplification of cDNA ends technique. The goldfish IGFBP-2 cDNA sequence was 1,513 bp long and had an open reading frame of 825 bp encoding a predicted polypeptide of 274 amino acid residues. Semi-quantitative RT-PCR results revealed that goldfish IGFBP-2 mRNA was expressed in all detected tissues. In liver, central nervous system and pituitary gland, goldfish IGFBP-2 expressed at high levels, followed by anterior intestine, middle intestine and kidney. In posterior intestine, ovary, skin, fat, spleen, muscle and gill, the goldfish IGFBP-2 expression levels were very low. Fasting and refeeding experiment showed that the mRNA expression of goldfish IGFBP-2 was up-regulated significantly in liver compared to the fed group and restored rapidly to normal level after refed. However, the mRNA expressions of IGFBP-2 in hypothalamus and pituitary of goldfish were insensitive to fasting. Furthermore, the mRNA expressions of IGFBP-2 in hypothalamus, pituitary and liver were varied in periprandial changes and significantly down-regulated at 2 and 4 h after meal. These results imply that the IGFBP-2 mRNA expression may be associated with anabolic and catabolic metabolism and regulated by metabolic factors in goldfish.     

Molecular cloning,characterization, and expression analysis of luteinizing hormone receptor gene in turbot (Scophthalmus maximus)

The luteinizing hormone receptor (LHR) plays a crucial role in female reproduction. In the present study, full-length sequence coding for the LHR was obtained from female turbot (Scophthalmus maximus) by homology cloning and a strategy based on rapid amplification of cDNA end-polymerase chain reaction. The full-length LHR cDNA was 3,184 bp long and contained a 2,058-bp open reading frame which encoded a protein of 685 amino acids. Multiple sequence alignments of the turbot LHR manifested high homologies with the corresponding sequences of available teleosts and representative vertebrates, and significant homology with that of Hippoglossus hippoglossus. In addition, the turbot LHR showed typical characteristics of glycoprotein receptors, including a long N-terminal extracellular domain, seven transmembrane domains, and a short C-terminal intracellular domain. LHR mRNA was abundant in the ovary, but was deficient in extra-ovarian tissues. Furthermore, LHR mRNA gradually developed from previtellogenesis to migratory nucleus stage, with the highest values observed in migratory nucleus stage during reproductive cycle. However, LHR mRNA sharply decreased in atresia stage. These results suggested that LHR is a typical G protein-coupled receptor that is involved in the promotion of turbot ovarian development and may be related to the final maturation and ovulation of oocyte. These findings contribute to the understanding of the potential roles of LHR in controlling the fish reproductive cycle.     

Molecular cytogenetic study of genome ploidy in the German mirror carp Cyprinus carpio

We examined the polyploidy of Cyprinus carpio, the German mirror carp. Specimens were collected in the field in Hulan, China, and treated with phytohemagglutinin and colchicine before the chromosome spreads and the karyotype of kidney cells were examined using silver staining, chromomycin A₃ (CMA₃)/distamycin (DA)/4,6-diamidino-2-phenylindole (DAPI) staining, and fluorescence in situ hybridization (FISH) with a 5.8S + 28S rDNA probe. One to twosilver stained (Ag) nucleolus organizing regions (NORs) were detected during metaphase and interphase events, with 80 % of the metaphase spreads and 69 % of the interphase nuclei showing two Ag-NORs signals. One CMA₃ signal was detected on the terminus of the short arm of each submetacentric chromosome 8 (SMC8) homolog (n = 2). The 5.8S + 28S rDNA probe hybridized at the terminus of the short arm of each SMC8 homolog (n = 2). The results of the Ag-NORs, CMA₃/DA/DAPI, and 5.8S + 28S rDNA FISH analyses were consistent with regard to the total number and location of the SMC8 NORs in the chromosome spreads and karyotype, indicating that, at the molecular cytogenetic level, the German mirror carp is an evolutionary tetraploid with two sets of chromosomes after diploidization.     

Responses to ROS inducer agents in zebrafish cell line: differences between copper and UV-B radiation

Fish are commonly exposed to environmental pollutants, which in turns could induce an oxidative stress. So, it is important to understand the effects and the responses elicited by these toxicants in fish species, being fish cell lines important tools for this purpose. Thus, the aim of the present study was to compare the effects of copper and UV-B radiation exposure on zebrafish hepatocytes (ZFL lineage) in terms of reactive oxygen species (ROS) levels, sulfhydril groups content and mRNA levels of important genes related to cellular response to toxic agents. Exposure of ZFL cells to UV-B radiation (23.3 mJ/cm²) significantly increased levels of intracellular ROS and mRNA of both superoxide dismutase isoforms (sod1 and sod2), three glutathione S-transferase isoforms (gstα, gstµ and gstπ) and a heat shock protein (hsp70). However, no changes in nonprotein sulfhydryl groups (NP-SH) content, as well as in the mRNA levels of genes related to glutathione (GSH) synthesis and recycling, were observed. Contrary to this, copper exposure (20 mg/L) diminished NP-SH content and increased the levels of mRNA of genes related to GSH synthesis (gclc and gs). Moreover, copper exposure increases the mRNA levels of some genes related to antioxidant defenses (gpx and gstπ), biotransformation reactions (cyp1a1) and protein repair (hsp70). In conclusion, these results demonstrated that both toxicants could increase ROS levels in ZFL cell line, but the responses are different, which could be related to activation of different signaling pathways.     

Binding profiles and cytokine-inducing effects of fish rhamnose-binding lectins on Burkitt’s lymphoma Raji cells

Rhamnose-binding lectin (RBL) is one of the animal lectin categories which take part in the innate immune responses of fish. Osmerus lanceolatus lectin (OLL) from shishamo smelt eggs is an RBL composed of two tandem-repeated domains, both of which are considered to be a carbohydrate-recognition domain. SAL, catfish (Silurus asotus) egg RBL composed of three domains, binds to Burkitt’s lymphoma Raji cells through globotriaosylceramide (Gb3) carbohydrate chain and to reduce cell size and growth by altering membrane composition without causing cell death. In this experiment, we tried to compare the binding effects of these two RBLs on Raji cells. Flow cytometric and fluorescence microscopic analyses revealed that OLL also directly bound to and shrunk Raji cells with ten times less reactivity than SAL but reduced cell growth with decreasing cell viability. Anti-Gb3 antibody completely blocked the binding of SAL to Raji cells but not that of OLL. In addition, the direct bindings of OLL and SAL to Raji cells were comparably inhibited by melibiose, but lactose was more effective inhibitor for the binding of OLL than that of SAL. These results suggest that OLL has slightly different cell-binding property compared with SAL and binds not only to Gb3 but also to the other carbohydrate receptor-bearing β-galactoside chains. The quantitative RT-PCR analysis revealed that SAL induced the expression of TNF-α but not of IFN-γ, IL-1β, and IL-10. Thus, SAL-induced cytostatic effect on Raji cells might be partially caused by TNF-α-mediated signaling pathway.     

Heat-shock protein 70 modulates apoptosis signal-regulating kinase 1 in stressed hepatocytes of Mugil cephalus

Oxidative stress causes damage at the cellular level and activates a number of signaling pathways. Heat-shock proteins (HSPs) play an important role in repair and protective mechanisms under cell response to stress conditions. HSP70 has been shown to act as an inhibitor of apoptosis. Apoptosis signal-regulating kinase-1 (ASK1) activity is regulated at multiple levels, one of which is through inhibition by cytosolic chaperons HSP70. The current study was aimed to investigate the alteration in signaling molecules that allow the fish to survive under stressed natural field conditions. The study also investigates the variation in biomolecular composition of hepatocytes by using Fourier transform infrared spectroscopy. The impact of stress on hepatocytes was assessed by measuring the level of lipid peroxides (LPO), catalase activity (CAT) and assessing the changes in hepatocytes of Mugil cephalus inhabiting Kovalam and Ennore estuaries. The expression of HSP70 and ASK1 were analyzed by immunoblot analysis and ELISA, respectively. The spectral analysis showed variations in biomolecular composition of hepatocytes at a wave number region of 4,000–400 cm^?1. There was significant decrease of CAT activity (p < 0.01) (25 %) with significant increase of LPO (p < 0.001) (35 %) and HSP70 (p < 0.001) and insignificant increase of ASK1 (p < 0.05) (16 %) in fish hepatocytes inhabiting Ennore estuary than Kovalam estuary. In conclusion, the present study suggests that the survival of fish in the Ennore estuary under stressed condition may be due to the upregulation of HSP70 that mediates the altered signal pathway which promotes cellular resistance against apoptosis.     

Identifying the origin of Corbicula clams using trace element analysis

The trace element contents of Corbicula clam shells collected from Japan, Russia, China, and the Republic of Korea were analyzed to determine their geographic origin. The crushed shells were decomposed with nitric acid–hydrogen peroxide, and the concentrations of 14 elements (Li, Mg, V, Mn, Co, As, Rb, Mo, Ba, Ce, Pb, U, Sr, and Ca) were measured by inductively coupled plasma mass spectrometry and inductively coupled plasma optical emission spectrometry. Some of the elements identified in samples displayed a geographic trend. The average content of manganese in Japanese samples was twice that of Russian samples. Conversely, the arsenic content in Japanese samples was approximately half of that in Russian samples. Linear discriminant analysis was applied to the data from Japanese and Russian samples, and a discriminant model was constructed. The discriminant model was used to determine the geographic origin of Corbicula clams produced in Japan, with 89.8 % of those identified as Japanese and 92.2 % of those identified as Russian being classified correctly. Therefore, trace element analysis of the shells of Corbicula clams is a useful technique for the identification of their country of origin.     

Profiles of thyrotropin,thyroid hormones,follicular cells and type I deiodinase gene expression during ontogenetic development of tilapia larvae and juveniles

The aims of the present study are to determine whether triiodothyronine (T3) and/or thyroxine (T4) in tilapia larvae is gifted through the mother, and to investigate the change profiles of thyrotropin (TSH), thyroid follicular cells and type I deiodinase (D1) gene expression following larval development. T3 and T4 contents were measured using radioimmunoassay, thyrotropin was observed using immunocytochemistry, and the D1 gene was cloned and measured using real-time PCR. Results indicated that the β-TSH-immunoreactive cells (thyrotropin ICC) signals were detected at 9 dph (i.e., 9 days of post-hatching). Thyroid follicular cells were observed first at 3 dph, while the T3 contents of the whole body gradually decreased before 11 dph. T4 contents were detected until 13 dph, with higher secretion during 19–21 dph. In addition, the T3 synthesis was not inhibited by thiourea (TU) before 13 dph, but the TU response in the larvae appeared after 13 dph. Type I deiodinase (D1: GenBank accession number KC591724) was found to contain 2444 bases and encoded 248 amino acids. The D1 mRNA expression began to increase at 13 dph, with a higher expression during 15–19 dph. These results suggested that the T3 contents were maternally derived before 13 dph. Both thyroid hormonal changes and some parameters related to thyroid hormone synthesis in ontogenetic tilapia are discussed.     

Dynamic expression pattern of corticotropin-releasing hormone,urotensin I and II genes under acute salinity and temperature challenge during early development of zebrafish

Corticotropin-releasing hormone (CRH), urotensin I (UI) and urotensin II (UII) are found throughout vertebrate species from fish to human. To further understand the role of crh, uI and uII in teleosts during development, we investigated the expression pattern of crh, uI, uIIα and uIIβ genes, and their response to acute salinity and temperature challenge during early development of zebrafish, Danio rerio. The results reveal that crh, uI, uIIα and uIIβ mRNA are detected from 0hpf, and the expression levels increase to a maximum at 6 days post fertilization (dpf), with the exception of uIIα that peak at 5dpf. Exposure of zebrafish embryos and larvae to acute osmotic (30ppt) stress for 15 min failed to modify expression levels of crh, uI, uIIα and uIIβ mRNA from levels in control fish except at 6dpf when uIIα and uIIβ were significantly (P < 0.05) modified. Exposure of embryos and larvae to a cold (18 °C) or hot stress (38 °C) generally down-regulated mRNA levels of crh, uI, uIIα and uIIβ apart from at 3dpf. The results indicate that the contribution of crh, uI, uIIα and uIIβ genes to the stress response in zebrafish may be stressor-specific during early development. Overall, the results from this study provide a basis for further research into the developmental and stressor-specific function of crh, uI, uIIα and uIIβ in zebrafish.     

Cloning,expression, and ligand-binding characterization of two neuropeptide Y receptor subtypes in orange-spotted grouper,Epinephelus coioides

As one of the most important multifunctional peptides, neuropeptide Y (NPY) performs its physiological functions through different subtype receptors. In this study, full-length cDNAs of two NPY receptors (YRs) in orange-spotted grouper (Epinephelus coioides) were cloned and named npy8br (y8b) and npy2r (y2). Phylogenetic analysis indicated that the Y8b receptor is an ortholog of the teleostean Y8b receptor, which belongs to the Y1 subfamily, and the Y2 receptor is an ortholog of the teleostean Y2 receptor, which belongs to the Y2 subfamily. Both of the YRs have G protein-coupled receptor family profiles. Multiple alignments demonstrate that the extracellular loop regions of YRs have distinctive residues of each species. Expression profile analysis revealed that the grouper Y8b receptor mRNA is primarily expressed in the brain, stomach and intestine, while the grouper Y2 receptor mRNA is primarily expressed in the brain, ovary, liver and heart. Double immunofluorescence analysis determined that the grouper YRs interact with the grouper NPY around the human embryonic kidney 293T cell surface. Furthermore, site-directed mutagenesis in a phage display system revealed that Asp^6.59 might be a common NPY-binding site, while Asp^2.68 of the Y8b receptor and Glu^5.24 of the Y2 receptor could be likely involved in subtype-specific binding. Combining the expression profile and ligand-binding feature, the grouper Y8b receptor could be involved in regulating food intake via the brain-gut axis and the grouper Y2 receptor might play a role in balancing the regulatory activity of the Y8b receptor and participate in metabolism in the liver and ovary.     

The bioactivity of gonadotropin releasing hormones and its regulation in the gilthead seabream,Sparus aurata: in vivo andin vitro studies

Thein vivo andin vitro potency of native and modified forms of gonadotropin releasing hormone (GnRH) to release gonadotropin (GtH) was studied inSparus aurata and correlated with their relative susceptibility to degradation by cytosolic-bound enzymes of the pituitary, kidney, and liver. Salmon (s) GnRH and luteinizing hormone-releasing hormone (LHRH) are equipotent whereas analogs of these peptides ((D-Arg⁶-Pro⁹NET)-sGnRH, (D-Ala⁶-Pro⁹NET)-LHRH, (D-Trp⁶)-LHRH) are superactive in inducingin vivo GtH release (at 10 μg/kg body weight). In anin vitro superfusion system of pituitary fragments all analogs are equipotent to the native peptides (at 10⁻¹⁰ to 2.5 × 10⁻⁷M). sGnRH and LHRH are rapidly degraded by cytosolic peptidases of the pituitary, liver, and kidney. The preferred site of cleavage is the Tyr⁵-Gly⁶ bond. Substitution of the position 6 glycine by D-amino acids renders the 5–6 bond resistant to degradation and shifts the main site of cleavage to the Pro⁹-Gly¹⁰NH₂ bond. Substitution at position 6 (as above) and at position 10 with Pro⁹NET results in analogs that are resistant to degradation. We propose that enzymatic cleavage terminates GnRH bioactivityin vivo and thus increased resistance to degradation is a major determinant of GnRH analog superactivity.     

The role of Ca2+ and Na+ membrane transport in brook trout (Salvelinus fontinalis) spermatozoa motility

The role of environmental ion composition and osmolality in Ca²⁺ signaled activation was assessed in spermatozoa of brook trout Salvelinus fontinalis. Milt from ten mature males was obtained by abdominal massage. Spermatozoa motility was evaluated in 0, 100, and 300 mOsm/kg NaCl or sucrose solutions, buffered by 10 mM Tris–HCl pH 8.5. For investigation of spermatozoa reaction to external Ca²⁺ concentration, 2 mM ethylene glycol tetraacetic acid (EGTA) was added to the activation media as a calcium ions chelator. For investigation of the effect of external Na⁺ concentration in conditions of low external Ca²⁺, 100 µM amiloride was added to the EGTA-containing solutions as a Na⁺ transport blocker. Low motility was observed in sucrose (Na⁺ free) solutions containing 2 mM EGTA but not in Na⁺ solutions containing 2 mM EGTA. Addition of amiloride led to significantly increased motility (P < 0.05) compared with sucrose (Na⁺ free) solutions containing 2 mM EGTA. We conclude that Na⁺ transport in Ca²⁺-free solutions plays a regulatory role in brook trout spermatozoa activation. The influence of competitive Na⁺ and Ca²⁺ transport on the control of spermatozoa activation requires further study with respect to its application for improvement of artificial activation and storage media.     

Exploration of the mechanisms of protein quality control and osmoregulation in gills of Chromis viridis in response to reduced salinity

Fish gills are the vital multifunctional organ in direct contact with external environment. Therefore, activation of the cytoprotective mechanisms to maintain branchial cell viability is important for fish upon stresses. Salinity is one of the major factors strongly affecting cellular and organismal functions. Reduction of ambient salinity may occur in coral reef and leads to osmotic stress for reef-associated stenohaline fish. However, the physiological responses to salinity stress in reef-associated fish were not examined substantially. With this regard, the physiological parameters and the responses of protein quality control (PQC) and osmoregulatory mechanisms in gills of seawater (SW; 33–35 ‰)- and brackish water (BW; 20 ‰)-acclimated blue-green damselfish (Chromis viridis) were explored. The results showed that the examined physiological parameters were maintained within certain physiological ranges in C. viridis acclimated to different salinities. In PQC mechanism, expression of heat-shock protein (HSP) 90, 70, and 60 elevated in response to BW acclimation while the levels of ubiquitin-conjugated proteins were similar between the two groups. Thus, it was presumed that upregulation of HSPs was sufficient to prevent the accumulation of aggregated proteins for maintaining the protein quality and viability of gill cells when C. viridis were acclimated to BW. Moreover, gill Na⁺/K⁺-ATPase expression and protein amounts of basolaterally located Na⁺/K⁺/2Cl^? cotransporter were higher in SW fish than in BW fish. Taken together, this study showed that the cytoprotective and osmoregulatory mechanisms of blue-green damselfish were functionally activated and modulated to withstand the challenge of reduction in salinity for maintaining physiological homeostasis.     

Immunoadjuvant effect of garlic (Allium sativum)–mineral oil suspension on immunity and resistance to Aeromonas hydrophila infection in rohu,Labeo rohita

The immunomodulatory effect of garlic–mineral oil as a modified adjuvant was investigated in rohu, Labeo rohita along with formalin-killed Aeromonas hydrophila. The garlic extract was not having any proteolytic effect on the tested antigen as marked in SDS-PAGE. The efficacy of graded levels of modified adjuvant formulation was assessed by measuring serum hemolysin and agglutination titers as well as level of protection against A. hydrophila challenge in the immunized fish at days 10 and 30 post-vaccination (dpv). The higher hemolysin and bacterial agglutinin titers along with significantly lower mortality against A. hydrophila challenge in modified adjuvant-based vaccinated fish, particularly at low level of garlic incorporation, indicated the pronounced immunoadjuvant role of this modified adjuvant formulation in rohu.