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PubMed2006

Structural conservation and food habit-related liver expression of uncoupling protein 2 gene in five major Chinese carps.

The full-length cDNA of grass carp (Ctenopharyngodon idellus) and silver carp (Hypophthalmichthys molitrix) uncoupling protein 2 (UCP2) was obtained from liver. The grass carp UCP2 cDNA was determined to be 1152 bp in length with an open reading frame that encodes 310 amino acids. Five introns (Intron 3, 4, 5, 6 and 7) in the translated region, and partial sequence of Intron 2 in the untranslated region of grass carp UCP2 gene were also obtained. Gene structure comparison between grass carp and mammalian (human and mouse) UCP2 gene shows that, the UCP2 gene structure of grass carp is much similar to that of human and mouse. Partial UCP2 cDNA sequences of bighead carp (Aristichthys nobilis) and mud carp (Cirrhinus molitorella), were further determined. Together with the common carp (Cyprinus carpio) UCP2 sequence from GenBank (AJ243486), multiple alignment result shows that the nucleotide and amino acid sequences of the UCP2 gene, were highly conserved among the five major Chinese carps that belong to four subfamilies. Using beta-actin as control, the ratio UCP2/beta-actin mRNA (%) was determined to be 149.4 +/- 15.6 (common carp), 127.4 +/- 22.1(mud carp), 96.7 +/- 12.7 (silver carp), 94.1 +/- 26.8 (bighead carp) and 63.7 +/- 16.2 (grass carp). The relative liver UCP2 expression of the five major Chinese carps, shows a close relationship with their food habit: benthos and detritus-eating fish (common carp and mud carp) > planktivorious fish (silver carp and bighead carp) > herbivorous fish (grass carp). We suggest that liver UCP2 might be important for Chinese carps to detoxify cyanotoxins and bacteria in debris and plankton food.

Amino Acid Sequence↗
PubMed1993

Plasmodium falciparum: the immune response in rabbits to the clustered asparagine-rich protein (CARP) after immunization in Freund's adjuvant or immunostimulating complexes (ISCOMs).

The Plasmodium falciparum clustered asparagine-rich protein (CARP) is a merozoite-associated antigen which contains approximately 30% asparagine. Analysis of the DNA sequences located 5' of the cloned 1.4-kb CARP gene in the P. falciparum genome suggests that this gene fragment may encode the complete CARP and that the gene product is a protein of M(r) 50,000. To analyze the immunogenicity of CARP, the gene was expressed as a fusion protein with staphylococcal protein A (SpA-CARP). Immunization of rabbits with SpA-CARP in Freund's complete adjuvant (FCA) resulted in a strong antibody response against CARP as measured in ELISA. This response was efficiently boosted and sustained over a long time while that induced by two immunizations with SpA-CARP in ISCOMs was weak and of shorter duration. In both instances, the antibody levels against CARP were further increased by a second booster injection consisting of either SpA-CARP or CARP fused to the serum albumin-binding region (BB) of streptococcal protein G (BB-CARP) in PBS, indicating that immunizations with SpA-CARP in FCA or ISCOMs had induced a CARP-specific immunological memory. Boosting with BB-CARP in PBS was more efficient than boosting with SpA-CARP in PBS. In all rabbits, the antibodies obtained after the booster with CARP in PBS were the most efficient inhibitors of merozoite invasion in vitro. The antisera reacted with the intracellular parasite in immunofluorescence and with a band of M(r) 50,000 in immunoblotting while several high-molecular-weight components as well as the one of M(r) 50,000 were immunoprecipitated. The specificity of the antibody responses varied between the different rabbits as indicated in ELISA, with short synthetic peptides representing different CARP sequences. Taken together, the results suggest that a previously cloned genomic DNA fragment may encode the complete P. falciparum blood-stage antigen CARP and that CARP is immunogenic in rabbits both when administered in FCA or ISCOMs.

Amino Acid Sequence↗
PubMed2003

Genetic divergence between Cyprinus carpio carpio and Cyprinus carpio haematopterus as assessed by mitochondrial DNA analysis, with emphasis on origin of European domestic carp.

Although common carp is the major fish species in Asian and European aquaculture and many domestic varieties have occurred, there is a controversy about the origination of European domestic common carp. Some scientists affirmed that the ancestor of European domestic common carp was Danube River wild common carp, but others considered it might be Asian common carp. For elucidating origination of European domestic common carp, we chose two representative European domestic common carp strains (German mirror carp and Russian scattered scaled mirror carp) and one wild common carp strain of Cyprinus carpio carpio subspecies (Volga River wild common carp) and two Asian common carp strains, the Yangtze River wild common carp (Cyprinus carpio haematopterus) and traditionally domestic Xingguo red common carp, as experimental materials. ND5-ND6 and D-loop segments of mitochondrial DNA were amplified by polymerase chain reaction and analyzed through restriction fragment length polymorphism (RFLP) and sequencing respectively. The results revealed that HaeIII and DdeI digestion patterns of ND5-ND6 segment and sequences of control region were different between European subspecies C. carpio carpio and Asian subspecies C. carpio haematopterus. Phylogenetic analysis showed that German mirror carp and Russian scattered scaled mirror carp belonged to two subspecies, C. carpio carpio and C. carpio haematopterus, respectively. Therefore, there were different ancestors for domestic carp in Europe: German mirror carp was domesticated from European subspecies C. carpio carpio and Russian scattered scaled mirror carp originated from Asian subspecies C. carpio haematopterus.

Animals↗
PubMed2004

[The analysis of genetic variability of mtDNA 12S rRNA genes in the allotetraploid, triploid crucian carp and their parents].

The complete 12S rRNA genes from the five F11 allotetraploids, two diploid gynogens, two triploid crucian carps, one red crucian carp, one xiangjiang common carp, and one Japanese crucian carp were successfully cloned and sequenced. Sequence compositions and variations were analyzed with MEGA 1.0 software. Five F11 allotetraploids shared two haplotypes, and two diploid gynogens, two triploid crucian carps, one red crucian carp, one xiangjiang common carp, one Japanese crucian carp shared one haplotype, respectively. The similarities among red crucian carp, xiangjiang common carp, allotetraploid, diploid gynogen, Japanese crucian carp, and triploid crucian carp varied from 95% to 99%. The similarities between allotetraploid, triploid crucian carp and their female parents (red crucian carp and Japanese crucian carp, respectively) were higher than those between allotetraploid, triploid crucian carp and their male parents (xiangjiang common carp and allotetraploid, respectively). Such result indicated that mitochondrial 12S rRNA genes of the allotetraploid and triploid crucian carp were inherited maternally. A phylogenetic tree was reconstructed using the neighbor joining method of the MEGA package. It was noticeable that after nine generations from F3 to F11 hybrids, the five F11 allotetraploids shared two haplotypes (AT-1 and AT-2), indicating the allotetraploid stock had the genetic diversity. The results provided some valuable information for the breeding, genetic conservation, and population rejuvenation of the tetraploid stock.

Animals↗
PubMed2004

[Triploid crucain carp-allotetraploid hybrids (male) x goldfish( hermaphrodite)].

A new type of triploid crucian carp was generated by mating the males of the allotetraploid hybrids of red crucian carp (Carassius auratus red var.) (female) x common carp (Cyprinus carpio L.) (male), with the females the goldfish( Carassius auratus red var.) which was red in color and possessed the biforked tails. The biological characteristics of one-year old triploid crucian carp including the number and karotype of chromosome, gonadosomatic indices and gonadal structures, appearance and growth rate were investigated. The results indicated that the triploid crucian carp possessed 150 chromosomes with the karyotype: 33m + 51sm + 33st + 33t. In the breeding season, the gonadosomatic indices (GSI) of the ovaries and testes in the triplpoid crcuian carp were, respectively, lower than those in the diploid red crucian carp, and the tissue sections showed that both the ovaries and the testes in the triploid cruican carp were not able to produce the mature ova and sperm, respectively, indicating that they were sterile during one year. The triploid crucian carp was gray in color and had a single fork tail. They had other morphological traits intermediate to the paternal allotetraploid hybrids and the maternal goldfish, for example, one pair of very short and not readily visible barbells, the formula of lateral line scales: 31-6/7, 18 dorsal fin spines, and 0.5 in ratio of the width to the length of the body which was evidently higher than that (0.41) in another kind of triploid crucian carp called Xiangyun crucian carp, generated by crossing the males of the allotetraploid hybrids with the females of the Japanese crucain carp (Carassius auratus cuvieri T. et S.). Following 8-month culture, the triploid crucian carp averagely reached 350 gram in which the maximum individual weighed 550 gram. Among one- year old triploid crucian carp, the females grew faster than the males. Compared with Xiangyun crucian carp, the new type of triploid crucian carp not only presented some advantages the Xiangyun crucian carp had, for example, fast growth rate and potential sterility, but also got some new good performances like the high ratio of the width to the length of the body and the sweet flesh, suggesting they will be very potential in the commercial interest.

Animals↗
PubMed2004

Genetic variation analysis within and among six varieties of common carp (Cyprinus carpio L.) in China using microsatellite markers.

Five microsatellites were used to study the genetic diversity and genetic structure of one wild and five domestic varieties of common carp in China (the Yangtze River wild common carp, Xingguo red carp, purse red carp, Qingtian carp, Russian scattered scaled mirror carp and Japanese decorative carp). All loci in this study showed marked polymorphism with the number of alleles from 4 to 13. Domestic varieties (except Xingguo red carp) showed less genetic diversity than the Yangtze River wild common carp in terms of allelic diversity. Population differentiation was assessed and each combination of populations displayed significant differentiation (p < 0.05) with the exception of that between the Yangtze River wild common carp and Xingguo red carp. Genetic distance analysis (Nei's standard genetic distance and pairwise F(st) distance) showed that the largest distance was between Russian scattered scaled mirror carp and the Yangtze River wild common carp and the smallest distance was between the Yangtze River wild common carp and Xingguo red carp. However, among six populations Japanese decorative carp displayed the highest level of variability in terms of heterozygosity.

Animals↗
PubMed1998

[Comparative studies on gene expression of isozymes in 4 types of various ploidy crucian carp during embryogenesis].

Isozyme zymograms of esterase (EST), lactate dehydrogenase (LDH), malate dehydrogenase (MDH) and superoxide dismutase (SOD) were analysed by polyacrylamide gradient gel electrophoresis at different developmental stages of embryogenesis in 4 types of various ploidy crucian carp embryos, including haploids, diploids, natural triploids, and multiple tetraploids, and 2 types of haploid and diploid common carp embryos. Haploid embryos of crucian carp (Carassius auratus) and common carp (Cyprinus carpio) were produced by treating eggs with UV-irradiated milt from blunt snout bream (Megalobrama amblycephala). Natural triploid embryos were obtained from the eggs of gynogenetic silver crucian carp (Carassius auratus gibelio) inseminated with milt from red common carp. Multiple tetraploid embryos were also produced by gynogenesis from eggs of the newly discovered multiple tetraploid females inseminated with milt from red common carp. Gradient gel electrophoresis indicated that the band types and staining intensity of 4 isozymes expressed in haploid embryos of crucian carp and red common carp were similar to that in the correlative diploid embryos. In natural triploid silver crucian carp embryos, the zymograms of MDH and SOD isozymes were identical with that of diploid crucian carp embryos, but the EST and LDH isozymes manifested more new enzyme bands in comparison with diploid embryos. The corresponding expressed products of some bands in the triploid embryos, such as EST5 and EST6, could be observed also in red common carp embryos, which provided evidence for hybrid origin about the gynogenetic fish. The multiple tetraploids incorporated one foreign genome of red common carp, therefore, the effects of genes from the foreign genome could be observed in the multiple tetraploid embryos. Gene expression of the isozymes in the tetraploid embryos was somewhat similar to that in hybrids. Owing to interaction of triploid silver crucian carp genomes and common carp haploid genome, some isozyme bands, such as EST5 and EST6, changed in quantity, and some bands increased, such as s-SOD1, s-SOD2, s-SOD3 and s-SOD4 in the tetraploid embryos. Moreover, the heterogeneity was revealed among embryos developed from gynogenetic eggs of 3 different multiple tetraploid individuals.

Animals↗
PubMed2006

[Genetic diversity and differentiation of Russian common carp (Cyprinus carpio L.) breeds inferred from RAPD markers].

Polymorphic components of the common carp Cyprinus carpio L. genome were examined by means of polymerase chain reaction with random primers (RAPD-PCR). Using four primers, genetic diversity estimates were obtained for 12 populations and seven strains of Russian common carp breeds, as well as for European Hungarian common carp and Amur wild common carp (N = 87). The highest number of polymorphic loci was revealed in Angelinskii common carp, as well as in the samples of Altai common carp and Amur wild common carp (P = 23.8-18.7%), while the lowest number of polymorphic loci was in the BB strain of Ropsha common carp. The index of genetic diversity, H, was high (11%) in Amur wild common carp, as well as in Altai and Angelinskii common carps. In the remaining breeds, the value of this index varied from 4 to 8%. Based on summarized RAPD profile (132 bands), a dendrogram of genetic differences was constructed. In this dendrogram, all breeds examined grouped into two clusters. One of the clusters was formed by Hungarian and Angelinskii common carps, and the three samples of Altai common carp. The second cluster was formed by the group consisting of the representatives of Cherepetskskii, Stavropol, and Ropsha common carps, along with the differing from them Amur wild common carp. The observed differentiation was confirmed by the analysis of the polymorphic markers variance by the method of principle components. Evolutionary history and the reasons for genetic differentiation of Russian common carp breeds are discussed.

Animals↗
PubMed1997

CARP, a cardiac ankyrin repeat protein, is downstream in the Nkx2-5 homeobox gene pathway.

To identify the molecular pathways that guide cardiac ventricular chamber specification, maturation and morphogenesis, we have sought to characterize factors that regulate the expression of the ventricular myosin light chain-2 gene, one of the earliest markers of ventricular regionalization during mammalian cardiogenesis. Previously, our laboratory identified a 28 bp HF-la/MEF-2 element in the MLC-2v promoter region, which confers cardiac ventricular chamber-specific gene expression during murine cardiogenesis, and showed that the ubiquitous transcription factor YB-1 binds to the HF-la site in conjunction with a co-factor. In a search for interacting co-factors, a nuclear ankyrin-like repeat protein CARP (cardiac ankyrin repeat protein) was isolated from a rat neonatal heart cDNA library by yeast two-hybrid screening, using YB-1 as the bait. Co-immunoprecipitation and GST-CARP pulldown studies reveal that CARP forms a physical complex with YB-1 in cardiac myocytes and immunostaining shows that endogenous CARP is localized in the cardiac myocyte nucleus. Co-transfection assays indicate that CARP can negatively regulate an HF-1-TK minimal promoter in an HF-1 sequence-dependent manner in cardiac myocytes, and CARP displays a transcriptional inhibitory activity when fused to a GAL4 DNA-binding domain in both cardiac and noncardiac cell context. Northern analysis revealed that carp mRNA is highly enriched in the adult heart, with only trace levels in skeletal muscle. During murine embryogenesis, endogenous carp expression was first clearly detected as early as E8.5 specifically in heart and is regulated temporally and spatially in the myocardium. Nkx2-5, the murine homologue of Drosophila gene tinman was previously shown to be required for heart tube looping morphogenesis and ventricular chamber-specific myosin light chain-2 expression during mammalian heart development. In Nkx2-5(-/-)embryos, carp expression was found to be significantly and selectively reduced as assessed by both whole-mount in situ hybridizations and RNase protection assays, suggesting that carp is downstream of the homeobox gene Nkx2-5 in the cardiac regulatory network. Co-transfection assays using a dominant negative mutant Nkx2-5 construct with CARP promoter-luciferase reporter constructs in cardiac myocytes confirms that Nkx2-5 either directly or indirectly regulates carp at the transcriptional level. Finally, a carp promoter-lacZ transgene, which displays cardiac-specific expression in wild-type and Nkx2-5(+/-) background, was also significantly reduced in Nkx2-5(-/-) embryos, indicating that Nkx2-5 either directly or indirectly regulates carp promoter activity during in vivo cardiogenesis as well as in cultured cardiac myocytes. Thus, CARP is a YB-1 associated factor and represents the first identified cardiac-restricted downstream regulatory gene in the homeobox gene Nkx2-5 pathway and may serve as a negative regulator of HF-1-dependent pathways for ventricular muscle gene expression.

Amino Acid Sequence↗
PubMed1998

Somatostatin inhibits (d-Arg6, Pro9-NEt) salmon gonadotropin-releasing hormone- and dopamine D1-stimulated growth hormone release from perifused pituitary cells of chinese grass carp, ctenopharyngodon idellus.

In this study, a heterologous radioimmunoassay (RIA) for grass carp GH has been validated and used to monitor the kinetics of GH release from perifused grass carp pituitary cells. To establish the anatomical specificity of GH antiserum used in this RIA, immunohistochemical staining was performed in grass carp pituitary sections. Somatotrophs recognized by this GH antiserum were located mainly in the proximal pars distalis without overlapping with gonadotrophs located in the same area or with lactotrophs located in the rostral pars distalis. The immunoreactivity of somatotrophs was abolished by preabsorbing GH antiserum with purified grass carp GH, suggesting that the possibility of a cross-reactivity of antiserum with other grass carp pituitary hormones is unlikely. Using 125I-labeled carp GH as the RIA tracer, parallelism was observed among the displacement curves of grass carp GH standard, grass carp serum, and culture medium conditioned by grass carp pituitary cells, suggesting that this RIA can be used to quantitate grass carp GH levels in biological samples. Using an in vitro column perifusion system, a superactive gonadotropin-releasing hormone (GnRH) analog (d-Arg6, Pro9-NEt)-sGnRH(sGnRHa, 0.3-30 nM), dopamine (DA, 0.1-10 muM), and the nonselective DA agonist apomorphine (0.1-10 muM) stimulated GH release from grass carp pituitary cells in a dose-dependent manner. The GH-releasing effect of DA was mimicked by the D1 agonists SKF38393 (0.1-10 muM) and SKF77434 (0.1-10 muM), but not by the D2 agonist LY171555 (3 muM). In addition, the GH response to DA (1 muM) was blocked by the D1 antagonist SCH23390 (5 muM) but not by the D2 antagonist (+/-) sulpiride (5 muM), suggesting that the GH-releasing action of DA is mediated through receptors resembling mammalian D1 receptors. Somatostatin-14 (SRIF14, 0.01-100 nM), unlike sGnRHa and DA, induced a dose-dependent suppression on basal GH release. At a high dose (100 nM), SRIF14 also abolished the GH responses to sGnRHa (100 nM), DA (10 muM), and the D1 agonist SKF38393 (3 muM). These results, as a whole, provide evidence that GH release in the grass carp is under the direct regulation of GnRH, DA, and SRIF at the pituitary cell level. The present study also suggests that DA D1 receptors are present in grass carp pituitary cells mediating the GH-releasing action of DA.

Animals↗
PubMed1997

A novel cardiac-restricted target for doxorubicin. CARP, a nuclear modulator of gene expression in cardiac progenitor cells and cardiomyocytes.

Doxorubicin (Dox), a cardiotoxic antineoplastic drug, disrupts the cardiac-specific program of gene expression (Kurabayashi, M., Dutta, S., Jeyaseelan, R., and Kedes, L. (1995) Mol. Cell. Biol. 15, 6386-6397; Jeyaseelan, R., Poizat, C., Wu, H. Y., and Kedes, L. (1997) J. Biol. Chem. 272, 5828-5832). To determine whether this drug might interfere with the function of cardiac-specific regulatory pathways, we used a differential display strategy to clone from neonatal rat cardiomyocyte candidate mRNAs that were rapidly sensitive to Dox. We report here the identification of a constitutively expressed, cardiac-restricted, nuclear protein whose mRNA level is exquisitely sensitive to Dox. Hence we have named this protein cardiac adriamycin-responsive protein (CARP). CARP mRNA is present at the earliest stages of cardiac morphogenesis. It was detected by in situ hybridization within the cardiogenic plate of 7. 5-day post coitum (p.c.) embryos, and in 8.5-day p.c. embryos CARP transcripts are present in uniformly high levels in the myocardium. Throughout cardiac development, CARP expression is specific for the myocardium; endocardial cushions and valves exhibit only background levels of signal. Transcript levels persist but gradually decrease in neonatal, 2-week-old, and adult hearts. There were no stages when CARP mRNA could not be detected. The pattern and timing of CARP mRNA expression, including transient expression in the tongue at 14.5 days p.c., coincides with that of Nkx2.5/Csx (a putative homolog of tinman, the Drosophila melanogaster gene responsible for cardiac development). The cloned full-length 1749 nucleotide CARP cDNA encodes a 319-amino acid 40-kDa polypeptide containing five tandem ankyrin repeats. CARP appears to be the rat homolog of a previously reported human single-copy gene (C-193; Chu, W., Burns, D. K., Swerlick, R. A., and Presky, D. H. (1995) J. Biol. Chem. 270, 10236-10245), whose mRNA is inducible by cytokines only in human endothelial cells. CARP appears to function as a negative regulator of cardiac-specific gene expression. Overexpression of CARP in cardiomyocytes suppresses cardiac troponin C and atrial natriuretic factor transcription. Cotransfection experiments in HeLa cells indicate that CARP inhibits Nkx2.5 transactivation of atrial natriuretic factor promoter. When fused to a GAL4 DNA-binding domain, CARP has transcriptional inhibitory properties in noncardiac cells. CARP thus represents the first example of a cardiac-restricted transcriptional regulatory protein that is sensitive to Dox.

Amino Acid Sequence↗
PubMed1989

Purification and properties of pituitary gonadotropic hormone from Indian teleosts: freshwater murrel (Channa punctatus) and carp (Catla catla).

Gonadotropic hormone (GtH) from the pituitaries of two widely different Indian teleosts, a freshwater murrel and a carp, was purified by solvent fractionation, Sephadex G-100 gel filtration, affinity chromatography on concanavalin A-Sepharose (Con A-Sepharose), and immunoaffinity chromatography. Elution profile from gel filtration showed three peaks in both cases, peak I and II were clearly separated in murrel but not in carp. Peak II demonstrated strong GtH activity in both murrel and carp but this activity could also be detected in peak I and III of carp. TSH activity was restricted to peak I in murrel and was distributed in all three peaks in carp. Chromatography on Con A-Sepharose was useful in harvesting carp glycoprotein hormones, but gave no advantage in murrel. A pure homogenous GtH from murrel and carp could be obtained by using immunoaffinity chromatography. Polyacrylamide gel electrophoresis of murrel and carp GtH showed a single discrete band. Determination of molecular weight (MW) by Sephadex G-100 gel filtration indicated that murrel and carp GtH were 42,000 and 40,000 Da, respectively. SDS-polyacrylamide gel electrophoresis of murrel and carp GtH revealed two dissimilar subunits, alpha and beta. MW of murrel and carp alpha-subunits were 18,000 and 16,000 while those of beta were 27,000 and 26,000 Da, respectively. Comparison of electrophoretic patterns of murrel and carp GtH alpha and beta with ovine LH subunits reveal distinct teleostean GtH subunits.

Animals↗
PubMed2005

Soluble expression, purification, and stabilization of a pro-apoptotic human protein, CARP.

CARP is a novel pro-apoptotic protein that has been cloned and characterized in our previous report. Previous studies showed that suppression of CARP expression results in cell proliferation in several mammalian cell lines and over-expression of CARP leads to apoptosis and inhibition of proliferation in seven tumor cell lines [Liu et al., CARP is a novel caspase recruitment domain containing pro-apoptotic protein, Biochem. Biophys. Res. Commun. 293 (2002) 1396]. To obtain soluble and active form of CARP protein for further functional and structural studies, we have expressed CARP in Escherichia coli by using Gateway cloning system. Optimal induction and expression conditions were also studied. Recombinant histidine-tagged CARP was expressed in E. coli when the carp gene was subcloned into a Gateway expression vector pET21-DEST. The partially soluble recombinant CARP protein was purified to near homogeneity by a two-step FPLC procedure, first by Ni2+ affinity chromatography followed by a gel-filtration chromatography, which yielded about 10 mg protein/L culture with at least 95% purity. Two peaks were detected in the analytical gel-filtration chromatograph while only one peak corresponding to monomer of the CARP protein was left after adding 2 mM dithiothreitol (DTT). The polymers observed are likely due to the formation of intermolecular disulfide bridges. These results suggest that adding DTT is a good solution to prevent the formation of disulfide bonds and to stabilize the protein. Successfully growing crystals of the purified CARP protein also proved that we can produce well folded CARP protein in E. coli.

Apoptosis↗
PubMed1983

High mutagenic potency of several polycyclic aromatic hydrocarbons induced by liver postmitochondrial fractions from control and xenobiotic-treated immature carp.

The metabolism of carcinogens in fish was examined by measuring the activation of different polycyclic aromatic hydrocarbons (PAH) by carp (Cyprinus carpio L.) liver post-mitochondrial fractions (S9) using the Salmonella typhimurium TA100 reverse mutation assay. For this study, 1 non-carcinogen, anthracene (AN), and 4 carcinogens, chrysene (CHR), benzo[a]pyrene (BaP), 3-methylcholanthrene (3MC) and 7,12-dimethylbenzanthracene (DMBA), were chosen. The bioactivating potency of the metabolic systems of carp pretreated with phenobarbital (PB), 3MC or Aroclor 1254 (ARO) were compared to uninduced carp liver. The results show that carp liver has the ability to metabolize carcinogenic PAH into mutagenic metabolites, which is enhanced when carp are pretreated with 3MC or ARO, but not with PB. A positive correlation between the induction of aryl hydrocarbon hydroxylase (AHH) activity in carp liver and the mutagenic potencies of CHR, BaP, DMBA and 3MC, has been observed. The bioactivating ability of carp liver S9 was compared with the ability of the same fractions from female Wistar rats (this study) as well as from Sprague-Dawley rats (literature data). When the mutagenic potencies of selected PAH had been normalized on the activity of BaP, the following order of mutagenic activities with S9 fractions from ARO-treated animals was obtained: (1) BaP (1) greater than DMBA (0.26) greater than 3MC (0.22) greater than CHR (0.05) greater than AN (0) for carp; (2) BaP (1) greater than 3MC (0.48) greater than CHR (0.31) greater than DMBA (0.16) greater than AN (0) for Sprague-Dawley rats; and (3) BaP (1) greater than 3MC (0.17) greater than DMBA (0.11) greater than CHR (0) = AN (0) for female Wistar rats. We conclude that carp and rats are very similar in their ability to activate carcinogenic PAH into mutagenic metabolites, which suggests that carp may be very susceptible to the carcinogenic activity of these compounds. According to our results from the mutagenicity study, as well as from the enzyme induction study, we propose the use of carp as a suitable model system for the study of chemical carcinogens.

Animals↗
PubMed2003

Common carp insulin-like growth factor-I gene: complete nucleotide sequence and functional characterization of the 5'-flanking region.

Insulin-like growth factor-I (IGF-I) plays an important role in the growth and development of fish. To understand the molecular mechanism which controls the transcription of the IGF-I gene in common carp, we have cloned and completely sequenced the IGF-I gene and the 5'-flanking region from a local tropical fish, the common carp (Cyprinus carpio), and characterized its promoter activity by transfection into human embryonic kidney (293GHR) cells which express the human growth hormone (GH) receptor. The common carp gene is the smallest IGF-I gene known so far, spanning approximately 13 kb, and is consisted of five exons and four introns. The sequence of the gene is consistent with the single type of IGF-I cDNA that we have isolated previously from a common carp liver cDNA library. The expression pattern of IGF-I is similar between juvenile carp and adult carp. While liver was found to be the major site of IGF-I gene expression in common carp, the expression levels in other tissues are relatively low. Like many other IGF-I gene promoters, there are no apparent TATA box and CCAAT box upstream of the transcription initiation site. However, sequence analysis of the common carp IGF-I promoter region identified several consensus liver-enriched transcription factor binding sites, including HNF-1alpha, HNF-3beta, C/EBP, and one STAT5. We have analyzed the promoter activity of the 5'-flanking region of the common carp IGF-I gene by performing luciferase reporter assays in transfected 293GHR cells. Addition of human GH to the transfected cells led to an increased expression of the reporter gene, indicating that the cloned genomic fragment possessed promoter activity. This was confirmed by the lack of promoter activity of a construct in which the putative promoter was cloned in a reverse orientation upstream of the reporter gene. The liver-specific transcription factor, hepatic nuclear factor (HNF)-1alpha, was also found to be involved in the regulation of the common carp IGF-I transcription. Transfection results from a set of deletion mutants helped to map the locations of the responsive elements on the promoter responsible for the GH effect and for the interaction with HNF-1alpha. These observations provide information for delineating the transcriptional regulation of IGF-I gene expression in common carp.

5' Flanking Region↗
PubMed2002

Aquacultural and socio-economic aspects of processing carps into some value-added products.

Carps are the mainstay of Indian aquaculture, contributing over 90% to the total fish production, which was estimated to be 1.77 million metric tonnes in 1996. Carp culture has a great potential for waste utilization and thus for pollution abatement. Many wastes such as cow, poultry, pig, duck, goat, and sheep excreta, biogas slurry, effluents from different kinds of factories/industries have been efficiently used for enhancing the productivity of natural food of carps and related species. Besides, several organic wastes/byproducts such as plant products, wastes from animal husbandry, and industrial by-products have been used as carp feed ingredients to lower the cost of supplementary feeding. However, to ensure the continued expansion of fish ponds and the pollution control, there must be a market for the fish (carps) produced in these ponds. The carps have, however, a low market value due to the presence of intra-muscular bones, which reduces their consumer acceptability. Thus, a need was felt to develop some boneless convenience products for enhancing the consumer acceptability of the carps. Efforts were made to prepare three value-added fish products, namely fish patty, fish finger and fish salad from carp flesh and were compared with a reference product ('fish pakoura'). Sensory evaluation of these products gave highly encouraging results. The methods of preparation of these products were transferred to some progressive farmers of the region who prepared and sold these products at very attractive prices. Carp processing has a great potential for the establishment of a fish ancillary industry and thus for boosting the production of these species. In Punjab alone, there is a potential of consuming 32,448 metric tonnes per annum of such value-added products (which would require 54,080 metric tonnes of raw fish). The development of value-added products has a significant role in raising the socio-economic status of the people associated with carp culture. The average cost of production of these products was estimated to be INR 80 per kg. With a sale price of INR 110 per kg, and a sale of 50 kg per day of the value-added products (26 days a month), the average monthly income of a carp-processing unit comes to be INR 39,000 (929 USD, approximately).

Animals↗
PubMed2005

Evidence for recombination of mitochondrial DNA in triploid crucian carp.

In this study, we report the complete mitochondrial DNA (mtDNA) sequences of the allotetraploid and triploid crucian carp and compare the complete mtDNA sequences between the triploid crucian carp and its female parent Japanese crucian carp and between the triploid crucian carp and its male parent allotetraploid. Our results indicate that the complete mtDNA nucleotide identity (98%) between the triploid crucian carp and its male parent allotetraploid was higher than that (93%) between the triploid crucian carp and its female parent Japanese crucian carp. Moreover, the presence of a pattern of identity and difference at synonymous sites of mitochondrial genomes between the triploid crucian carp and its parents provides direct evidence that triploid crucian carp possessed the recombination mtDNA fragment (12,759 bp) derived from the paternal fish. These results suggest that mtDNA recombination was derived from the fusion of the maternal and paternal mtDNAs. Compared with the haploid egg with one set of genome from the Japanese crucian carp, the diploid sperm with two sets of genomes from the allotetraploid could more easily make its mtDNA fuse with the mtDNA of the haploid egg. In addition, the triple hybrid nature of the triploid crucian carp probably allowed its better mtDNA recombination. In summary, our results provide the first evidence of mtDNA combination in polyploid fish.

Animals↗
PubMed2004

Some immune parameters in carp cyprinus Carpio susceptible and resistant to the haemoflagellate Trypanoplasma borreli.

The present study addresses aspects of the (specific) immune response of carp to the haemoflagellate Trypanoplasma borreli. Sera of resistant carp contained antibodies, which agglutinated the flagellates in vitro. When flagellates were incubated in fish sera from resistant carp, binding of antibodies to flagellates could be demonstrated by flow cytometry, and T. borreli were effectively killed. Heat-treatment of the sera prevented killing, indicating that complement activation is important for the control of a T. borreli infection. Sera of carp that were highly susceptible to infection with T. borreli contained no antibodies capable of binding to or killing the parasite. Furthermore, specific antibodies were not generated after experimental infection. This lack of antibody production in susceptible carp is not due to a general unresponsiveness of lymphoid cells, since peripheral blood leukocytes (PBL) from susceptible and resistant carp responded to mitogenic stimuli in vitro with lymphocyte proliferation in a similar manner. However, viable flagellates were significantly less able to stimulate proliferation of PBL from susceptible carp. In vitro-produced culture supernatants of freshly isolated PBL from both carp lines (but not those of head kidney cells) differentially modulated the mitogen-induced proliferation of PBL from susceptible and resistant carp. The supernatants enhanced the proliferation of leukocytes obtained from individuals from the same carp line, but suppressed the mitogen-induced proliferation of PBL from the other line tested. This indicates that lymphoid cells from susceptible and resistant carp differ in their spectrum of spontaneously produced immunomodulatory mediators. Whether this is decisive for a T. borreli-specific and successful immune response is discussed.

Analysis of Variance↗