Characterization and expression pattern of a novel β-defensin in common carp (Cyprinus carpio L.): implications for its role in mucosal immunity

β-defensins are a group of cysteine-rich cationic antimicrobial peptides that play antibacterial and antiviral roles in immune systems of vertebrates. Here, we report the cloning and identification of a β-defensin 3 cDNA sequence from the common carp (Cyprinus carpio L.). Sequence alignment and phylogenetic analysis indicated that this β-defensin 3 belonged to the BD-2 group of fish. Real-time PCR showed that the β-defensin 3 mRNA was expressed in all the tissues of normal common carp that we examined and was highly expressed in the spleen and gills. When challenged with Vibrio anguillarum, the expression level of common carp β-defensin 3 mRNA was quickly upregulated in various tissues. Our results indicate that the β-defensin 3 showed markedly high constitutive expression in the gills, and significantly upregulated expression in the hindgut of the common carp after infection, suggesting it plays an important role in the innate and mucosal immunity of common carp.     

Reduced inflammatory response to Aeromonas salmonicida infection in common carp (Cyprinus carpio L.) fed with β-glucan supplements

The objective of the present study was to determine the action of β-glucans as feed additives on the gene expression profile of some inflammatory-related cytokines from common carp (Cyprinus carpio L.) during the early stages of a non-lethal bacterial infection with Aeromonas salmonicida. β-glucan (MacroGard^®), was administered daily to carp (6 mg per kg body weight) in the form of supplemented commercial food pellets for 14 days prior to infection. Control and treated fish were then intraperitoneally injected with PBS or 4 × 10⁸ bacteria per fish and were sampled at time 0 and 6 h, 12 h, 1 day, 3 days and 5 days post-injection. Head kidney and gut were collected and the gene expression patterns for tnfα1, tnfα2, il1β, il6 and il10 were analyzed by quantitative PCR. Results obtained showed that treatment with β-glucans generally down-regulated the expression of all measured genes when compared to their corresponding controls. After injection, highest changes in the gene expression levels were obtained at 6 h; particularly, in head kidney there was higher up-regulation of tnfa1 and tnfa2 in infected fish fed β-glucans in comparison to control feed; however, in gut there was a significant down-regulation of tnfα1, tnfα2, il1β and il6 in infected fish fed β-glucans. Analysis of carp specific antibodies against A. salmonicida 30 days after injection revealed their levels were reduced in the infected β-glucan group. In conclusion, a diet supplemented with β-glucan (MacroGard^®) reduced the gene expression levels of some inflammation-related cytokines in common carp. Such a response appears to be dependent of organ studied and therefore the immunostimulant may be preventing an acute and potential dangerous response in gut, whilst enhancing the inflammatory response in head kidney when exposed to A. salmonicida.     

Comparative study of β-glucan induced respiratory burst measured by nitroblue tetrazolium assay and real-time luminol-enhanced chemiluminescence assay in common carp (Cyprinus carpio L.)

The respiratory burst is an important feature of the immune system. The increase in cellular oxygen uptake that marks the initiation of the respiratory burst is followed by the production of reactive oxygen species (ROS) such as superoxide anion and hydrogen peroxide which plays a role in the clearance of pathogens and tissue regeneration processes. Therefore, the respiratory burst and associated ROS constitute important indicators of fish health status. This paper compares two methods for quantitation of ROS produced during the respiratory burst in common carp: the widely used, single-point measurement based on the intracellular reduction of nitroblue tetrazolium (NBT) and a real-time luminol-enhanced assay based on the detection of native chemiluminescence. Both assays allowed for detection of dose-dependent changes in magnitude of the respiratory burst response induced by β-glucans in head kidney cells of carp. However, whereas the NBT assay was shown to detect the production of only superoxide anions, the real-time luminol-enhanced assay could detect the production of both superoxide anions and hydrogen peroxide. Only the chemiluminescence assay could reliably record the production of ROS on a real-time scale at frequent and continual time intervals for time course experiments, providing more detailed information on the respiratory burst response. The real-time chemiluminescence assay was used to measure respiratory burst activity in macrophage and neutrophilic granulocyte-enriched head kidney cell fractions and total head kidney cell suspensions and proved to be a fast, reliable, automated multiwell microplate assay to quantitate fish health status modulated by β-glucans.     

Cloning and expression analysis in mature individuals of two chicken type-Ⅱ GnRH (cGnRH-Ⅱ) genes in common carp (Cyprinus carpio)

Gonadotropin-releasing hormone (GnRH), the pivotal signal molecular of hypothalamic-pituitary- gonad (HPG) axis, plays a crucial role in gonadal de-velopment and maintenance of reproduction function of vertebrates by stimulating the anterior pituitary re-leasing gonadotropin (GtH). Mammal GnRH (mGn- RH) was firstly identified from porcine and ovine in the 1970s[1,2], which was originally named luteinizing hormone releasing hormone, viz LHRH. To date, 16 GnRH variants have been characte…     

Characterisation of the cDNA clones of two β-tubulin genes and their expression in the potato plant (Solanum tuberosum L.)

The cDNA clones of two potato -tubulin genes were isolated from a tuberising stolon tip library. Analysis of 20 positive clones showed that they represented one or another of two different but very similar -tubulin genes, designated TUBST1 and TUBST2. The expression pattern of -tubulin genes in the potato plant was investigated by RNA blot analysis and by RT-PCR. Southern analysis of potato genomic DNA with coding and non-coding -tubulin probes revealed that there are multiple -tubulin genes in the potato genome and that there is likely to be considerable divergence in the 3 non-coding sequences. Phylogenetic analysis of plant -tubulin genes is described.     

Effect of β-1/3,1/6-glucan upon immune responses and bacteria in the gut of healthy common carp (Cyprinus carpio)

β-glucans are frequently included in the diet of healthy common carp Cyprinus carpio as a pre-emptive measure for combatting disease. In order to study the effect this has on the relationship between the gut bacteria and host immune response, carp were maintained on either a β-glucan free diet or feed containing 0.1% MacroGard®, a β-1/3, 1/6-glucan, for up to 7 weeks and analysis of innate immune gene expression and molecular analysis of the gut bacteria was performed. The data reveals feeding of MacroGard® to healthy carp does not induce bactericidal innate immune gene expression in the gut but does appear to alter bacterial species richness that did not have a negative effect on overall health. Analysis of innate immune gene expression within the upper midgut revealed that there were significant changes over time in the expression of Interleukin (il)-1β, inducible nitric oxide synthase (inos), mucin (muc2) and C-reactive protein (crp2). Diet did not affect the number of copies of the bacterial 16s rDNA gene in the gut, used as a as a measure of total bacteria population size. However, PCR-denaturing gradient gel electrophoresis (DGGE) analysis revealed a shift in bacterial species richness with MacroGard feeding. Bactericidal immune gene expression of crp2, muc2 and il-1β was weakly correlated with gut bacteria population size indicating a potentially limited role of these genes in interacting with the gut bacteria in healthy carp in order to maintain gut homeostatic conditions. These findings highlight the importance of considering both host immunity and the microbiome together in order to fully elucidate the effeect of immunomodulants, such as β-glucans, upon gut health.     

Dietary β-glucan stimulate complement and C-reactive protein acute phase responses in common carp (Cyprinus carpio) during an Aeromonas salmonicida infection

The effect of β-glucans as feed additive on the profile of C-reactive protein (CRP) and complement acute phase responses was studied in common carp Cyprinus carpio after exposition to a bacterial infection with Aeromonas salmonicida. Carp were orally administered with β-glucan (MacroGard^®) for 14 days with a daily β-glucan intake of 6 mg per kg body weight. Fish were then intraperitoneally injected with either PBS or 1 × 10⁸ bacteria per fish and sampled at time 0, 6, 12, 24, 48, 72, 96 and 120 h post-injection (p.i.) for serum and head kidney, liver and mid-gut tissues. CRP levels and complement activity were determined in the serum samples whilst the gene expression profiles of CRP and complement related genes (crp1, crp2, c1r/s, bf/c2, c3 and masp2) were analysed in the tissues by quantitative PCR. Results obtained showed that oral administration of β-glucan for 14 days significantly increased serum CRP levels up to 2 fold and serum alternative complement activity (ACP) up to 35 fold. The bacterial infection on its own (i.e. not combined with a β-glucan feeding) did have significant effects on complement response whilst CRP was not detectably induced during the carp acute phase reaction. However, the combination of the infection and the β-glucan feeding did show significant effects on both CRP and complement profiles with higher serum CRP levels and serum ACP activity in the β-glucan fed fish than in the control fed fish. In addition, a distinct organ and time dependent expression profile pattern was detected for all the selected genes: a peak of gene expression first occurred in the head kidney tissue (6 h p.i. or 12 h p.i.), then an up-regulation in the liver several hours later (24 h p.i.) and finally up- or down-regulations in the mid-gut at 24 h p.i. and 72 h p.i. In conclusion, the results of this study suggest that MacroGard^® stimulated CRP and complement responses to A. salmonicida infection in common carp.     

Molecular cloning ofClostridium thermocellum genes involved in β-glucan degradation in bacteriophage lambda

A genomic library ofClostridium thermocellum DSM 1237 was constructed in a bacteriophage lambda vector and screened for hydrolysis of carboxymethyl cellulose (CMC), lichenan and methylumbelliferyl--glucoside. Recombinant clones expressing four -glucanases and two distinct -glucosidases were obtained. The -glucanase activities could be classified with respect to their substrate specificities as -1, 4-endoglucanase (CMCase), -1, 3-endoglucanase (laminarinase), and -1, 3-1, 4-endoglucanases (lichenanase). The -glucosidases were identified as cellobiases hydrolyzing both aryl--glucosides and cellobiose.     

Bovine β-defensins: Identification and characterization of novel bovine β-defensin genes and their expression in mammarygland tissue

-Defensin genes code for multifunctional peptides with a broad-range antimicrobial activity. In this project we hypothesized that -defensin genes may be candidate genes for resistance to mastitis. In this article we describe the identification and genomic characterization of eight bovine -defensin genes, including six novel defensin genes and two pseudogenes. Expression in the bovine mammary gland of one of the novel genes, DEFB401, has been demonstrated, as well as the expression of LAP, TAP, DEFB1, BNBD3, BNBD9, and BNBD12. For genomic characterization, 20 BACs from two different bovine BAC libraries (RZPD numbers 750 and 754) were isolated by PCR screening with -defensin consensus primers derived from published sequences. PCR products from BACs generated with consensus primers have been subcloned and sequenced, revealing a total of 16 genes and two pseudogenes. Six novel -defensin genes share the typical exon–intron structure and are highly homologous to published bovine -defensin genes. They are named DEFB401–DEFB405 and LAP-like, and two novel pseudogenes are named EBD-P and EBD-P2. Analysis of mammary gland tissue-derived cDNA from nine cows with different clinical findings demonstrated the expression of several -defensin genes mentioned above. First results indicate that the lactational status of the cow presumably has no influence on gene expression. Competent knowledge of antimicrobial activity of -defensins from literature, the abundance of -defensin mRNA in the bovine mammary gland, and the inducibility of some genes give first evidence that -defensins may play a role in local host defense during udder infections.The nucleotide sequence data reported in this article have been submitted to EMBL and have been assigned the accession numbers AJ563279–AJ563283, AJ567353–AJ567365, AJ567990–AJ567993, and AJ620296.     

Characterization of transgene integration pattern in F4 hGH-transgenic common carp （Cyprinus carpio L.）

The integration pattern and adjacent host sequences of the inserted pMThGH-transgene in the F4 hGH-transgenic common carp were extensively studied. Here we show that each F4 transgenic fish contained about 200 copies of the pMThGH-transgene and the transgenes were integrated into the host genome generally with concatemers in a head-totail arrangement at 4-5 insertion sites. By using a method of plasmid rescue, four hundred copies of transgenes from two individuals of F4 transgenic fish, A and B, were recovered and clarified into 6 classes. All classes of recovered transgenes contained either complete or partial pMThGH sequences. The class I, which comprised 83% and 84.5% respectively of the recovered transgene copies from fish A and B, had maintained the original configuration, indicating that most transgenes were faithfully inherited during the four generations of reproduction. The other five classes were different from the original configuration in both molecular weight and restriction map, indicating that a few transgenes had undergone mutation, rearrangement or deletion during integration and germline transmission. In the five types of aberrant transgenes, three flanking sequences of the host genome were analyzed. These sequences were common carp β-actin gene, common carp DNA sequences homologous to mouse phosphoglycerate kinase-1 and human epidermal keratin 14, respectively.     

Bioenergetics and RNA/DNA ratios in the common carp (Cyprinus carpio ) under hypoxia

Hypoxia caused by eutrophication occurs over large areas in aquatic systems worldwide. Common carp (Cyprinus carpio) exposed to hypoxia (1 mg · O₂ · l⁻¹ and 2 mg · O₂ · l⁻¹) for 1 week showed a significant reduction in feeding rate, respiration rate, faecal production and nitrogenous excretion compared to those maintained at normoxia (7 mg · O₂ · l⁻¹). Fish exposed to hypoxia showed negative scope for growth (SfG), but no significant difference in the specific growth rate was revealed after 1 week in both hypoxic groups. A significant reduction in RNA/DNA ratio was, however, clearly evident in the white muscle of the 1 mg · O₂ · l⁻¹ treatment group, but not in the 2 mg · O₂ · l⁻¹ treatment group. Both specific growth rate and RNA/DNA ratio were significantly reduced when fish were exposed to severe hypoxia (0.5 mg · O₂ · l⁻¹) for 4 weeks. At all levels of hypoxia, growth reduction was accompanied by a significant decrease in RNA/DNA ratio in white muscle. Covariance analysis showed no significant difference between the slope of RNA/DNA ratio and growth rate under normoxic conditions and 0.5 mg · O₂ · l⁻¹ for 4 weeks (F=1.036, P > 0.326), as well as 1.0 mg · O₂ · l⁻¹ and 2.0 mg · O₂ · l⁻¹ for 1 week (F = 0.457, P > 0.5), indicating that the RNA/DNA ratio serves as a biomarker of growth under all oxygen levels, at least under controlled experimental conditions. SfG also appears to be more sensitive than the RNA/DNA ratio in responding to hypoxia in fish. Accepted: 15 September 2000     

Biochemical characterization of two novel β-glucosidase genes by metagenome expression cloning

Two novel β-glucosidase genes designated as bgl1D and bgl1E, which encode 172- and 151-aa peptides, respectively, were cloned by function-based screening of a metagenomic library from uncultured soil microorganisms. Sequence analyses indicated that Bgl1D and Bgl1E exhibited lower similarities with some putative β-glucosidases. Functional characterization through high-performance liquid chromatography demonstrated that purified recombinant Bgl1D and Bgl1E proteins hydrolyzed D-glucosyl-β-(1-4)-D-glucose to glucose. Using p-nitrophenyl-β-D-glucoside as substrate, K(m) was 0.54 and 2.11 mM, and k(cat)/K(m) was 1489 and 787 mM(-1) min(-1) for Bgl1D and Bgl1E, respectively. The optimum pH and temperature for Bgl1D was pH 10.0 and 30°C, while the optimum values for Bgl1E were pH 10.0 and 25°C. Bgl1D exhibited habitat-specific characteristics, including higher activity in lower temperature and at high concentrations of AlCl(3) and LiCl. Bgl1D also displayed remarkable activity across a broad pH range (5.5-10.5), making it a potential candidate for industrial applications.     

Adaptation of fatty acid composition to temperature—a study on carp (Cyprinus carpio L.) liver slices

1. Liver slices obtained from warm-, and cold-adapted carp were incubated in the presence of [1-¹⁴C]sodium acetate, -stearate, -linoleate, and -linolenate at various temperatures and the distribution of radioactivity among different phospholipid fatty acids was determined.2. Relative labelling of saturated fatty acids is reduced, while that of long chain polyunsaturated fatty acids, especially of docosahexanoate, is increased with decreasing temperatures.3. Liver slices of cold adapted carp produced a fatty acid population at 25°C indistinguishable from that produced by warm adapted ones at the same temperature.4. Liver slices obtained from cold-, and warm-adapted animals start to reorganise the pattern of labelling immediately after the exposure to the opposite temperatures as evident from pulse-chase labelling experiments.5. Desaturation of saturated and various unsaturated fatty acids is initiated immediately after down-shift of the temperature. This cold induced increase in desaturase activity is prevented by cycloheximide in the incubation medium.6. It is concluded that phospholipid fatty acid composition is continuously adjusted to the temperature and is governed partly by temperature coefficient of fatty acid synthetase and partly by induction or deactivation of desaturases in cold and warm, respectively.     

Molecular cloning and expression analysis of three genes encoding starch synthase II in rice

Three starch synthase (SS) genes, OsSSII-1, OsSSII-2 and OsSSII-3, were identified in rice (Oryza sativa L.) and localized to chromosomes 10, 2 and 6, respectively. The three OsSSII full-length cDNAs were cloned, and the predicted amino acid sequences were found to share 52–73% similarity with other members of the plant SSII family. The SS activity of each OsSSII was confirmed by expression and enzyme activity assay in Escherichia coli. Expression profile analysis revealed that OsSSII-1 was expressed in endosperms, leaves and roots; OsSSII-2 was mainly expressed in leaves, while OsSSII-3 was mainly expressed in endosperms. Similar to the OsSSI proteins, the OsSSII-2 and OsSSII-3 proteins were found in the soluble as well as the starch-granule-bound fractions in rice. The roles of the OsSSII proteins in starch biosynthesis in rice and the evolutionary relationships of the genes encoding monocotyledonous and dicotyledonous class-II SS enzymes are discussed.Abbreviations CDS Coding domain sequence - EST Expressed sequence tag - GB Granule-bound - Glc Glucose - SS Starch synthase     

Molecular cloning and biochemical characterization of α- and β-tubulin from potato plants (Solanum tuberosum L.)

Few studies have investigated microtubules from plants that host pathogenic fungi. Considerable efforts are underway to find an antimitotic agent against plant pathogens like Phytophthora infestans. However, screening the effects of antifungal agents on plant tubulin in vivo or using purified native microtubule in vitro is a time consuming process. A recombinant, correctly folded, microtubule-like structure forming tubulin could accelerate research in this area. In this study, we cloned full length cDNAs isolated from potato leaves using reverse-transcribed polymerase chain reaction (RT-PCR). Solanum tuberosum (Stub) α-tubulin and β-tubulin were predicted to encode 449 and 451 amino acid long proteins with molecular masses of 57 kDa and 60 kDa, respectively. Average yields of α- and β-tubulin were 2.0–3.5 mg l^?1 and 1.3–3.0 mg l^?1 of culture, respectively. The amino acids, His6, Glu198, and Phe170 involved in benomyl sensitivity were conserved in Stub tubulin. The dimerization of tubulin monomers was confirmed by western blot analysis. When combined under appropriate conditions, these recombinant α- and β-tubulins were capable of polymerizing into microtubules. Accessibility of cysteine residues of tubulin revealed that important ligand binding sites were folded correctly. This recombinant tubulin could serve as a control of phytotoxicity of selected antimitotic fungicide compounds during in vitro screening experiments.     

Molecular weight and subunit composition of GABAA-ergic compound-sensitive Cl−, HCO 3 − -Stimulated Mg2+ ATPase from the plasma membrane of carp brain (Cyprinus carpio L.)

Molecular weight and subunit composition of Cl^?, HCO ₃ ^? , and picrotoxin-stimulated Mg²⁺-ATPase solubilized with sodium deoxycholate from the plasma membrane fraction of fish brain were studied by gel filtration. These enzymes were eluted from a Sephacryl S-300 column as a single peak and corresponded to a ～300 kDa protein with a Stokes radius of 5.4 nm. The enzyme-enriched fraction concentrated and denatured by SDS was eluted from a Sephacryl S-200 column as a single subunit with a molecular weight of ～56 kDa. SDS-PAGE also revealed a single major protein band with a molecular weight of ～56 kDa. It was concluded that the molecular weight and subunit composition of Cl^?, HCO ₃ ^? -stimulated Mg²⁺-ATPase isolated from the plasma membrane of fish brain are similar to those of GABA_A/benzodiazepine receptor complex from fish brain but differ from those of P-type transport ATPases.