Molecular and functional characterization of channel catfish (Ictalurus punctatus) neutrophil collagenase

Channel catfish (Ictalurus punctatus) neutrophils, like mammalian neutrophils, contain a variety of enzymes and lytic peptides that participate in pathogen destruction. We have identified and characterized from a channel catfish anterior kidney cDNA library a 1.6 kb cDNA that encodes for channel catfish neutrophil collagenase. The deduced amino acid sequence has a predicted molecular mass of 53 kDa. The putative catfish collagenase has nucleotide and amino acid homology of 51.4% and 45.1%, respectively, with human neutrophil collagenase and 50.4% and 47.1%, respectively, with mouse neutrophil collagenase. Certain regions of the molecule, including the cysteine switch and the putative zinc binding sites, were identical to those in the human and mouse genes. Polyclonal antiserum, prepared to the fusion protein, recognizes proteins from channel catfish neutrophil supernatants with molecular masses of approximately 63, 53 and 28 kDa. Supernatants from phorbol dibutyrate stimulated neutrophils were capable of degrading type I collagen. In addition, the polyclonal antiserum prevented the collagenase activity of the supernatants from stimulated catfish neutrophils; whereas, preimmune serum had no effect on collagenase activity of supernatants. Supernatants from unstimulated cells or the fusion protein did not possess the ability of degrading type I collagen. These results indicate that channel catfish neutrophil collagenases share molecular and functional features with mammalian neutrophil collagenase.     

Molecular cloning and characterization of beluga whale (Delphinapterus leucas) interleukin-1beta and tumor necrosis factor-alpha.

Interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha) are cytokines produced primarily by monocytes and macrophages with regulatory effects in inflammation and multiple aspects of the immune response. As yet, no molecular data have been reported for IL-1beta and TNF-alpha of the beluga whale. In this study, we cloned and determined the entire cDNA sequence encoding beluga whale IL-1beta and TNF-alpha. The genetic relationship of the cytokine sequences was then analyzed with those from several mammalian species, including the human and the pig. The homology of beluga whale IL-1beta nucleic acid and deduced amino acid sequences with those from these mammalian species ranged from 74.6 to 86.0% and 62.7 to 77.1%, respectively, whereas that of TNF-alpha varied from 79.3 to 90.8% and 75.3 to 87.7%, respectively. Phylogenetic analyses based on deduced amino acid sequences showed that the beluga whale IL-1beta and TNF-alpha were most closely related to those of the ruminant species (cattle, sheep, and deer). The beluga whale IL-1beta- and TNF-alpha-encoding sequences were thereafter successfully expressed in Escherichia coli as fusion proteins by using procaryotic expression vectors. The fusion proteins were used to produce beluga whale IL-1beta- and TNF-alpha-specific rabbit antisera.     

Molecular cloning, expression pattern, and 3D structural analysis of the MHC class IIB gene in the Chinese longsnout catfish (Leiocassis longirostris)

Major histocompatibility complex (MHC) class I and class II molecules encode glycoproteins which mediate the specificity of the vertebrate adaptive immune response. In this study, MHC class IIB gene from the Chinese longsnout catfish (Leiocassis longirostris) was cloned and sequenced, which encoded a predicted protein of 248 amino acids (28.06 kDa) containing a signal peptide, a beta 1 domain, a beta 2 domain, a connecting peptide, a transmembrane region, and a cytoplasmic tail. Using PCR with primers designed from known fish MHC class IIB sequences followed by elongation of the 5' and 3' ends using rapid amplification of cDNA ends (RACE), the full-length cDNA of longsnout catfish MHC class IIB was identified to be 1293 bp, consisting of a 26 bp 5'-terminal untranslated region (UTR), a 520 bp 3'-UTR, and a 747 bp open reading frame (ORF) bearing characteristics of the immunoglobulin C-type 1 (IGc1) family. The deduced amino acid sequences of the Chinese longsnout catfish MHC class IIB gene had 58-75% identity with those of other fishes. Six class IIB alleles were identified from five individuals. At most two different alleles observed in each individual may infer the existence of a single locus of class IIB gene in the Chinese longsnout catfish genome. An extensive study of polymorphism was examined in 60 individuals. A total of 11 haplotypes of exon 2 were detected in the sampled Chinese longsnout catfish. The rates of nonsynonymous substitutions (d(N)) occurred at a higher frequency than that of synonymous substitutions (d(S)), suggesting the polymorphism of exon 2 seemed to be maintained by the balancing selection. By using long PCR technique, the genomic sequence was further identified to be 2345 bp in length, which contained six exons and five introns. Interestingly, a 98 bp intron 5 cut the 3'-UTR into two parts. Real-time quantitative RT-PCR demonstrated high expression of MHC IIB in gills, spleen, head kidney, and intestine, moderate expression in liver and stomach, and low or negligible expression in heart. Homology modelling has been applied to create a 3D model of longsnout catfish MHC class IIB, with features evaluated and discussed to investigate its interaction with CD4 participating in antigen recognition. The present findings will provide valuable information for understanding structure, function, expression, and molecular polymorphism of MHC class IIB in adaptive immunity of the Chinese longsnout catfish and teleost.     

Characterization of a mannose-binding lectin from channel catfish (Ictalurus punctatus)

Mannose-binding lectin (MBL) is an important component of innate immunity capable of activating the lectin pathway of the complement system. A MBL gene was isolated from channel catfish (Ictalurus punctatus). The deduced protein contains a canonical collagen-like domain, a carbohydrate recognition domain (CRD), and a neck region similar to mammalian mannose-binding lectin. The catfish mannose-binding lectin CRD contains the EPN motif shown previously to mediate mannose specificity. The catfish mannose-binding lectin showed 30-43% identity with MBL protein sequences of rainbow trout, zebrafish, common carp, and goldfish, and 33-35% identity with sequences of mammalian species. In this study, while liver was the predominant source of mannose-binding lectin gene expression in healthy tissues, mannose-binding lectin expression in spleen rose sharply following challenge with a Gram-negative bacterium.     

Molecular cloning and phylogenetic analysis of inflammatory cytokines of the ferret (Mustela putorius furo)

The present study determined the cDNA and deduced amino acid sequences of ferret (Mustela putorius furo) inflammatory cytokines, interferon (IFN)-gamma, interleukin (IL)-1beta, IL-6, IL-8 and tumor necrosis factor (TNF)-alpha. The homologies of the nucleotide sequences of IFN-gamma, IL-1beta, IL-6, IL-8 and TNF-alpha of the ferret to those from other mammalian species ranged from 64.3-92.9%, 73.0-83.9, 58.1-84.8%, 58.1-89.7% and 79.0-95.0%, respectively. As distinctive amino acid residues constituting various motifs and ligand-binding sites and cysteine residues were highly conserved in ferret inflammatory cytokine proteins, ferret cytokines may have fundamentally similar functions to those of other mammals. Phylogenetic analyses based on the deduced amino acid sequences revealed that all ferret inflammatory cytokines were more closely related to those of the Carnivora order, specifically dog and cat, than to other species.     

Sphaerospora infection of American catfishes (Ictalurus punctatus and I. nebulosus) in Europe.

The occurrence in Europe of a Sphaerospora species described in North America is reported. Based upon its morphological characteristics, the parasite could be identified with the species S. hankai described from brown bullhead in Canada. This parasite was found to infect channel catfish (Ictalurus punctatus) cultured in farm ponds in Italy and brown bullhead (Ictalurus nebulosus) living in the supply channels of fish ponds in Hungary. The spores and sporogonic developmental stages were situated in the lumen of the renal tubules. In the authors' opinion, S. ictaluri described from channel catfish can be considered synonymous with S. hankai.     

Pharmacokinetics of gentamicin in channel catfish (Ictalurus punctatus)

The pharmacokinetics of gentamicin were determined in male and female Ictalurus punctatus weighing between 0.3 kg and 1.7 kg. Plasma gentamicin concentrations were measured by radioimmunoassay technique. In the 1st experiment, an intracardiac bolus dosage of gentamicin (1 mg/kg) was given to 10 channel catfish. Samples of blood were obtained (by cardiac puncture) immediately before gentamicin was given and at various times over a 24-hour period. The gentamicin half-life was 770 minutes (beta = 0.0009 +/- 0.0003 minute-1). The maintenance therapeutic IV dosage of gentamicin was calculated to be 1.6 mg/kg given at 33.2-hour intervals. In the 2nd experiment, an IM dosage of the drug (1 mg/kg) was given to 9 channel catfish, and samples of blood were obtained at various times over a 24-hour period. The gentamicin half-life was 770 minutes (beta = 0.0009 +/- 0.0002 minutes-1). The maintenance therapeutic IM dosage of gentamicin was calculated to be 3.5 mg/kg given at 33.2-hour intervals. The IM bioavailability of gentamicin in channel catfish was estimated to be 60%. Other pharmacokinetic values were also determined. It was concluded that the therapeutic regimen of choice for gentamicin in channel catfish was 3.5 mg/kg given IM with 33-hour intervals between doses.     

Characterization of the hepcidin gene in eight species of bats

Hemochromatosis, or iron storage disease, has been associated with significant liver disease and mortality in captive Egyptian fruit bats (Rousettus aegyptiacus). The physiologic basis for this susceptibility has not been established. In humans, a deficiency or resistance to the iron regulatory hormone, hepcidin has been implicated in the development of hereditary hemochromatosis. In the present study, we compared the coding sequence of the hepcidin gene in eight species of bats representing three distinct taxonomic families with diverse life histories and dietary preferences. Bat hepcidin mRNA encoded a 23 amino acid signal peptide, a 34 or 35 amino acid pro-region, and a 25 amino acid mature peptide, similar to other mammalian species. Differences in the sequence of the portion of the hepcidin gene that encodes the mature peptide that might account for the increased susceptibility of the Egyptian fruit bat to iron storage disease were not identified. Variability in gene sequence corresponded to the taxonomic relationship amongst species.     

Effects of fasting on circulating IGF-binding proteins, glucose, and cortisol in channel catfish (Ictalurus punctatus)

The effects of fasting on IGF-binding proteins (IGFBPs), glucose, and cortisol in channel catfish were examined. Fed fish (controls) were compared to 14-, 30-, and 45-day fasted fish and 45-day fasted fish refed for 15 additional days. Body length and weight changes, condition factor (CF), hepatosomatic index (HSI), and plasma glucose and cortisol were assessed to determine growth and metabolic status. Body length and growth rates were inhibited (P<0.05) after 14, 30, and 45 days of fasting. The 14-, 30-, and 45-day fasted fish exhibited hypoglycemia and reduced CF and HSI. Cortisol levels were increased (22.8 +/- 15.2 ng/ml versus 4.7 +/- 3.9 ng/ml) in 30-day fasted fish compared to fed controls (P<0.05). Associated with the increase in cortisol in fasted fish was a concomitant increase in plasma levels of a 20-kDa IGFBP through day 45. A 35- and a 45-kDa IGFBP were also identified but were similar between fed and unfed fish throughout the experiment. At the end of 15 days of refeeding, 20-kDa IGFBP, glucose, and cortisol levels were similar to fed controls. Refeeding also caused an increase in growth rates. These results suggest the existence of a catfish counter part to mammalian IGFBP-1, similar to lower molecular mass IGFBPs reported in other species of fish. These results also suggest that a 20-kDa IGFBP is upregulated during fasting-induced growth inhibition of channel catfish and provide additional evidence of the conserved nature of the IGF-IGFBP-growth axis in fish.     

Molecular cloning and phylogenetic analysis of inflammatory cytokines of Camelidae (llama and camel)

We cloned, sequenced and analyzed the cDNAs encoding Camelidae inflammatory cytokines, including llama (lama glama) interleukin (IL)-1alpha, IL-1beta, IL-6, tumor necrosis factor (TNF)-alpha and camel (Camelus bactrianus) IL-6 and TNF-alpha. The similarity levels of the deduced amino acid sequences of IL-1alpha, IL-1beta, IL-6 and TNF-alpha from llama (camel) to those from other mammalian species, ranged from 60.7% to 87.7%, 52.8% to 75.3%, 41.4% to 98.6%, and 72.9% to 99.6%, respectively. Phylogenetic analyses based on nucleic acid sequences showed that llama IL-1alpha, IL-1beta, IL-6 and TNF-alpha were more closely related to those of camel, pig, cattle, sheep and horse than to those of human, dog, cat, mouse and rat.     

Characterization of anti-channel catfish MHC class IIbeta monoclonal antibodies

This study characterizes four monoclonal antibodies (mAb) developed against the major histocompatibility complex (MHC) class II beta chain of the channel catfish, Ictalurus punctatus. Immunoprecipitations using catfish clonal B cells revealed that each of these mAbs immunoselected proteins of approximately 32 and 36 kD, which are of the appropriate sizes for MHC class II alpha and beta chains, respectively. Cell distribution studies using a fluorescence-activated cell sorter (FACS) combined with RT-PCR analyses demonstrated that MHC class II beta is expressed at a high density on catfish clonal macrophage, B and T cell lines, on alloantigen stimulated leukocytes, and on lipopolysaccharide-induced B-cell blasts. Collectively, these results demonstrate the potential importance of these antibodies as reagents in future studies dealing with the functional role of MHC class II molecules in immune recognition of self from non-self.     

Differential cytochemical staining characteristics of channel catfish leukocytes identify cell populations in lymphoid organs

This is one of the first characterizations of channel catfish (Ictalurus punctatus) leukocytes by enzyme cytochemistry. Leukocytes demonstrated cytoplasmic staining patterns very similar to mammalian leukocytes when stained with acid phosphatase, alpha-naphthyl butyrate esterase, beta-glucuronidase, alpha-naphthyl acetate esterase, Sudan Black B and anti-immunoglobulin specific immunohistochemistry. Lymphocytes, monocytes, macrophages, neutrophils, and surface immunoglobulin positive (surface Ig+) cells were present in channel catfish renal hematopoietic tissue and spleen and demonstrated distinctive cytoplasmic foci staining patterns, cytoplasmic blushing or cell membrane staining. Monocytes, macrophages, lymphocytes and surface Ig+ cells were present in the thymus. Thymic and splenic cellular organization appeared very similar to these same mammalian tissues. In the thymus, acid phosphatase positive cells were distributed throughout the parenchyma, while alpha-naphthyl butyrate esterase and beta-glucuronidase positive cells were concentrated in the cortex and the medulla, respectively. Surface immunoglobulin positive cells occurred in the cortex. In the spleen, acid phosphatase positive cells were scattered throughout the parenchyma, while alpha-naphthyl butyrate esterase positive cells were scattered throughout the parenchyma and adjacent to splenic arterioles. Beta-glucuronidase and surface immunoglobulin positive cells were restricted to immediately adjacent to splenic arterioles. Sudan Black B positive cells were scattered throughout the parenchyma, while alpha-naphthyl acetate esterase positive cells occurred adjacent to peri-arteriole lymphoid sheaths and appear very similar to mammalian metallophils.     

Communications: Specificity of the Channel Catfish Antibody to Edwardsiella ictaluri

Abstract

The specificity of channel catfish Ictalurus punctatus serum antibody to Edwardsiella ictaluri was characterized by microtiter agglutination assay. There was no correlation between antibody titer to Aeromonas hydrophila and antibody titer to E. ictaluri in wild or feral channel catfish. Anti-E. ictaluri antibodies in naturally infected channel catfish were not removed by adsorption by nine other species of bacteria found in the channel catfish intestine and fish ponds. Channel catfish immunized with nine other species of bacteria did not develop substantial antibody titer to E. ictaluri. The antibody response of channel catfish to E. ictaluri is highly specific, and the microtiter agglutination test is a specific indicator of previous exposure to E. ictaluri     

Nutrition of aquaculture species.

Dietary requirements for amino acids and fatty acids have been reported for channel catfish (Ictalurus punctatus), salmonids (Oncorhynchus spp.), common carp (Cyprinus carpio), tilapias (Oreochromis spp.), and eel (Anguilla japonicus). Most of the vitamin and mineral requirements are available for channel catfish and salmonids, and some are available for common carp, tilapia, eel, and other finfish and crustaceans. From this available information, cost-effective feeds can be formulated for the major commercial aquaculture species. Major differences in nutrient requirements between fish and mammals or birds are as follows: fish have a lower digestible energy:protein ratio (8 to 10 kcal of DE/g of CP for fish vs 15 to 20 kcal of DE/g of CP for livestock); fish require n-3 fatty acids and land animals require n-6; fish can absorb minerals from the water, which negates the need for some minerals in the diet; and fish have limited ability to synthesize vitamin C and must depend on a dietary source. Areas for further research include 1) refinement of nutrient requirements of the major culture species considering effects of fish size, temperature, and management; 2) nutrient requirements of crustaceans; 3) effects of nutrition on fish health and product quality; and 4) feeding technology.     

ghrelin对生殖系统的调节作用

ghrelin是新近发现的一个含有28个氨基酸残基的多肽,是生长激素促分泌素受体(GHS-R)的天然配体,除具有调节GH分泌和能量平衡的功能之外,尚有其他许多功能。近年来体外或体内试验研究表明,ghrelin对生殖激素如LH、PRL具有一定的调节作用;另外,ghrelin及其受体系统广泛存在于生殖系统中。提示这一新发现的激素可能对生殖系统具有重要的调节作用。文章就ghrelin对生殖系统调节作用的研究进展加以综述。