Response of toll-like receptors, lysozyme, and IGF-I in back-cross hybrid (F1 male (blue x channel) x female channel) catfish challenged with virulent Edwardsiella ictaluri

Responses of toll-like receptors (TLR3 and TLR5), lysozyme, and insulin-like growth factor-I (IGF-I) to experimental challenge with virulent Edwardsiella ictaluri were measured in back-cross hybrid (F1 male (blue x channel) x female channel) catfish. The resistance levels to E. ictaluri and host response mechanisms of back-cross hybrids are unknown. Fish were challenged with virulent E. ictaluri and sampled pre-challenge, 2 h and 2, 5, 8, 14, and 21 days post-challenge. Levels of mRNA expression of two toll-like receptors (TLR3 and TLR5) in liver, kidney, spleen, and stomach, plasma lysozyme activity, and circulating IGF-I levels were measured at each timepoint. Throughout challenge, TLR3 was expressed at higher levels than TLR5 in liver (P=0.0011) and kidney (P=0.0007) whereas TLR5 was more highly expressed than TLR3 in stomach (P=0.0032). TLR3 was upregulated in comparison to non-exposed controls in liver (P=0.0015) and stomach (P<0.0001) on day 14 and TLR5 was upregulated in liver (P=0.0175) on days 2 through 8. Plasma lysozyme activity peaked on day 5 (P<0.001) and IGF-I levels significantly decreased on days 2 through 14 (P<0.0001). TLR expression patterns suggest that both TLR3 and TLR5 may play a role in host response to bacterial challenge. Plasma lysozyme activity also increased and circulating IGF-I decreased in response to the presence of the pathogen.     

IGF-I and IGF-II mRNA expression in slow and fast growing families of USDA103 channel catfish (Ictalurus punctatus)

The objective of this study was to examine insulin-like growth factor (IGF)-I and IGF-II mRNA levels in fast and slow growing families of catfish. Relative levels of IGF-I and IGF-II mRNA were determined by real-time PCR. Family A exhibited a specific growth rate (SGR) of 3.6 and was designated as fast growing, while family H exhibited a SGR of 3.1 and was designated as slow growing (P=0.017). Levels of IGF-II mRNA were 3.3-fold greater (P=0.006) in muscle for the fast growing family compared to the slow growing family. Levels of IGF-II mRNA were 1.8-fold greater (P=0.049) in liver for the fast growing family compared to the slow growing family. Levels of IGF-II mRNA from both fast and slow families were 12.2-fold greater (P<0.001) in muscle and 5.8-fold greater (P=0.021) in liver, respectively, compared to levels of IGF-I mRNA. Muscle and liver levels of IGF-I mRNA were similar between families. Elevated levels of IGF-II mRNA in muscle and liver compared to IGF-I mRNA, as well as differences in levels of IGF-II mRNA between fast and slow growing families of fish suggests a role of IGF-II in growth of channel catfish.     

Molecular characterization of the insulin-like growth factor-I (IGF-I) gene in channel catfish (Ictalurus punctatus)

The insulin-like growth factor I (IGF-I) gene was characterized in channel catfish. Partial cDNA sequence, missing exon 1 and part of exon 2, was obtained in 5'- and 3'-RACE experiments. Direct sequencing of two bacterial artificial chromosome clones revealed gene structure and provided sequence from 640 bp upstream of the initiator methionine to 136 bp beyond the polyadenylation site. Genomic sequence contained a putative TATA box 506 bp upstream of the initiator methionine. The 477-bp reading frame within five exons encoded a 159-amino acid (aa) pre-propeptide highly similar to IGF-I in higher vertebrates. The sequence encoding the signal peptide was unique in catfish and contained 70% G+C content with the potential for a stable stem-loop structure. Full-length cDNA was only maintained in recombination-deficient (DH10B) strain E. coli. Levels of IGF-I mRNA were highest in liver, followed by brain and muscle, then heart and kidney (P<0.05). A CT/GA dinucleotide microsatellite in intron 1 was highly polymorphic in commercial channel catfish, and permitted placement of the IGF-I gene on the catfish genetic map. However, specific IGF-I alleles were not correlated with differences in growth rate from 100 to 130 days post-hatch in USDA103 line catfish.     

Insulin and IGF-I action on insulin receptors, IGF-I receptors, and hybrid insulin/IGF-I receptors in vascular smooth muscle cells

Insulin and insulin-like growth factor I (IGF-I) are known to affect cardiovascular disease. We have investigated ligand binding and the dose-response relationship for insulin and IGF-I on vascular smooth muscle cells (VSMCs) at the receptor level. VSMCs from rat thoracic aorta were serum starved, stimulated with IGF-I or insulin, lysed, immunoprecipitated, and analyzed by Western blot. d-[U-(14)C]Glucose accumulation and [6-(3)H]thymidine incorporation into DNA were also measured. Specific binding of both insulin and IGF-I was demonstrated, being higher for IGF-I. Both IGF-I receptor (IGF-IR) and insulin receptor (IR) beta-subunits were detected and coprecipitated after immunoprecipitation (IP) against either of the two. No coprecipitation was found after reduction of disulphide bonds with dithiotreitol before IP. After stimulation with 10(-10)-10(-9) M IGF-I, IP of the IGF-IR, or IR beta-subunit and immunoblot with anti-phosphotyrosine antibody, we found two distinct bands indicating phosphorylation of both the IGF-IR and the IR beta-subunit. Stimulation with 10(-10)-10(-9) M insulin and IP against the IGF-IR did not show phosphorylation of either beta-subunit, whereas after IP of the IR we found phosphorylation of the IR beta-subunit. [(14)C]Glucose accumulation and [(3)H]thymidine incorporation were elevated in cells stimulated with IGF-I at 10(-10)-10(-7) M, reaching maximum by 10(-9) M. Insulin stimulation showed measurable effects only at supraphysiological concentrations, 10(-8)-10(-7) M. In conclusion, coprecipitation of both the IGF-IR and the IR beta-subunit indicates the presence of hybrid insulin/IGF-I receptors in VSMC. At a physiological concentration, insulin activates the IR but does not affect either glucose metabolism or DNA synthesis, whereas IGF-I both activates the receptor and elicits biological effect.     

Sequence of IGF-I, IGF-II, and HGF expression in regenerating skeletal muscle

Various cytokines are thought to play a role in muscle regeneration, however, the interaction and mechanisms of action of these cytokines remains largely unknown. In this study, we investigated the role of HGF, IGF-I, and IGF-II during myogenesis using the regeneration model of skeletal muscle as well as myoblast culture. RT-PCR analysis revealed that HGF and IGF-I expressions were markedly upregulated, in regenerating muscle. In contrast, there was no significant difference in IGF-II expression between normal and regenerating muscle. Immunohistochemical analysis demonstrated that HGF was expressed mostly by myocytes during the early stages of muscle regeneration. Additionally, HGF inhibited the formation of myotubes by myoblasts, but promoted cellular proliferation. Otherwise, IGF-I and IGF-II were expressed by myocytes through the early to middle stages of muscle regeneration. The addition of HGF to myoblast growing in vitro significantly increased the number of cells. These findings indicate that these three cytokines have pleiotropic effects in regenerating skeletal muscle.     

Cellular pattern of insulin-like growth factor-I (IGF-I) and type I IGF receptor gene expression in early organogenesis: comparison with IGF-II gene expression

To investigate the potential role(s) of the insulin-like growth factors (IGFs) in embryogenesis, we have used in situ hybridization histochemistry to localize mRNAs for IGF-I, IGF-II, and the type I IGF receptor during an early period in rat embryonic development (embryonic days 14 and 15). IGF-I and IGF-II mRNAs were found in distinctly different patterns of cellular distribution. IGF-I mRNA was particularly abundant in undifferentiated mesenchymal tissue in the vicinity of sprouting nerves and spinal ganglia, and in circumscribed regions of the developing face that corresponded to the target zones of the trigeminal nerve. IGF-I mRNA was also found in aggregations of mesenchyme surrounding, but not in developing muscle and cartilage. IGF-I mRNA was selectively concentrated in areas of active tissue remodeling, such as the cardiac outflow tract, and was undetectable in liver, pituitary, and nervous system at this early stage of organogenesis. IGF-II mRNA was abundant in developing muscle, cartilage, and vascular tissue, and in the embryonic liver and pituitary. IGF-II mRNA was also conspicuous in areas of vascular interface with the brain, such as the choroid plexus and the organum vasculosum of the lamina terminalis. Messenger RNA for the type I IGF receptor was widely distributed in embryonic tissues, but the highest level were seen in the ventral floorplate of the hindbrain, where specialized neuroepithelial cells act as guides for axonal targeting. In conclusion, the different cellular patterns of expression of genes for IGF-I and IGF-II indicate that these two IGFs are differently regulated and, thus, may have significantly different roles in the process of embryonic development. Furthermore, the early and widespread expression of the type-I IGF receptor gene, in contrast to the relatively limited and localized pattern of IGF-I gene expression, is consistent with the view that this receptor may mediate the effects of IGF-II as well as IGF-I during embryogenesis.     

Sequence of IGF-I, IGF-II, and HGF expression in regenerating skeletal muscle

Various cytokines are thought to play a role in muscle regeneration, however, the interaction and mechanisms of action of these cytokines remains largely unknown. In this study, we investigated the role of HGF, IGF-I, and IGF-II during myogenesis using the regeneration model of skeletal muscle as well as myoblast culture. RT-PCR analysis revealed that HGF and IGF-I expressions were markedly upregulated, in regenerating muscle. In contrast, there was no significant difference in IGF-II expression between normal and regenerating muscle. Immunohistochemical analysis demonstrated that HGF was expressed mostly by myocytes during the early stages of muscle regeneration. Additionally, HGF inhibited the formation of myotubes by myoblasts, but promoted cellular proliferation. Otherwise, IGF-I and IGF-II were expressed by myocytes through the early to middle stages of muscle regeneration. The addition of HGF to myoblast growing in vitro significantly increased the number of cells. These findings indicate that these three cytokines have pleiotropic effects in regenerating skeletal muscle.     

Structural characterisation and expression analysis of toll-like receptor 2 gene from catfish

Toll-like receptors (TLRs) are important components of innate immunity. They were found to recognise specific structures on pathogens termed pathogen-associated molecular patterns (PAMPs) and utilise conserved signaling pathways to activate pro-inflammatory cytokines and type-1 interferons. In spite of much understanding gained from the mammalian systems, many fish TLRs are unknown. Recent studies in Japanese flounder as well as in zebrafish suggested that the ligand binding and activation of inflammatory responses in fish may be different from and more complex than those found in mammals. In channel catfish, the major aquaculture species in the United States, only partial sequences of TLR3 and TLR5 were reported. As a part of efforts to characterise the innate immune components in channel catfish, here we cloned and sequenced both the cDNA and the gene for TLR2, a receptor believed mostly responsible for recognition of lipopeptides on the surface of most Gram-positive bacteria. However, expression analysis after infection with a Gram-negative bacterium, Edwardsiella ictaluri indicated that TLR2 was modestly down-regulated in the head kidney tissue of blue catfish, and with a similar pattern in the head kidney of channel catfish though the down-regulation in channel catfish was not statistically significant. In the spleen, an insignificant down-regulation was initially observed early after infection, with an increase of TLR expression later after infection. These results suggest the involvement of TLR2 in the responses after the bacterial infection. As LPS is believed to be the major PAMP for Gram-negative bacteria, additional research is warranted to determine the functions and mechanisms of TLR2 in infections of Gram-negative bacteria.     

Structure and expression of transferrin gene of channel catfish,Ictalurus punctatus

Transferrin is important in iron metabolism and has been reported to be involved in disease defence responses after bacterial infection. In this study, we identified, sequenced, and characterized the transferrin gene from channel catfish, Ictalurus punctatus. The catfish transferrin gene was similar to those of other vertebrate species with 17 exons and 16 introns. Sequence analysis indicated the presence of the two duplicated lobes, each containing two sub-domains separated by a cleft harboring the iron-binding site, suggesting their structural conservation. The channel catfish transferrin cDNA encodes 679 amino acids with 42–56% similarity to known transferrin genes from various species. Southern blot analysis suggested the presence of two copies of the transferrin gene in the catfish genome, perhaps arranged in a tandem fashion. The catfish transferrin gene was mapped to a catfish BAC-based physical map. The catfish transferrin gene was highly expressed in the liver, but expression was low in most other tested tissues. Transferrin expression was significantly up-regulated after infection with Edwardsiella ictaluri, the causative agent of enteric septicemia of catfish. Such induction was also found with co-injection of iron-dextran and E. ictaluri, while transferrin expression was not significantly induced with the injection of iron-dextran alone.     

Fibroblast growth factor inhibits insulin-like growth factor-II (IGF-II) gene expression and increases IGF-I receptor abundance in BC3H-1 muscle cells.

Muscle is an important target tissue for insulin-like growth factor (IGF) action. We have previously reported that muscle cell differentiation is associated with down-regulation of the IGF-I receptor at the level of gene expression that is concomitant with an increase in the expression and secretion of IGF-II. Furthermore, treatment of myoblasts with IGF-II resulted in a similar decrease in IGF-I receptor mRNA abundance, suggesting an autocrine role of IGF-II in IGF-I receptor regulation. To explore further the role of IGF-II in IGF-I receptor regulation, BC3H-1 mouse muscle cells were exposed to differentiation medium in the presence of basic fibroblast growth factor (FGF), a known inhibitor of myogenic differentiation. FGF treatment of cells resulted in a 50% inhibition of IGF-II gene expression compared to that in control myoblasts and markedly inhibited IGF-II secretion. Concomitantly, FGF resulted in a 60-70% increase in IGF-I binding compared to that in control myoblasts. Scatchard analyses and studies of gene expression demonstrated that the increased IGF-I binding induced by FGF reflected parallel increases in IGF-I receptor content and mRNA abundance. These studies indicate that FGF may up-regulate IGF-I receptor expression in muscle cells through inhibition of IGF-II peptide expression and further support the concept of an autocrine role of IGF-II in IGF-I receptor regulation. In addition, these studies suggest that one mechanism by which FGF inhibits muscle cell differentiation is through inhibition of IGF-II expression.     

Comparative study of mannose-binding C-type lectin isolated from channel catfish (Ictalurus punctatus) and blue catfish (Ictalurus furcatus)

Mannose-binding C-type lectin (MBL) was isolated from channel catfish (Ictalurus punctatus) NWAC 102 and 103 strains, blue catfish (Ictalurus furcatus) D+B and Rio Grande strains, hybrid catfish (channel catfish female NWAC 103 x blue catfish male D+B) sera, and purified by affinity chromatography from channel catfish Norris strain serum. Reduction of purified channel catfish MBL with 2-ME yielded a single band of 62 kDa by SDS-PAGE and Western blot analysis using guinea pig anti-MBL IgG as primary antibody. Channel catfish NWAC 102 strain, channel catfish NWAC 103 strain and hybrid catfish sera had molecular masses of 63 kDa for MBL. Blue catfish (D+B strain) serum MBL had a molecular mass of 66 kDa. Rio Grande blue catfish serum MBL had a molecular mass of 65 kDa. Amino acid composition analysis (mol%) of the affinity-purified channel catfish MBL found a high content of serine present. Functional binding studies of channel catfish and blue catfish MBLs binding to Edwardsiella ictaluri were done using a dot-immunoblot ELISA method. A dot-immunoblot ELISA binding assay was done to compare nine different strains and species of channel catfish and blue catfish for their levels of serum MBL. Blue catfish had higher levels of MBL than did the various strains of channel catfish tested. MBL could be used as a genetic marker for selection of disease resistance in the different strains of catfish used in aquaculture. This study describes the presence of serum MBL in catfish and evidence for a C-type lectin complement pathway of innate immunity.     

Three novel mammalian toll-like receptors: gene structure, expression, and evolution

We describe three novel genes, encoding members of the Toll-like receptor (Tlr) family (TLR7, TLR8, and TLR9). These Tlr family members, unlike others reported to date, were identified within a genomic database. TLR7 and TLR8 each have three exons, two of which have coding function, and lie in close proximity to one another at Xp22, alongside a pseudogene. The remaining gene (TLR9) resides at 3p21.3 (in linkage with the MyD88 gene), and is expressed in at least two splice forms, one of which is monoexonic and one of which is biexonic, the latter encoding a protein with 57 additional amino acids at the N-terminus. The novel Tlrs comprise a cluster as nearest phylogenetic neighbors. Combining all sequence data related to Toll-like receptors, we have drawn several inferences concerning the phylogeny of vertebrate and invertebrate Tlrs. According to our best estimates, mammalian TLRs 1 and 6 diverged from a common mammalian ancestral gene 95 million years ago. TLR4, which encodes the endotoxin sensor in present-day mammals, emerged as a distinct entity 180 million years ago. TLRs 3 and 5 diverged from a common ancestral gene approximately 150 million years ago, as did Tlr7 and Tlr8. Very likely, fewer Tlrs existed during early vertebrate evolution: at most three or four were transmitted with the primordial vertebrate line. Phylogenetic data that we have adduced in the course of this work also suggest the existence of a Drosophila equivalent of MyD88, and indicate that the plasma membrane protein SIGIRR is close functional relative of MyD88 in mammals. Finally, a single present-day representative of the Toll-like proteins in Drosophila has striking cytoplasmic domain homology to mammalian Tlrs within the cluster that embraces TLRs 1, 2, 4, and 6. This would suggest that an ancestral (pre-vertebrate) Tlr may have adopted a pro-inflammatory function 500 million years ago.