A technical report provides and discusses experimental data concerning the carcinogenicity of several plant constituents. These include certain plant pyrrolizidine alkaloids, flavonoids, and the ...bracken fern containing toxic and carcinogenic constituents. Carcinogenicity test data developed in various studies are summarized and compared
We have investigated the protective effect of immunization of juvenile red seabream,
Pagrus major, with DNA plasmids encoding the viral major capsid protein (MCP) and an open reading frame (ORF) ...containing a transmembrane domain against red seabream iridovirus (RSIV). The expression of the MHC class I transcript in the DNA-vaccinated fish was significantly upregulated at the 15th day post-vaccination and the relative level of expression was maintained until the 30th day post-vaccination. This pattern of expression was similar in fish vaccinated with a commercially prepared formalin-inactivated RSIV vaccine. In vaccine efficiency tests, the relative percentage survival (RPS) of fish receiving the DNA vaccines and their combination ranged from 42.8 to 71.4% in two experimental runs, and these were significantly different from the control groups. Our results clearly demonstrate that DNA vaccines are able to induce robust protection in fish against RSIV infection, and a cellular immune response as shown by the upregulation of the MHC class I transcript after vaccination, which may be associated with such protection.
We cloned the cDNAs and genes of two different types of toll-like receptors from Japanese flounder. The results of homology searches suggested that these genes (designated JF-TLR2 and JF-TLR22) are ...homologues of human TLR2 and fugu TLR22, respectively. The cDNAs of JF-TLR2 and JF-TLR22 encoded 818 and 961 amino acid residues, respectively. JF-TLR2 and JF-TLR22 contained two distinct structural/functional motifs of the TLR family, such as a leucine-rich repeat (LRR) domain in the extracellular portion and a toll/interleukin-1 receptor (TIR) domain in the intracellular portion. The genes of JF-TLR2 and JF-TLR22 consisted of 12 exons (4.9 kb in total length) and four exons (4.3 kb in total length), respectively. The first exon of each gene is a non-coding exon. Southern blot hybridization indicated that both JF-TLR2 and JF-TLR22 exist as single copies in the genome. These genes were mainly expressed in peripheral blood leukocytes (PBLs) and weakly expressed in PBL-rich organs such as kidney, spleen and gill. Expression of these genes was induced by both peptidoglycan and polyI:C, although the number of JF-TLR-expressing cells were not changed after induction. All of these results suggest that they are involved in the innate immune system.
Streptococcosis caused by
Streptococcus iniae, a Gram-positive bacterium, is one of the diseases often found in the culture of Japanese flounder (
Paralichthys olivaceus). Inactivated bacterial-whole ...cells of
S. iniae have been used as a prophylactic treatment to prevent the disease. To understand the molecular mechanisms involved in activation of the fish immune system upon formalin-killed cell (FKC) treatment, we have investigated the gene transcription profile of Japanese flounder head kidney cells after intraperitoneal injection of
S. iniae FKC. Three known genes (C3 complement, calmodulin, microtubule aggregate protein) and 6 unknown genes were specifically up-regulated more than 10-fold by
S. iniae FKC. RT-PCR analysis of differential expression of the 3 unknowns and interleukin-1β confirmed the result of the microarray analysis. From this study, we conclude that 6 up-regulated unknown clones represent novel genes that are involved against Gram-positive FKC immune response.
We established a transgenic zebrafish strain expressing chicken lysozyme gene under the control of the Japanese flounder keratin gene promoter, and investigated its resistance to a pathogenic ...bacterial infection. To generate the lysozyme transgenic construct, Japanese flounder keratin promoter was linked to both the hen egg white (HEW) lyoszyme gene and green fluorescence protein (GFP) gene used as a selection marker for the transgenic strains, in a recombinant plasmid. The recombinant plasmid was microinjected into fertilized zebrafish eggs. In F2 transgenic zebrafish, GFP expression was strong in the epithelial tissues, liver and gill from the embryonic stage to the adult stage. The expressions of HEW lysozyme and GFP mRNA were confirmed in the liver and skin by RT-PCR. Western blot analysis showed that both HEW lysozyme and GFP were present in protein extracts from the liver of transgenic zebrafish, but not in protein extracts from the muscle. The lytic activity of protein extracts from the liver (assessed by a lysoplate assay using Micrococcus lysodeikticus as a substrate) was 1.75 times higher in F2 transgenic zebrafish than in the wild type. In a challenge experiment, 65% of the F2 transgenic fish survived an infection of Flavobacterium columnare and 60% survived an infection of Edwardsiella tarda, whereas 100% of the control fish were killed by both pathogens. However, the survival rates of the transgenic fish were not significantly higher when higher concentrations of bacteria were used.
Virulence factors were compared in Aeromonas species isolated from clinically normal and septicaemic farmed frogs from Thailand. Haemolysin activities against frog erythrocytes were significantly ...different within the collection of aeromonads. Groups of high haemolytic activity (unspeciated Aeromonas, Au), moderate haemolytic activity (A. hydrophila), and low haemolytic activity (A. veronii biovar sobria, A. veronii biovar veronii, A. caviae, A. schubertii) were noted. DNA colony hybridisation studies revealed that Au isolates possessed a haemolysin gene (ASH1) which was not present in any of the other Thai aeromonads or type strains tested. Elastinolytic activity was demonstrated in 90% of the Au isolates, 60% of the A. hydrophila isolates and in none of the other motile aeromonads. The cytotoxic activity of the Aeromonas isolates varied according to the source of cells used in the assays. Cells from rainbow trout were extremely sensitive to Au toxins but less so to toxins produced by other species. In contrast mammalian cells showed very little sensitivity to Au toxins but were more sensitive to toxins produced by A. hydrophila. Selection of suitable assay substrates is therefore important.
A loop-mediated isothermal amplification (LAMP) procedure is described to detect the genomic DNA molecule of red seabream iridovirus (RSIV), a fish iridovirus belonging to the Iridoviridae family. ...The RSIV DNA was amplified using DNA extracts obtained from spleen of infected red seabream,
Pagrus major and from various RSIV isolates. The method was at least 10 times more sensitive than conventional PCR in detecting for the presence of RSIV. A striking feature of the LAMP reaction is its ability to synthesize a large amount of DNA leading to the production of a white precipitate, magnesium pyrophosphate, as a by-product. The presence or absence of this white precipitate facilitates easy detection of the RSIV genomic DNA without the use of gel electrophoresis. A strong correlation exists between the amount of input viral DNA copy and the corresponding turbidity reading at the end of the reaction; hence, the LAMP reaction may be used potentially to quantify RSIV particles in the infected fish.
We generated 196 expressed sequence tags (EST) from a Japanese eel Anguilla japonica spleen cDNA library. Of these, 90 EST (46%) showed no homology to known genes, while 106 EST (54%) showed homology ...to known genes and transcripts. Of the 106 homologous EST, 13 were homologous to 12 different immune‐related genes, 46 were homologous to 33 different ribosomal proteins, three were homologous to the 28S rRNA gene, 10 were homologous to eight different mitochondrial genes and the remaining 34 encoded 28 different identified genes. Six identified EST are similar to previously reported EST of Japanese flounder Paralichthys olivaceus spleen cDNA. These analyses demonstrate the utility of EST and sequence‐tagged clones in comparative studies of gene expression in the fish spleen.
Intramuscular injection of Japanese flounder,
Paralichthys olivaceus (average weight approximately 2 g) with 1 and 10 μg of a plasmid DNA vaccine encoding the hirame rhabdovirus (HIRRV) glycoprotein ...gene (pCMV-HRVg) was found to provide strong protection against HIRRV. We also conducted a real-time PCR analysis to quantify immune-related genes, e.g. MHC class Iα, IIα, IIβ, TCR-α, β1, β2 and δ, to characterize the immune response at 1 and 7 days after DNA vaccination. In general, the copy numbers were at least 2-fold higher than those of the non-vaccinated fish. Interestingly, the gene expression of TCR β1 and β2 increased 1 day post-DNA vaccination, after which their copy numbers returned to levels similar to those before vaccination. These results suggest that the immune system of Japanese flounder was activated immediately after DNA immunization.
An interleukin 8 (IL-8) homologue has been identified in the rainbow trout
Oncorhynchus mykiss. The transcript contains an open reading frame of 294 nucleotides that translates into a 97 amino acid ...putative peptide, with 5′ and 3′ untranslated regions (UTR) of 171 and 453 nucleotides, respectively. As with previously sequenced lamprey and flounder genes, the trout amino acid sequence lacks the typical ELR motif upstream of the first pair of cysteines, where DLR is present. The trout IL-8 gene contains four exons divided by three short introns of 341, 247 and 292
bp, and occupies 1824
bp of genomic DNA. RT–PCR reveals a low level constitutive expression of the IL-8 homologue in many tissues, including spleen, heart, liver, head kidney and gill. Expression was not detectable in the brain. Whilst no apparent affect of lipopolysaccharide (LPS) on IL-8 expression was observed in vivo, stimulation of a trout macrophage cell line (RTS-11) with either LPS or poly I:C did result in clear up-regulation of IL-8 expression, detectable by RT–PCR and Northern blot analysis.
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