Pristimerin Inhibits MMP-9 Expression and Cell Migration Through Attenuating NOX/ROS-Dependent NF-κB Activation in Rat Brain Astrocytes Challenged with LPS.

PURPOSE: Neuroinflammation plays a crucial role in neurodegenerative diseases. Matrix metalloproteinases (MMPs) are a landmark of neuroinflammation. Lipopolysaccharide (LPS) has been demonstrated to induce MMP-9 expression. The mechanisms underlying LPS-induced MMP-9 expression have not been completely elucidated in astrocytes. Nuclear factor-kappaB (NF-κB) is well known as one of the crucial transcription factors in MMP-9 induction. Moreover, reactive oxygen species (ROS) could be an important mediator of neuroinflammation. Here, we differentiated whether ROS and NF-κB contributed to LPS-mediated MMP-9 expression in rat brain astrocytes (RBA-1). Besides, pristimerin has been revealed to possess antioxidant and anti-inflammatory effects. We also evaluated the effects of pristimerin on LPS-induced inflammatory responses. METHODS: RBA-1 cells were used for analyses. Pharmacological inhibitors and siRNAs were used to evaluate the signaling pathway. Western blotting and gelatin zymography were conducted to evaluate protein and MMP-9 expression, respectively. Real-time PCR was for mRNA expression. Wound healing assay was for cell migration. 2',7'-dichlorodihydrofluorescein diacetate (H2DCF-DA) and dihydroethidium (DHE) staining were for ROS generation. Immunofluorescence staining was conducted to assess NF-κB p65. Promoter-reporter gene assay and chromatin immunoprecipitation (ChIP) assay were used to detect promoter activity and the association of nuclear proteins with the promoter. RESULTS: Our results showed that the increased level of ROS generation was attenuated by edaravone (a ROS scavenger), apocynin (APO; an inhibitor of p47Phox), diphenyleneiodonium (DPI; an inhibitor of NOX), and pristimerin in RBA-1 cells exposed to LPS. Besides, pretreatment with APO, DPI, edaravone, Bay11-7082, and pristimerin also inhibited the phosphorylation, nuclear translocation, promoter binding activity of NF-κB p65 as well as upregulation of MMP-9 expression-mediated cell migration in RBA-1 cells challenged with LPS. CONCLUSION: These results suggested that LPS enhances the upregulation of MMP-9 through nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX)/ROS-dependent NF-κB activity. These results also provide new insights into the mechanisms by which pristimerin attenuates LPS-mediated MMP-9 expression and neuroinflammatory responses.

Ferritin administration effectively enhances immunity, physiological responses, and survival of Pacific white shrimp (Litopenaeus vannamei) challenged with white spot syndrome virus.

We examined the physiological (hemolymph glucose, lactate, and lipid) and innate non-specific immune responses (total hemocyte count (THC), phenoloxidase (PO) activity, respiratory bursts (release of superoxide anion, O(2)(-)) and superoxide dismutase (SOD) activity) to white spot syndrome virus (WSSV) in white shrimp (Litopenaeus vannamei) that were individually injected with 0.1, 0.5, and 1 ng g(-1) ferritin. Results showed that the THC, PO activity, and respiratory bursts of L. vannamei obviously increased (p < 0.05) 12 h after being injected with any dose of ferritin. However, the THC, PO activity, and respiratory bursts of L. vannamei that had received 0.5 and 1 ng g(-1) ferritin were significant higher than those of the other groups at 36-60, 60-72, and 36-60 h, respectively. SOD activities of L. vannamei 12 h after receiving 0.1, 0.5, and 1 ng g(-1) ferritin were significantly higher than those receiving saline. L. vannamei injected with ferritin at any dose maintained lower glucose, lactate, and lipid levels in response to WSSV challenge after 12-36, 24-48, and 36-60 h, respectively. The survival of shrimp that had received 0.5 and 1 ng g(-1) ferritin was significantly higher than that of shrimp that received saline and of control shrimp after 72 h. The ferritin messenger RNA transcripts of shrimp that had received 0.5 and 1 ng g(-1) ferritin were significantly higher than that of shrimp that received saline after 36 h. It was, therefore, concluded that the immune ability and resistance against WSSV infection increased in L. vannamei that had received > 0.5 ng g(-1) ferritin. Ferritin does play important roles in the innate immunity of the white shrimp. We observed higher SOD activities of L. vannamei that had received 0.1, 0.5, and 1 ng ferritin after 12 h than those that had received only saline (control), and the high SOD expression remained at the same levels even after 72 h of treatment.

A second proPO present in white shrimp Litopenaeus vannamei and expression of the proPOs during a Vibrio alginolyticus injection, molt stage, and oral sodium alginate ingestion.

Prophenoloxidase (proPO) is a melanin-synthesising enzyme that plays important roles in immune responses by crustaceans. Previously, we cloned and characterized proPO-I from white shrimp, Litopenaeus vannamei. In the present study, a novel prophenoloxidase-II (proPO-II) cDNA was also cloned from haemocytes of L. vannamei using oligonucleotide primers and reverse-transcriptase polymerase chain reaction (RT-PCR). Both 3'- and 5'-regions were isolated by the rapid amplification of complementary (c)DNA end (RACE) method. The 2504-bp cDNA contained an open reading frame (ORF) of 2073 bp, an 84-bp 5'-untranslated region, and a 347-bp 3'-untranslated region containing the poly A tail. The molecular mass of the deduced amino acid sequence (691 amino acids) was 78.8 kDa with an estimated pI of 6.07. It contains two putative tyrosinase copper-binding motifs and a conserved C-terminal region common to all known proPOs. Comparisons of the amino acid sequences showed that white shrimp proPO-II is more closely related to the proPO of other penaeids than to that of crayfish, lobsters, crab, or a freshwater prawn, and is the ancestor type of known penaeid proPOs. proPO-I and proPO-II messenger (m)RNAs of shrimp were located on different loci, and were constitutively expressed mainly in haemocytes. The transcriptional regulation of these two proPOs in shrimp at different molt stages, those administered dietary sodium alginate, and those challenged with Vibrio alginolyticus were surveyed. The results showed that the proPOs may be directly involved in the acute-phase immune defence, and proPO-II may contribute earlier to immune defence in shrimp injected with V. alginolyticus, and it may be regulated by ecdysone. However, a similar effect was found by stimulating proPO-I and proPO-II mRNA expression in shrimp fed a sodium alginate-containing diet. Results of this study provide a basis for developing a comprehensive understanding of expression/function relationships of individual proPOs in shrimp.

Effects of rutin from Toona sinensis on the immune and physiological responses of white shrimp (Litopenaeus vannamei) under Vibrio alginolyticus challenge.

Rutin is a bioflavonoid with strong antioxidant activity. To investigate the regulatory roles of rutin in various functions in crustaceans, we examined physiological (haemolymph glucose, lactate, and lipid) and innate non-specific immune responses (total haemocyte count (THC), phenoloxidase activity (PO), respiratory bursts (release of superoxide anion, O(2)(-)) and superoxide dismutase (SOD) activity) to the pathogen Vibrio alginolyticus in white shrimp (Litopenaeus vannamei) that were individually injected with rutin extracted from Toona sinensis at 10, 20, or 50 microg g(-1). Results showed that PO activity and respiratory burst of L. vannamei increased obviously (P<0.05) when injected with rutin at a dose of 20 and 50 microg g(-1) after 12 and 24 h, respectively. Both the THC and SOD activities of experimental and control groups revealed no significant difference at all doses. L. vannamei injected with rutin at either dose maintained lower glucose, lactate, and lipid levels in response to V. alginolyticus challenge after 12-36, 24-48, and 24-60 h, respectively. The survival rate of L. vannamei that received rutin at either dose was significantly higher than that received saline after 48-72 h. It was, therefore, concluded that the immune ability and resistance against V. alginolyticus infection of L. vannamei receiving rutin at > or = 10 microg g(-1) increased.

Application of RNAi technology to the inhibition of zebrafish GtHalpha, FSHbeta, and LHbeta expression and to functional analyses.

Zebrafish (Danio rerio) were used as a model fish, and the technique of RNA interference (RNAi) was employed to knockdown three subunits of the gonadotropin alpha (GtHalpha, common alpha), follicle-stimulating hormone beta (FSHbeta), and luteinizing hormone beta (LHbeta) genes. Three short-hairpin RNA (shRNA) expression vectors and three mismatched shRNA expression vectors as controls for each subunit gene were constructed, and the depression efficiency was tested in vivo by microinjection; the RNA or protein expression levels of the GtH genes were monitored by RT-PCR, Southern blotting, and green fluorescent protein (GFP) analyses. Expression of GtH mRNA was obviously and more efficiently depressed by GtHalpha RNAi expression compared with the other two subunits. A GtHalpha morpholino analysis showed that the GtHalpha morpholino led to suppression of embryonic development and the production of embryonic mutants as a result of an injection of GtHalpha -shRNA. Taken together, these results show that GtHalpha-shRNA, which more efficiently targets RNAi, may have an essential role in the further development of sterility technology of transgenic fish for biosafety purposes.

Organization and promoter analysis of the grouper (Epinephelus coioides) epinecidin-1 gene.

Epinecidin-1, an antimicrobial peptide documented in some fish, is an essential component of the innate immune response in fish, but little is known about its gene regulation. To better understand the molecular mechanism controlling transcription of the epinecidin-1 gene, we cloned and sequenced the genes and promoter regions of three epinecidin-1 peptides from the grouper (Epinephelus coioides). These genes have the potential to encode three putative epinecidin-1 peptides with either a short or a long 5'-untranslated region (UTR). These epinecidin-1 genes, numbered 124-1 (gene structure: 5 exons), 124-2 (gene structure: 5 exons), and 961 (gene structure: 4 exons), have 3' UTR sequences that dramatically differ by being located on different exons in clones 124 and 961. To address the roles of lipopolysaccharide (LPS) and poly(I):poly(C) in regulating epinecidin-1 expression, serial deletions were prepared in the promoter region of two clones that contained three genes. Different fragments of the epinecidin-1 5'-flanking region were transfected into U937 (human histiocytic lymphoma) and ZFL (zebrafish liver) cells and then treated with 0, 1, 10, and 100 mug/mL LPS or poly(I):poly(C). The results showed that after treatment with 10 mug/mL LPS, high promoter activity was observed in the 0.6-kb promoter fragment (of clone 961). Promoter deletions showed that hepatocyte nuclear factor (HNF)-1 was required for a maximal response of epinecidin-1 961 promoter activity after LPS treatment in ZFL cells. Morphological studies of transgenic zebrafish indicated that the 2-kb epinecidin-1 124-1 promoter-driven GFP transcripts appeared in the eye and skin as confirmed by immunohistochemical staining. These results indicate that the 2-kb epinecidin-1 124-1 promoter is active in a tissue-specific manner.

Functional analysis of mitogen-activated protein kinase-3 (MAPK3) and its regulation of the promoter region in zebrafish.

Mitogen-activated protein kinase (MAPK) plays a pivotal role in intracellular actions in response to a variety of extracellular stimuli. Real-time reverse-transcription polymerase chain reaction analysis of MAPK3 tissue distribution in zebrafish showed significant differences in the fin and liver compared with muscle. A 1.2-kilobase (kb) pair and a 2.3-kb fragment of the 5'-flanking region displayed minimal promoter activity in the zebrafish liver (ZFL) and HeLa cell lines after treatment with insulin-like growth factors (IGF-I and IGF-II). Targeted knockdown of the MAPK3 gene by two antisense morpholino oligonucleotides revealed that although the zebrafish MAPK3 MO 1-targeted sequence was located at 5' untranslated region and the zebrafish MAPK3 MO 2-targeted sequence was located in the mature peptide region, similar results were shown in zebrafish for disruption of notochord development, with the whole body exhibiting distortion. From a comparative point of view, this study of the MAPK3 gene in zebrafish might not correlate well with previously published studies on mice. These molecular results suggest that MAPK3 plays an important role in whole-body development and is required for general embryonic development. Finally, MAPK3 may play important roles in fish cell growth.

Gene expression and localization of the epinecidin-1 antimicrobial peptide in the grouper (Epinephelus coioides), and its role in protecting fish against pathogenic infection.

Epinecidin-1 is an antimicrobial peptide and plays a vital role in protecting fish against pathogenic infection. As a mimic of a grouper epinecidin-1 peptide, it has tertiary structures that closely resemble those of pleurocidin found in the winter flounder (Pleuronectes americanus). The tissue-specific, lipopolysaccharide (LPS)-stimulation-specific, and poly(I):poly(C)-stimulation-specific expressions of the grouper (Epinephelus coioides) epinecidin-1 antimicrobial peptide were determined using a comparative reverse-transcription polymerase chain reaction. Results of the tissue distribution analysis revealed high levels of epinecidin-1 messenger RNA (mRNA) in the head kidneys, intestines, and skin. Expression of epinecidin-1 mRNA was dose-dependently stimulated by both LPS and poly(I):poly(C). Immunohistochemical analysis with the polyclonal antiserum of a grouper epinecidin-1 peptide (rabbit polyclonal antibody) showed that the peptide was localized with the epinecidin-1 antibody in the gills and intestines. Two synthetic peptides of the grouper epinecidin-1 peptide (g-ple 22-51 and g-ple 22-42) and one winter flounder pleurocidin as a control exhibited high antimicrobial activities against gram-negative or gram-positive bacteria. In addition, peptide treatment was effective in promoting a significant increase in fish survival after the injection of Vibrio vulnificus in tilapia (Oreochromis mossambicus) and grouper. These results are relevant to the design of prophylactic and therapeutic strategies to counter bacterial infections, especially for preventing or ameliorating immune defects in fish during bacterial infections.

Cloning and expression analysis of a protein kinase C gene, PKCmu, and its regulation of the promoter region in zebrafish.

The cDNA and genomic DNA of zebrafish (Danio rerio) protein kinase Cmu (PKCmu), with its promoter region, were obtained. The 508-amino acid zebrafish PKCmu has 86.17% similarity to human PKCmu. Real-time reverse-transcription polymerase chain reaction analysis with starvation and hormonal treatment found significant differences between the control group and the experimental group after 14 days of starvation. After injecting insulin-like growth factor II (IGF-II), growth hormone (GH), insulin, or human chorionic gonadotropin, significant differences were observed between the control and experimental groups 24 h after treatment. After injecting the gonadotropin-releasing hormone or luteotropin-releasing hormone, significant differences were seen between the control and experimental groups 15 h after treatment. These results suggest that in vivo PKCmu expression is regulated by the insulin family or by the GH, but other sex hormones produced a significant expression level more quickly than the insulin family and GH. The zebrafish PKCmu gene is located on zebrafish chromosome 17 and consists of 16 exons. A 2.6 kilobase pair on the 5' flanking region displayed maximal promoter activity in the zebrafish liver (ZFL) cell line after treatment with IGF-I, IGF-II, and GH. However, a 1.6 kilobase pair on the 5' flanking region displayed maximal promoter activity in the HeLa cell line after treatment with IGF-I, IGF-II, and GH. Finally, PKCmu may have important nuclear effects on cell growth and may involve nuclear localization. By transiently transfecting ZFL cells with various zebrafish PKCmu segments, we identified a nuclear localization signal: the amino acid sequence between amino acids 206 and 209 was able to predominantly direct enhanced green fluorescence protein (EGFP) into the nucleus, whereas a deletion of this motif abrogated the nuclear localization property.

Shrimp (Penaeus monodon) anti-lipopolysaccharide factor reduces the lethality of Pseudomonas aeruginosa sepsis in mice.

We investigated the efficacy of amino acids 55-76 of the synthetic shrimp anti-lipopolysaccharide factor peptide (SALF(55-76) cyclic peptide), the C-terminal part of the shrimp anti-lipopolysaccharide factor. This study was conducted to elucidate the effects of the antiseptic action of this peptide. The SALF(55-76) cyclic peptide was tested against bacterial clinical isolates and showed broad-spectrum antimicrobial activity. Transmission electron microscopic (TEM) examination of SALF(55-76) cyclic peptide-treated Pseudomonas aeruginosa showed that severe swelling preceded cell death and breakage of the outer membrane; the intracellular inclusion was found to have effluxed extracellularly. When mice were treated with the SALF(55-76) cyclic peptide before bacterial challenge with P. aeruginosa, the peptide highly protected mice against death by sepsis. The P. aeruginosa recovered from SALF(55-76) cyclic peptide-treated mice after 4 h exhibited reduced bacterial growth similar to that recovered from vancomycin-treated mice. In addition, the syntheses of inflammatory cytokines, such as interleukin (IL)-2, IL-4, IL-10, IL-12, IL-13, interferon-gamma, and tumor necrosis factor [TNF]-alpha, were significantly upregulated 4 h after SALF(55-76) cyclic peptide treatment except for IL-4 in the liver. The expressions of Toll-like receptor 4 (Tlr4), Irf3, myd88, and Tram, were considerably elevated, but only Tlr4 existed in the spleen 4 h after SALF(55-76) cyclic peptide treatment. The prophylactic administration of SALF(55-76) cyclic peptide was begun the TNF-alpha response in comparison to untreated mice by an ELISA analysis. Due to its multifunctional properties, the SALF(55-76) cyclic peptide may become an important prophylaxis against and therapy for bacterial infectious diseases, as well as for septic shock.

Dopamine depresses immunity in the tiger shrimp Penaeus monodon.

The total haemocyte count (THC), differential haemocyte count (DHC), phenoloxidase activity, respiratory bursts (release of superoxide anion), superoxide dismutase activity, phagocytic activity and clearance efficiency to the pathogen Photobacterium damsela were measured when tiger shrimp Penaeus monodon (13.5+/-1.5 g) were individually injected with saline or dopamine at 10(-8), 10(-7), or 10(-6)mol shrimp(-1). Results showed that a transient period of immunosuppression occurred between 2 and 8h after injection of dopamine for all immune parameters except circulating haemocytes, and all immune parameters had returned to control values within 8-16 h after receiving dopamine. The injection of dopamine also significantly increased the mortality of P. monodon challenged with the pathogen Pho. damsela. These results suggest that stress-inducing dopamine suppresses the immune system, which in turn promotes the susceptibility of P. monodon to Pho. damsela.

Three different hepcidins from tilapia, Oreochromis mossambicus: analysis of their expressions and biological functions.

Hepcidins are antimicrobial peptides that play important roles in resisting pathogenic infection. Through hybridization of a phage library, the cDNA sequences of three hepcidin-like antimicrobial peptides (named TH1-5, TH2-2, and TH2-3) in tilapia, Oreochromis mossambicus, were determined. The complete hepcidin cDNA sequences of TH1-5, TH2-2, and TH2-3 were respectively composed of 478, 533, and 583 bases, and contained a translated region of 88, 86, and 91 amino acids. An evolutionary assay of the three deduced amino acid sequences, which share eight cysteines at identical conserved positions, showed that tilapia TH2-3 is similar to Japanese flounder (Paralichthys olivaceus) JF2, tilapia TH2-2 is similar to Japanese flounder JF1, and tilapia TH1-5 is similar to seabream (Chrysophrys major) hepcidin. The predicted molecular weights of TH1-5, TH2-2, and TH2-3 are 9.5, 9.4, and 9.8 kDa, respectively. The predicted signal peptide cleavage sites in TH1-5 is between codons 24 and 25, in TH2-2, it is between codons 22 and 23, and in TH2-3, it is between codons 24 and 25. The structural models of tilapia hepcidins, constructed using the crystal structures of bass (Morone chrysopsx M. saxatilis) hepcidin as a respective template, showed that the positional cysteine residues form disulfide bonds with tilapia hepcidin, and the cysteines likely form disulfide bonds with the bass hepcidin cysteine. The tissue-specific, lipopolysaccharide (LPS) stimulation-specific, and polyinosinic-polycytidylic acid (poly I:poly C) stimulation-specific expressions of tilapia hepcidin mRNA were determined by a comparative reverse-transcription polymerase chain reaction. Results of the tissues distribution analysis revealed high expression levels of hepcidin messenger RNA (mRNA) in the liver and head kidneys for TH1-5. TH2-3 had high mRNA expression after LPS challenge in comparison to TH2-2 and TH1-5 in fish injected with 10mug/ml LPS. TH1-5 had high mRNA expression after poly I:poly C challenge in comparison to TH2-2 and TH2-3. Immunohistochemical analysis with the polyclonal antiserum of tilapia hepcidin TH1-5 (using a rabbit polyclonal antibody) showed that the peptide was localized in the spleen and head kidneys. Synthesized TH1-5 and TH2-3 peptides showed antimicrobial activity against several bacteria in this study, while the synthesized TH 2-2 peptide did not.

The physiological role of CTGF/CCN2 in zebrafish notochond development and biological analysis of the proximal promoter region.

During mouse embryogenesis, CTGF/CCN2 is expressed in zones containing hypertrophic chondroctyes and calcifying cartilage such as long bones, ribs, vertebral column, and phalanges. But in fish, its expression is yet unclear. Development of the vertebrae is morphologically similar among vertebrates, indicating that the underlying mechanism regulating the process is highly conserved during evolution. Analysis of 3.2kb of the CTGF/CCN2 proximal promoter sequence revealed a consensus TATAA box, putative AP1, Brn-2, CdxA, C/EBP alpha, C/EBP beta, C-Ets-, delta E, HFH-2, and HSF2 binding sites. Transient expression experiments with a 5'-deletion revealed at least 4 regulatory regions in the zebrafish CTGF/CCN2 gene, 2 with a stimulatory effect on transcription and 2 with an apparent inhibitory effect after IGF-I treatment in the ZFL cell line. To study the promoter-specific expression, we constructed a series of CTGF/CCN2 (3.0-, 2.5-, 2.0-, 1.5-, 1.0-, and 0.4-kb) promoter-driven green fluorescent protein (GFP) fragments encoding the GFP cDNA transgene which was microinjected into zebrafish embryos. Morphological studies of transgenic zebrafish indicated that the CTGF/CCN2 promoter-driven GFP transcripts appeared in the notochord. Targeted knockdown of the CTGF/CCN2 gene by two antisense morpholino oligonucleotides resulted in disruptions to notochord development. From a comparative point of view, this study of the CTGF/CCN2 gene in zebrafish may correlate well with those previously published on the mouse. These molecular results suggest that CTGF/CCN2 plays an important role in notochord development and is required for general embryonic development.

Molecular cloning and tissue distribution of ferritin in Pacific white shrimp (Litopenaeus vannamei).

Ferritin, a cytosolic iron storage protein composed of 24 subunits of heavy chain and light chain, is an intracellular protein primarily involved in iron metabolism. It can sequester up to 4500 ferric ions in its inner core to protect cells against toxicity of iron. Ferritin is known to play important roles in detoxification and is also involved in immunity processes. In this study, a full-length ferritin cDNA was cloned from the haemocyte of the Pacific white shrimp, Litopenaeus vannamei: it comprises 1249 bp, including 132 bp in the 5'-untranslated region, 510 bp in the open reading frame which encodes 170 amino acid residues, and 607 bp in the 3'-untranslated region. Alignments of the deduced amino acid sequence showed that the Pacific white shrimp ferritin shares 74%, 69%, 62%, 67%, 50% and 48% identity with crayfish, tick, brine shrimp, oyster, human and rat, respectively. The tissue-specific expression pattern was examined by reverse transcription polymerase chain reaction and real-time quantitative PCR. The ferritin mRNA is expressed in various tissues of the shrimp in the order of haemocyte, midgut gland, brain ganglion, gill, hepatopancreas, abdominal ganglion, eyestalk, muscle, thoracic ganglion, and heart.

Molecular cloning and characterisation of prophenoloxidase cDNA from haemocytes of the giant freshwater prawn, Macrobrachium rosenbergii, and its transcription in relation with the moult stage.

Expression of prophenoloxidase (proPO) cDNA was determined from haemocytes of the giant freshwater prawn Macrobrachium rosenbergii by a reverse-transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA using oligonucleotide primers based on the proPO sequence of tiger shrimp Penaeus monodon, freshwater crayfish Pacifastacus leniusculus, green tiger shrimp Penaeus semisulcatus, kuruma shrimp Marsupenaeus japonicus, and white shrimp Litopenaeus vannamei. The proPO of M. rosenbergii was constitutively expressed. The 2,547-bp cDNA contained an open reading frame (ORF) of 2,013 bp, a 96-bp 5'-untranslated region, and a 438-bp 3'-untranslated region containing the poly A tail. The molecular mass of the deduced amino acid (aa) sequence (671 aa) was 76.7 kDa with an estimated pI of 7.05. It contained putative copper-binding sites, a complement-like motif (GCGWPRHM), a proteolytic activation site, and a conserved C-terminal region common to all known proPOs. However, no signal peptide sequence was detected in giant freshwater prawn proPO. Comparison of amino acid sequences showed that prawn proPO is similar to the proPO of penaeid, crayfish and lobster. Prawn proPO was only synthesised in haemocytes. The proPO transcript was significantly increased in the A stage and achieved the highest level in the B stage, and then declined sharply in the C stage and reached the lowest level in the D(2)/D(3) stage.

Organization and promoter analysis of the zebrafish (Danio rerio) interferon gene.

Interferon plays important roles in confronting viral infections as the first line of defense. For the purpose of understanding the molecular mechanism which controls transcription of the interferon gene, we cloned and sequenced the interferon promoter region of the zebrafish interferon gene and characterized its activity using firefly luciferase transient transfection expression assays. Different fragments of the zebrafish interferon 5'-flanking region were transfected into ZFL cells. In these cell lines, maximum promoter activity was located in 2.2 kb of the zebrafish interferon 5' flanking region of the ZFL cell line. In this study, we investigated whether the viral replicative intermediate double-stranded RNA (herein we used synthetic polyinosinic-polycytidylic acid [poly(I):poly(C)] modifies the effects of interferon on gene expression. For this purpose, all zebrafish interferon promoter fragments were treated with either 1, 10, or 100 microg/ml poly(I):poly(C). The results showed that after treatment with 10 microg/ml poly(I):poly(C), high promoter activity was observed in the -2.2-kb interferon promoter fragment. Several putative transcription factors were shown in the promoter region, including IRF-1, C/EBP, NFkappaB, and GATA-1. Further study of the in vivo expression of the interferon promoter during development was carried out in transgenic zebrafish expressing an interferon promoter-driven green fluorescent protein (GFP) encoding the GFP cDNA transgene. Morphological studies of transgenic zebrafish indicated that the interferon promoter-driven GFP transcripts appeared in the yolk, head, and lymphoid organs. These results indicate that the interferon promoter is active in a tissue-specific manner, and suggest that the interferon promoter plays an important role in virus resistance during teleost growth.

Dopamine depresses the immune ability and increases susceptibility to Lactococcus garvieae in the freshwater giant prawn, Macrobrachium rosenbergii.

The total haemocyte count (THC), phenoloxidase activity, respiratory burst (release of superoxide anion), superoxide dismutase activity, and phagocytic activity and clearance efficiency to the pathogen Lactococcus garvieae were measured when freshwater giant prawns Macrobrachium rosenbergii (16.2 +/- 2.1 g) were individually injected with saline, or dopamine at 0.5, 5.0, or 50.0 pmol prawn(-1). The results show that a transient period of immunosuppression occurred between 2 and 8 h after injection of dopamine for all immune parameters except circulating haemocytes and all immune parameters returned to control values within 8-16 h after receiving dopamine. Injection of dopamine also significantly increased the mortality of M. rosenbergii challenged with the pathogen L. garvieae. These results suggest that stress-inducing dopamine suppresses the immune system, which in turn promotes the susceptibility to L. garvieae in M. rosenbergii.

Cloning and biological analysis of the zebrafish (Danio rerio) insulin-like growth factor binding protein-2 proximal promoter region.

Insulin-like growth factor binding protein 2 (IGFBP-2) plays an important role in the regulation of IGF's action and endocrinology in fish. To understand the molecular mechanism which controls transcription of the IGFBP-2 gene, we cloned and sequenced the IGFBP-2 proximal promoter region of the zebrafish IGFBP-2 gene and characterized its activity by firefly luciferase transient transfection expression assays. Different fragments of the zebrafish IGFBP-2 5'-flanking region were transfected into Hela and ZFL cells. In these cell lines, maximum promoter activity was located in the 900 base pairs (bp) of the zebrafish IGFBP-2 5' flanking region in the ZFL cell line and 318 bp of the zebrafish IGFBP-2 5' flanking region in the Hela cell line. The in vivo actions of the IGFBP-2 promoter on developmental stage expression were further investigated in transgenic zebrafish in which an IGFBP-2 (900-bp) promoter-driven green fluorescent protein encoding the GFP cDNA transgene was microinjected into zebrafish embryos. Morphological and RT-PCR studies of transgenic zebrafish indicated that the IGFBP-2 promoter-driven GFP transcripts appeared for the first time in the 32-cell stage. These results indicate that the IGFBP-2 promoter is active in a development-specific manner. These results suggest that the IGFBP-2 promoter plays an important role in teleost embryo growth.

Molecular cloning and characterisation of prophenoloxidase from haemocytes of the white shrimp, Litopenaeus vannamei.

cDNA encoding prophenoloxidase (proPO) of the white shrimp Litopenaeus vannamei was obtained from haemocytes by a reverse-transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA using oligonucleotide primers based on the proPO sequence of tiger shrimp Penaeus monodon, freshwater crayfish Pacifastacus leniusculus, green tiger shrimp Penaeus semisulcatus (accession no.: AF521949) and kuruma shrimp Marsupenaeus japonicus (accession no.: AB0733223). proPO of L. vannamei was constitutively expressed. The 2471-bp cDNA contained an open reading frame (ORF) of 2058 bp, a 96-bp 5'-untranslated region, and a 317-bp 3'-untranslated region containing the poly A tail. The molecular mass of the deduced amino acid sequence (686 amino acids) was 78.1 kDa with an estimated pI of 6.02. It contained putative copper binding sites, a complement-like motif (GCGWPQHM), a proteolytic activation site, and a conserved C-terminal region common to all known proPOs. However, no signal peptide sequence was detected in white shrimp proPO. Comparison of amino acid sequences showed that white shrimp proPO is more closely related to the proPO of another penaeid than to that of a freshwater crayfish. White shrimp proPO mRNA was synthesized in haemocytes and not in the hepatopancreas or muscle. The activation responses of the proPO of the white shrimp to an exogenous protease (trypsin), a detergent (sodium dodecyl sulphate), and algal and microbial cell wall components (laminarin, sodium alginate, zymosan, and lipopolysaccharide), and its susceptibility to protease inhibitors in vitro resemble the proPO activation system of other crustaceans. These facts suggest that the proPO system in haemocytes of the white shrimp Litopenaeus vannamei serves an important function in non-self recognition and host immune reactions.