Identification of an antibacterial protein as L-amino acid oxidase in the skin mucus of rockfish Sebastes schlegeli

Fish skin mucus contains a variety of antimicrobial proteins and peptides that seem to play a role in self defense. We previously reported an antibacterial protein in the skin secretion of the rockfish, Sebastes schlegeli, which showed selective antibacterial activity against Gram-negative bacteria. This study aimed to isolate and structurally and functionally characterize this protein. The antibacterial protein, termed SSAP (S. schlegeli antibacterial protein), was purified to homogeneity by lectin affinity column chromatography, anion-exchange HPLC and hydroxyapatite HPLC. It was found to be a glycoprotein containing N-linked glycochains and FAD. Its molecular mass was estimated to be 120 kDa by gel filtration HPLC and 53 kDa by SDS/PAGE, suggesting that it is a homodimer. On the basis of the partial amino-acid sequence determined, a full-length cDNA of 2037 bp including an ORF of 1662 bp that encodes 554 amino-acid residues was cloned by 3' RACE, 5' RACE and RT-PCR. A blast search showed that a mature protein (496 residues) is homologous to l-amino acid oxidase (LAO) family proteins. SSAP was determined to have LAO activity by the H(2)O(2)-generation assay and substrate specificity for only l-Lys with a K(m) of 0.19 mm. It showed potent antibacterial activity against fish pathogens such as Aeromonas hydrophila, Aeromonas salmonicida and Photobacterium damselae ssp. piscicida. The antibacterial activity was completely lost on the addition of catalase, confirming that H(2)O(2) is responsible for the growth inhibition. This study identifies SSAP as a new member of the LAO family and reveals LAO involvement in the innate immunity of fish skin.     

Antibacterial action of L-amino acid oxidase from the skin mucus of rockfish Sebastes schlegelii

L-amino acid oxidase (LAO) shows broadly antibacterial activity against Gram-positive and Gram-negative bacteria by H(2)O(2) generated in the oxidative process of L-amino acids. However, LAO (termed SSAP) isolated from the rockfish Sebastes schlegelii skin mucus acted selectively on Gram-negative bacteria. Therefore, this study was undertaken to clarify the antibacterial action of SSAP as compared with H(2)O(2). SSAP inhibited potently the growth of Aeromonas salmonicida, Photobacterium damselae subsp. piscicida and Vibrio parahaemolyticus with a minimum inhibitory concentration (MIC) of 0.078, 0.16 and 0.63 microg/mL, respectively. H(2)O(2) inhibited the growth of both Gram-positive and Gram-negative bacteria with an MIC ranging from 0.31 to 2.5 mM. When SSAP was incubated with P. damselae subsp. piscicida and Escherichia coli, SSAP was demonstrated to bind to P. damselae subsp. piscicida but not to E. coli by Western blotting and LAO activity measurement. These results show that the bacteria binding activity may be involved in the bacterial cell selectivity of SSAP. Electron microscopic observation of A. salmonicida, P. damselae subsp. piscicida and V. parahaemolyticus revealed that the treatments with SSAP and H(2)O(2) induced cell surface damage to A. salmonicida, remarkable elongation of P. damselae subsp. piscicida bodies and pores into V. parahaemolyticus cells.     

DNA polymerase beta mRNA and protein expression in Xiphophorus fish

Herein we report Xiphophorus DNA polymerase beta (XiphPolbeta) mRNA and protein expression levels in brain, liver, gill, and testes tissues from Xiphophorus maculatus, Xiphophorus helleri, and Xiphophorus couchianus parental line fish and two different tumor-bearing Xiphophorus interspecies hybrids. Polymerase beta protein levels in the Xiphophorus tissues were measured by Western blot, and mRNA was measured with a quantitative real time RT-PCR method which employed cRNA construction to produce accurate calibration curves. We found significant differences in both mRNA and protein levels between the tumor-bearing hybrid animals and the three parental species. However, there were no significant differences in either mRNA levels or protein expression observed between the parental species. Thus, interspecies hybridization results in dysregulation of Polbeta expression and this may manifest a modulation in DNA repair capability and susceptibility to latent tumorigenesis.     

Tandem repeat L-rhamnose-binding lectin from the skin mucus of ponyfish, Leiognathus nuchalis

Two lactose-binding lectins (PFL-1 and -2) were identified in the skin mucus of ponyfish, Leiognathus nuchalis. The molecular masses of PFL-1 and -2 were estimated to be 24 and 30kDa, respectively. Cloning of the PFL-1 cDNA demonstrated its unique tandem repeat structure composed of two homologous domains with 41.7% internal identity. Furthermore, PFL-1 exhibited homology with L-rhamnose-binding lectins previously purified from the eggs of fish and sea urchins. The N-terminal amino acid sequence of PFL-2 was similar to that of PFL-1, suggesting that this protein is an isotype of PFL-1. The PFL-1 gene was expressed in the skin, an important line of defense against pathogens in fish, but was not expressed in any of the other tissues tested here. PFL-1 is the fourth type of fish skin mucus lectin to be identified, suggesting that different species of fish express different types of lectin in their skin mucus.     

Molecular and cellular detection of expression of vitellogenin and <Emphasis Type="Italic">zona radiata</Emphasis> protein in liver and skin of juvenile salmon (<Emphasis Type="Italic">Salmo salar</Emphasis>) exposed to nonylphenol

In developing bioassays for estrogenic effects, vitellogenin (Vtg) induction and zona radiata protein (Zr-protein) induction in males and juveniles of oviparous vertebrates have been used as sensitive biomarkers for estrogenicity. Nonylphenol (NP) produces similar and parallel expression patterns of Vtg and Zr-protein levels in plasma and surface mucus of salmon, the response being concentration- and time-dependent. We have explored the potential mechanisms of Vtg and Zr-protein expression in surface mucus by comparative molecular and cellular approaches. Liver, skin, blood, and surface mucus samples were collected from fish exposed to a single waterborne concentration of NP (10 and 60 μg/l), 3, 7, and 10 days post-exposure, for gene expression analysis (liver and skin; quantitative real-time polymerase chain reaction) and protein analysis (blood and surface mucus; enzyme-linked immunosorbent assay). Protein expression was localized by immunohistochemistry. NP produced concentration- and time-dependent increases of hepatic estrogen receptors (ERα and ERβ), Vtg, and Zr-protein mRNA and plasma protein levels. These responses paralleled cellular detection of Vtg and Zr-protein in the liver with unique expression patterns in the cytoplasm of hepatocytes, hepatic sinusoids, and endothelial cells. ERα, Vtg, and Zr-protein mRNA were detectable in the skin. ERβ was the only skin response that was NP-concentration-dependent, especially at day 10 post-exposure. Immunohistochemistry for Vtg and Zr-protein in skin showed unique expression patterns in mucus vacuoles, epidermal cells, and scales in an NP-concentration- and time-specific manner. Thus, analysis of skin mRNA levels for xenoestrogen biomarker responses is a less-promising approach than protein analysis. The immunohistochemical localization of Vtg and Zr-protein levels in the skin further validates surface mucus as a sensitive biomarker source for estrogenic compounds. These responses represent an improvement for the detection of endocrine-disrupting compounds and related pollutants in the environment. The Norwegian Research Council (NFR) financially supported this study.     

Novel mannose-specific lectins found in torafugu, Takifugu rubripes: A review

In earlier work, we identified a novel mannose-specific lectin, termed pufflectin-s, from the skin mucus of torafugu, Takifugu rubripes. We here make a brief review on the lectin. The amino acid sequence of pufflectin-s shares sequence homology with mannose-binding lectins of monocotyledonous plants, and has conserved two of three carbohydrate recognition domains, QDNVY motifs, of these plant lectins. By site-directed mutagenesis, we verified that the QDNVY motif in the N-terminal region was critical to the mannose-binding function of pufflectin-s. RT-PCR and Northern blot analyses indicated that the pufflectin-s gene is expressed in the gill, oral cavity wall, esophagus, and skin. In addition, an isoform, pufflectin-i, which shares 91.4% amino acid identity with pufflectin-s, was isolated from the intestine. Using immunohistochemistry, pufflectin-s could be detected exclusively in the epithelial cells of the skin, gill, oral cavity wall and esophagus, whereas pufflectin-i was observed in both mucous and epithelial cells in the intestine. Nevertheless, mRNAs for both pufflectins were detected only in epithelial cells of these tissues with in situ hybridization. Pufflectin-s agglutinated some bacteria isolated from rearing water and from fish skin. This lectin also bound to a parasite, Heterobothrium okamotoi, suggesting that it may play an important role in the self-defense system of fugu.     

A unique epidermal mucus lectin identified from catfish (Silurus asotus): first evidence of intelectin in fish skin slime

The present study reports a new type of skin mucus lectin found in catfish Silurus asotus. The lectin exhibited calcium-dependent mannose-binding activity. When mannose eluate from chromatography with mannose-conjugated agarose was analysed by SDS-PAGE, the lectin appeared as a single 35-kDa band. Gel filtration showed that the lectin forms monomers and dimers. A 1216-bp cDNA sequence obtained by RACE-PCR from the skin encoded a 308 amino acid secretory protein with homology to mammalian and fish intelectins. RT-PCR demonstrated that the lectin gene was expressed in the gill, kidney and skin. Subsequent sequencing revealed the presence of an isoform in the gills. Antiserum detected the intelectin protein in club cells in the skin and gill, renal tubules and blood plasma. Although intelectin gene expression was not induced by in vivo bacterial stimulation, the intelectin showed agglutination activity against the pathogenic bacterium Aeromonas salmonicida, suggesting that the lectin plays an important role in self-defence against bacteria in the skin surface of the catfish. These findings represent one of the few examples of characterization and functional analysis of a fish intelectin protein.     

Differential expression of a hydroxyproline-rich cell-wall protein gene in embryonic tissues of Zea mays L.

A hydroxyproline-rich glycoprotein (HRGP) component of the maize cell wall was shown to be present in different organs of the plant by extraction of cell wall proteins and detection by Western blotting and immunocytochemistry. Antibodies raised against the protein or against synthetic peptides designed from the protein sequence immunoprecipitated a proline-rich polypeptide which was synthesized in-vitro from poly(A) ⁺ RNA extracted from different tissues of the plant and from the complete in-vitro-transcribed mRNA. A very low amount of the protein was found in immature embryos. In particular, the protein could not be detected in the scutellum either by Western blotting or by immunocytochemistry. In agreement with this finding, HRGP mRNA was barely detected in the scutellum, in contrast to its accumulation in the embryo axis. Our results indicate the existence of a unique cell wall structure in embryonic tissues from maize as well as a tissuespecific component of the control of maize HRGP gene expression, distinct to others already described such as cell division.Abbreviations HRGP(s) hydroxyproline-richglycoprotein(s) - DAP days after pollination The present work was supported by grants from Plan Nacional de Investigation Cientifica y Técnica (grant BI088-0242) and European Communities (grant BAP-374). L.R.-A. is the recipient of a fellowship from the Plan Nacional de Formación de Personal Investigador.     

生长激素mRNA在蓝太阳鱼垂体外组织中的表达分布

应用半定量RT PCR方法和Southern杂交技术,系统地研究了生长激素(GH)基因在蓝太阳鱼垂体外组织中的表达情况。在建立检测蓝太阳鱼GHmRNA表达的半定量RT PCR扩增条件之后,分别对蓝太阳鱼雄性幼鱼(6月龄)和雄性成鱼(1年龄)的12个组织部位中GHmRNA的表达进行了检测。结果表明,除了在垂体之外,还在肌肉、性腺、鳃、心脏、脑、肾脏6个组织检测到GHmRNA的表达,但各组织间的表达水平存在差异,而在脾脏、肝脏、胃3个组织未检测到表达;肌肉组织中的表达水平从幼鱼到成鱼后明显提高,同时观察到在幼鱼和成鱼的性腺组织中存在着较高水平的表达。本研究表明,GH基因在蓝太阳鱼的垂体外组织中存在着广泛的表达,由此提示,蓝太阳鱼GH可能以旁分泌或自分泌的方式对其生长繁殖起着重要的作用。     

Cloning and characterization of phospholipase D from olive flounder (Paralichthys olivaceus)

The phospholipase D1 (PLD1) cDNA, designated PoPLD, encoding a predicted protein of 1053 amino acids in olive flounder (Paralichthys olivaceus) has been cloned. The deduced amino acid sequence shares high identity with that of PLD1s and PLD2 in human, rat and mouse. The phylogenic analysis and sequence comparison of PoPLD with other PLD isozymes were found to be closely related to the PLD1 isozyme in primary structure. The tissue expression analysis of PoPLD showed that the mRNA of PoPLD was predominantly expressed in the brain, gullet, muscle, stomach, head kidney, pyloric caeca, intestine and gill. The expression of the PoPLD gene was examined in various tissues of flounder by RT-PCR following stimulation with LPS and compared also with that of the inflammatory cytokines IL-1beta and IL-8 in various tissues of the stimulated flounder. This provides indirect evidence that PLD1 might have a relevant role in immune responses against pathogens and in inflammation. In addition, the recombinant protein of PoPLD (GFP-PoPLD), which demonstrated a phosphatidylcholine (PC)-hydrolyzing activity, was partially localized as a distinct ring-shaped form surrounding the rim of the nucleus in EPC cells. Together, our results suggest that PoPLD is similar to the mammalian PLD1 isoform, is generally widespread within olive flounder tissue, might have a relevant role in the fish immune system against pathogens and specifically may be localized in the subcellular membranes of the nuclear rim in EPC cells.     

The mucous coat on gill lamellae of rainbow trout (Oncorhynchus mykiss)

The existence of a layer of mucus covering the gill lamellae of healthy rainbow trout (Oncorhynchus mykiss) was investigated. Using cryo-scanning electron microscopy, a smooth, undulating, thin layer was observed which completely covered gill filaments and lamellae, thereby obscuring epithelial microridges. After processing cryopreserved gill arches in glutaraldehyde for conventional scanning electron microscopy, the layer was no longer present and epithelial microridges were clearly visible. The identity of this layer was investigated using cryopreserved gills which were treated in one of two ways. First, gills were incubated with a rabbit antiserum to gill mucus, with normal rabbit serum, or with phosphate-buffered saline. Following fixation in glutaraldehyde and processing, only the gill tissue incubated with the mucus-specific antiserum was still covered with the smooth layer. The layer was also retained on the gills of fish anesthetized in a solution containing mucusspecific antiserum and then processes in glutaraldehyde for conventional scanning electron microscopy. The tenacious nature of the mucous layer was demonstrated by its stability following exposure to formalin and a cationic detergent. Second, the presence of this layer was confirmed on gill tissue which was cryopreserved, followed by freeze-substitution and vapor fixation, and then examined by transmission electron microscopy.     

Molecular cloning and gene expression of Foxl2 in the Nile tilapia, Oreochromis niloticus

A Foxl2 cDNA was cloned from the Nile tilapia ovary by RT-PCR and subsequent RACE. Alignment of known Foxl2 sequences from vertebrates confirmed the conservation of the Foxl2 open reading frame and protein sequences, especially the forkhead domain and C-terminal region, while some homopolymeric runs of amino acids are found only in mammals but not in non-mammalian vertebrates. RT-PCR revealed that Foxl2 is expressed in the tilapia brain (B), pituitary (P), gill, and gonads (G), with the highest level of expression in the ovary, reflecting the involvement of Foxl2 in B-P-G axis. Northern blotting and in situ hybridization also revealed an evident sexual dimorphic expression pattern in the gonads. Foxl2 mRNA was mainly detected in the granulosa cells surrounding the oocytes. The ovarian expression of Foxl2 in tilapia begins early during the differentiation of the gonads and persists until adulthood, implying the involvement of Foxl2 in fish gonad differentiation and the maintenance of ovarian function.     

Identification and molecular cloning of Xenopus laevis SP22, a protein associated with fertilization in mammals

SP22 is a novel sperm protein that has been shown to be highly correlated with fertility in rats. SP22 homologues have been studied in mouse and man but a definitive role for the protein has not yet been established. Using a polyclonal IgG to recombinant rat SP22, we detected the presence of this protein in Xenopus laevis tissues. Moreover, a Xenopus EST was found that shares a high degree of similarity with rat SP22 and the derived sequence codes for an 189 aa protein that is very similar to rat SP22. Finally, using Western blotting and RT-PCR analyses, we investigated the expression of Xenopus SP22 both in adult tissues and during embyronic development. SP22 protein expression predominated in the adult testis, and both mRNA and protein levels diminished subsequent to the initial day following fertilization.     

香鱼补体成分C9基因的克隆、序列分析及表达

补体成分C9是构成膜攻击复合体引起靶细胞溶解破坏的重要组成成分。该文测定了香鱼C9(aC9)基因的cDNA全序列,序列全长2125个核苷酸,编码一个由592个氨基酸组成、相对分子质量为6.56×104的前体蛋白,N端22个氨基酸为信号肽序列。序列分析表明,aC9与虹鳟C9的氨基酸同源性最高,达56.8%,与其它鱼类C9的同源性介于40.9%~53.8%之间。aC9在健康香鱼肝、脾、肠、鳃和肌肉有表达,其中在肝内的表达量最高。实时荧光定量PCR的结果显示,鳗利斯顿氏菌侵染4h后,肝中aC9mRNA表达量显著上调,并随着时间的推移在16h时达到峰值。Westernblotting分析的结果显示,鳗利斯顿氏菌侵染后香鱼血清中的aC9蛋白随着时间的推移呈显著上调。以上结果表明,香鱼肝组织C9基因表达变化与鳗利斯顿氏菌的侵染密切相关,揭示了C9在鱼类抗细菌免疫反应中具有重要的作用。     

Tissue expression of glandular kallikrein and its response to 17 beta-estradiol in the acclimatized carp

Cyprinus carpio skeletal muscle kallikrein was isolated to apparent homogeneity, and a polyclonal antiserum against the purified protein was generated. Glandular kallikrein expression and tissue distribution were assessed using both Western blots and immunohistochemistry. A 39-kDa protein was detected in skeletal muscle, the gill, kidney, and pituitary gland, where an additional 72-kDa immunoreactive band was observed. Immunohistochemistry revealed immunoreactive kallikrein in the intermuscle tissue, epithelial gill cells, apical portion of distal and proximal tubular cells in the kidney, mucus and epithelial cells of the skin, intestinal tube, and prolactin-producing cells of the pituitary gland. In addition, the effect of 17beta-estradiol on kallikrein expression was analyzed in three different tissues of winter- and summer-acclimatized male carps. A 2.5-fold (p<0.05) increase in kallikrein immunoreactivity due to estrogen treatment was observed in winter-acclimatized carp muscle, but not in summer-acclimatized fish. In contrast, the gill responded differently, since a 2-fold (p<0.05) increase was found only in summer-acclimatized carps. Kallikrein immunoreactivity in the kidney increased both in summer- (2.5 fold) and in winter-acclimatized carps (1.5 fold). The signals obtained demonstrate the existence of tissue-specific variable responses to estrogen treatment in vivo, between winter and summer-acclimatized carp.