Reducing sarcolipin expression improves muscle metabolism in mdx mice.

Duchenne muscular dystrophy (DMD) is an inherited muscle wasting disease. Metabolic impairments and oxidative stress are major secondary mechanisms that severely worsen muscle function in DMD. Here, we sought to determine whether germline reduction or ablation of sarcolipin (SLN), an inhibitor of sarco/endoplasmic reticulum (SR) Ca2+ ATPase (SERCA), improves muscle metabolism and ameliorates muscle pathology in the mdx mouse model of DMD. Glucose and insulin tolerance tests show that glucose clearance rate and insulin sensitivity were improved in the SLN haploinsufficient mdx (mdx:sln+/-) and SLN-deficient mdx (mdx:sln-/-) mice. The histopathological analysis shows that fibrosis and necrosis were significantly reduced in muscles of mdx:sln+/- and mdx:sln-/- mice. SR Ca2+ uptake, mitochondrial complex protein levels, complex activities, mitochondrial Ca2+ uptake and release, and mitochondrial metabolism were significantly improved, and lipid peroxidation and protein carbonylation were reduced in the muscles of mdx:sln+/- and mdx:sln-/- mice. These data demonstrate that reduction or ablation of SLN expression can improve muscle metabolism, reduce oxidative stress, decrease muscle pathology, and protects the mdx mice from glucose intolerance.

Endothermy in the smalleye opah (Lampris incognitus): A potential role for the uncoupling protein sarcolipin.

Sarcolipin (SLN) is a small transmembrane protein that in mice has been shown to uncouple the calcium ATPase pump of the sarcoplasmic reticulum, resulting in heat production. Mice up-regulate expression of SLN in response to cold challenge. This thermoregulatory mechanism is characterized as non-shivering muscle-based thermogenesis (NST). The current study was conducted to determine if the endothermic fish species, the smalleye opah (Lampris incognitus), has higher levels of sln transcription in tissues thought to be the main source of endothermic heat, namely the red aerobic pectoral fin musculature, which powers continuous swimming in this species. A search of the draft assembly of the opah genome reveals a single sln gene that is 95% identical to the zebrafish sln ortholog at the amino acid level. Quantitative PCR (qPCR) using opah-specific sln shows significantly higher sln transcript levels in the dark red pectoral fin muscle compared to both the light red pectoral muscle and white axial muscle tissues. The high ratio of sln transcripts to CaATPase (serca1) transcripts suggests that opah may utilize a futile calcium cycling NST mechanism in the dark red pectoral fin muscle to generate heat.

NK-lysin from skin-secreted mucus of Atlantic salmon and its potential role in bacteriostatic activity.

NK-lysin, despite being a direct effector of cytotoxic T and natural killer cells, is an antimicrobial peptide (AMP) with known antibacterial function in vertebrates and so in fish. Its presence has been described in different tissues of teleost fish. One of the strongest antimicrobial barriers in fish is skin-secreted mucus; however, this mucus has been found to contain only a small number of AMPs. The present study describes for the first time the constitutive expression of NK-lysin in Atlantic salmon (Salmo salar) mucus produced by the skin, recording the AMP at a higher concentration than in serum with greater bacteriostatic activity. Hepcidin may be involved to a greater extent in systemic responses since it was expressed to a higher degree in serum which was more potent for alternative complement and peroxidase activities.

MiR-422a weakened breast cancer stem cells properties by targeting PLP2.

OBJECTIVE: This study investigated miR-422a and PLP2 expressions in breast cancer cells and breast cancer stem cells (BCSCs). Besides, their influences on polymorphism changes were observed. METHODS: Flow cytometry and fluorescence-activated cell sorting was performed and CD24-/CD44+ cells were sorted from breast cancer cells and recognized as BCSCs. Microarray was applied to search for the differentially expressed miRNAs and mRNAs between MCF7 and BCSCs. The aberrant expression of miR-422a and PLP2 was further confirmed by RT-qPCR and the direct targeted relationship was verified by dual-luciferase reporter assay. After in vitro transfection, the expression of miR-422a and PLP2 were manipulated and biological functions of BMSCs were compared with CCK-8, colony formation and sphere formation assay. The tumorigenesis ability of transfected BMSCs was also investigated in NOD/SCID tumor mice models. RESULTS: BMSCs were successfully established from MCF7 cells and miR-422a expression was downregulated while PLP2 level decreased in BMSCs. MiR-422a directly targets the 3'UTR of PLP2 and suppressed its expression. Besides, the up-regulation of miR-422a contributed to weakened ability of proliferation and microsphere formation of BMSCs, while PLP2 overexpression facilitated those biological abilities. Tumorigenesis of BMSCs in mice models was impaired by either overexpression of miR-442a or silencing of PLP2. CONCLUSION: Up-regulation of miR-422a attenuated microsphere formation, proliferation and tumor formation of breast cancer stem cells via suppressing the PLP2 expression.

Both brown adipose tissue and skeletal muscle thermogenesis processes are activated during mild to severe cold adaptation in mice.

Thermogenesis is an important homeostatic mechanism essential for survival and normal physiological functions in mammals. Both brown adipose tissue (BAT) (i.e. uncoupling protein 1 (UCP1)-based) and skeletal muscle (i.e. sarcolipin (SLN)-based) thermogenesis processes play important roles in temperature homeostasis, but their relative contributions differ from small to large mammals. In this study, we investigated the functional interplay between skeletal muscle- and BAT-based thermogenesis under mild versus severe cold adaptation by employing UCP1-/- and SLN-/- mice. Interestingly, adaptation of SLN-/- mice to mild cold conditions (16 °C) significantly increased UCP1 expression, suggesting increased reliance on BAT-based thermogenesis. This was also evident from structural alterations in BAT morphology, including mitochondrial architecture, increased expression of electron transport chain proteins, and depletion of fat droplets. Similarly, UCP1-/- mice adapted to mild cold up-regulated muscle-based thermogenesis, indicated by increases in muscle succinate dehydrogenase activity, SLN expression, mitochondrial content, and neovascularization, compared with WT mice. These results further confirm that SLN-based thermogenesis is a key player in muscle non-shivering thermogenesis (NST) and can compensate for loss of BAT activity. We also present evidence that the increased reliance on BAT-based NST depends on increased autonomic input, as indicated by abundant levels of tyrosine hydroxylase and neuropeptide Y. Our findings demonstrate that both BAT and muscle-based NST are equally recruited during mild and severe cold adaptation and that loss of heat production from one thermogenic pathway leads to increased recruitment of the other, indicating a functional interplay between these two thermogenic processes.

Proteoliposome-based selection of a recombinant antibody fragment against the human M2 muscarinic acetylcholine receptor.

The development of antibodies against human G-protein-coupled receptors (GPCRs) has achieved limited success, which has mainly been attributed to their low stability in a detergent-solubilized state. We herein describe a method that can generally be applied to the selection of phage display libraries with human GPCRs reconstituted in liposomes. A key feature of this approach is the production of biotinylated proteoliposomes that can be immobilized on the surface of streptavidin-coupled microplates or paramagnetic beads and used as a binding target for antibodies. As an example, we isolated a single chain Fv fragment from an immune phage library that specifically binds to the human M2 muscarinic acetylcholine receptor with nanomolar affinity. The selected antibody fragment recognized the GPCR in both detergent-solubilized and membrane-embedded forms, which suggests that it may be a potentially valuable tool for structural and functional studies of the GPCR. The use of proteoliposomes as immunogens and screening bait will facilitate the application of phage display to this difficult class of membrane proteins.

Haploid genetic screens identify an essential role for PLP2 in the downregulation of novel plasma membrane targets by viral E3 ubiquitin ligases.

The Kaposi's sarcoma-associated herpesvirus gene products K3 and K5 are viral ubiquitin E3 ligases which downregulate MHC-I and additional cell surface immunoreceptors. To identify novel cellular genes required for K5 function we performed a forward genetic screen in near-haploid human KBM7 cells. The screen identified proteolipid protein 2 (PLP2), a MARVEL domain protein of unknown function, as essential for K5 activity. Genetic loss of PLP2 traps the viral ligase in the endoplasmic reticulum, where it is unable to ubiquitinate and degrade its substrates. Subsequent analysis of the plasma membrane proteome of K5-expressing KBM7 cells in the presence and absence of PLP2 revealed a wide range of novel K5 targets, all of which required PLP2 for their K5-mediated downregulation. This work ascribes a critical function to PLP2 for viral ligase activity and underlines the power of non-lethal haploid genetic screens in human cells to identify the genes involved in pathogen manipulation of the host immune system.

Increased sarcolipin expression and decreased sarco(endo)plasmic reticulum Ca2+ uptake in skeletal muscles of mouse models of Duchenne muscular dystrophy.

Abnormal intracellular Ca(2+) handling is an important factor in the progressive functional decline of dystrophic muscle. In the present study, we investigated the function of sarco(endo)plasmic reticulum (SR) Ca(2+) ATPase (SERCA) in various dystrophic muscles of mouse models of Duchenne muscular dystrophy. Our studies show that the protein expression of sarcolipin, a key regulator of the SERCA pump is abnormally high and correlates with decreased maximum velocity of SR Ca(2+) uptake in the soleus, diaphragm and quadriceps of mild (mdx) and severe (mdx:utr-/-) dystrophic mice. These changes are more pronounced in the muscles of mdx:utr-/- mice. We also found increased expression of SERCA2a and calsequestrin specifically in the dystrophic quadriceps. Immunostaining analysis further showed that SERCA2a expression is associated both with fibers expressing slow-type myosin and regenerating fibers expressing embryonic myosin. Together, our data suggest that sarcolipin upregulation is a common secondary alteration in all dystrophic muscles and contributes to the abnormal elevation of intracellular Ca(2+) concentration via SERCA inhibition.

Function of Shaker potassium channels produced by cell-free translation upon injection into Xenopus oocytes.

Voltage-gated ion channels are a class of membrane proteins that temporally orchestrate the ion flux critical for chemical and electrical signaling in excitable cells. Current methods to investigate the function of these channels rely on heterologous expression in living systems or reconstitution into artificial membranes; however these approaches have inherent drawbacks which limit potential biophysical applications. Here, we describe a new integrated approach combining cell-free translation of membrane proteins and in vivo expression using Xenopus laevis oocytes. In this method, proteoliposomes containing Shaker potassium channels are synthesized in vitro and injected into the oocytes, yielding functional preparations as shown by electrophysiological and fluorescence measurements within few hours. This strategy for studying eukaryotic ion channels is contrasted with existing, laborious procedures that require membrane protein extraction and reconstitution into synthetic lipid systems.

Tissue transglutaminase activity is involved in the differentiation of oligodendrocyte precursor cells into myelin-forming oligodendrocytes during CNS remyelination.

During normal brain development, axons are myelinated by mature oligodendrocytes (OLGs). Under pathological, demyelinating conditions within the central nervous system (CNS), axonal remyelination is only partially successful because oligodendrocyte precursor cells (OPCs) largely remain in an undifferentiated state resulting in a failure to generate myelinating OLGs. Tissue Transglutaminase (TG2) is a multifunctional enzyme, which amongst other functions, is involved in cell differentiation. Therefore, we hypothesized that TG2 contributes to differentiation of OPCs into OLGs and thereby stimulates remyelination. In vivo studies, using the cuprizone model for de- and remyelination in TG2(-/-) and wild-type mice, showed that during remyelination expression of proteolipid protein mRNA, as a marker for remyelination, in the corpus callosum lags behind in TG2(-/-) mice resulting in less myelin formation and, moreover, impaired recovery of motor behavior. Subsequent in vitro studies showed that rat OPCs express TG2 protein and activity which reduces when the cells have matured into OLGs. Furthermore, when TG2 activity is pharmacologically inhibited, the differentiation of OPCs into myelin-forming OLGs is dramatically reduced. We conclude that TG2 plays a prominent role in remyelination of the CNS, probably through stimulating OPC differentiation into myelin-forming OLGs. Therefore, manipulating TG2 activity may represent an interesting new target for remyelination in demyelinating diseases.

Decreased sarcolipin protein expression and enhanced sarco(endo)plasmic reticulum Ca2+ uptake in human atrial fibrillation.

Sarcolipin (SLN), a key regulator of cardiac sarco(endo)plasmic reticulum (SR) Ca(2+) ATPase, is predominantly expressed in atria and mediates ß-adrenergic responses. Studies have shown that SLN mRNA expression is decreased in human chronic atrial fibrillation (AF) and in aortic banded mouse atria; however, SLN protein expression in human atrial pathology and its role in atrial SR Ca(2+) uptake are not yet elucidated. In the present study, we determined the expression of major SR Ca(2+) handling proteins in atria of human AF patients and in human and in a mouse model of heart failure (HF). We found that the expression of SR Ca(2+) uptake and Ca(2+) release channel proteins are significantly decreased in atria but not in the ventricles of pressure-overload induced HF in mice. In human AF and HF, the expression of SLN protein was significantly decreased; whereas the expressions of other major SR Ca(2+) handling proteins were not altered. Further, we found that the SR Ca(2+) uptake was significantly increased in human AF. The selective downregulation of SLN and enhanced SR Ca(2+) uptake in human AF suggest that SLN downregulation could play an important role in abnormal intracellular Ca(2+) cycling in atrial pathology.

Expression of therapeutic proteins after delivery of chemically modified mRNA in mice.

Current viral vectors for gene therapy are associated with serious safety concerns, including leukemogenesis, and nonviral vectors are limited by low gene transfer efficiency. Here we investigate the therapeutic utility of chemically modified mRNA as an alternative to DNA-based gene therapy. A combination of nucleotide modifications abrogates mRNA interaction with Toll-like receptor (TLR)3, TLR7, TLR8 and retinoid-inducible gene I (RIG-I), resulting in low immunogenicity and higher stability in mice. A single intramuscular injection of modified murine erythropoietin mRNA raises the average hematocrit in mice from 51.5% to 64.2% after 28 days. In a mouse model of a lethal congenital lung disease caused by a lack of surfactant protein B (SP-B), twice weekly local application of an aerosol of modified SP-B mRNA to the lung restored 71% of the wild-type SP-B expression, and treated mice survived until the predetermined end of the study after 28 days.

Mammaglobin B is an independent prognostic marker in epithelial ovarian cancer and its expression is associated with reduced risk of disease recurrence.

BACKGROUND: Traditional prognostic factors in epithelial ovarian cancer (EOC) are inadequate in predicting recurrence and long-term prognosis, but genome-wide cancer research has recently provided multiple potentially useful biomarkers. The gene codifying for Mammaglobin B (MGB-2) has been selected from our previous microarray analysis performed on 19 serous papillary epithelial ovarian cancers and its expression has been further investigated on multiple histological subtypes, both at mRNA and protein level. Since, to date, there is no information available on the prognostic significance of MGB-2 expression in cancer, the aim of this study was to determine its prognostic potential on survival in a large cohort of well-characterized EOC patients. METHODS: MGB-2 expression was evaluated by quantitative real time-PCR in fresh-frozen tissue biopsies and was validated by immunohistochemistry in matched formalin fixed-paraffin embedded tissue samples derived from a total of 106 EOC patients and 27 controls. MGB-2 expression was then associated with the clinicopathologic features of the tumors and was correlated with clinical outcome. RESULTS: MGB-2 expression was found significantly elevated in EOC compared to normal ovarian controls, both at mRNA and protein level. A good correlation was detected between MGB-2 expression data obtained by the two different techniques. MGB-2 expressing tumors were significantly associated with several clinicopathologic characteristics defining a less aggressive tumor behavior. Univariate survival analysis revealed a decreased risk for cancer-related death, recurrence and disease progression in MGB-2-expressing patients (p < 0.05). Moreover, multivariate analysis indicated that high expression levels of MGB-2 transcript (HR = 0.25, 95%, 0.08-0.75, p = 0.014) as well as positive immunostaining for the protein (HR = 0.41, 95%CI, 0.17-0.99, p = 0.048) had an independent prognostic value for disease-free survival. CONCLUSION: This is the first report documenting that MGB-2 expression characterizes less aggressive forms of EOC and is correlated with a favorable outcome. These findings suggest that the determination of MGB-2, especially at molecular level, in EOC tissue obtained after primary surgery can provide additional prognostic information about the risk of recurrence.

Sarcolipin and ubiquitin carboxy-terminal hydrolase 1 mRNAs are over-expressed in skeletal muscles of alpha-tocopherol deficient mice.

The transcriptome of ataxic muscles from alpha-tocopherol transfer protein deficient (ATTP-KO), 23-month old, mice was compared with that of their normal littermates. Genes encoding sarcolipin (sln) and ubiquitin carboxyl-terminal hydrolase (uchl1) were over-expressed (> or =10-fold) in ataxic muscles. SLN is a 3.2 kDa membrane protein that binds to sarcoplasmic reticulum calcium ATPase, regulates Ca(+ +) transport and muscle relaxation-contraction cycles. UCHL1 is a 24.8 kDa member of proteosome proteins; it is over-expressed in myofibrillar myopathy and is associated with neurodegenerative diseases. Furthermore, six additional transcripts, three encoding thin-filament proteins and three encoding Ca(+ +) sensing proteins that participate in contraction-relaxation cycle, and eight transcripts that encode members of lysosomal proteins were also over-expressed in ataxic muscles. These observations suggest that chronic alpha-tocopherol (AT) deficiency activates critical genes of muscle contractility and protein degradation pathways, simultaneously. The magnitude of induction of sln and uchl1 was lower in asymptomatic, 8-month old, ATTP-KO mice and in 8-month old mice fed an AT-depleted diet. These studies suggest sln and uchl1 genes as novel targets of AT deficiency and may offer molecular correlates of well documented descriptions of neuromuscular dysfunctions in AT-deficient rodents. Since the neuromuscular deficits of ATTP-KO mice appear to be similar to those of patients with ATTP mutations, it is suggested that over-expression of sln and uchl1 may also contribute to AT-sensitive ataxia in humans.

MAL promoter hypermethylation as a novel prognostic marker in gastric cancer.

T-lymphocyte maturation associated protein, MAL, has been described as a tumour-suppressor gene with diagnostic value in colorectal and oesophageal cancers, and can be inactivated by promoter hypermethylation. The aim of this study was to analyse the prevalence of MAL promoter hypermethylation and the association with mRNA expression in gastric cancers and to correlate methylation status to clinicopathological data. Bisulphite-treated DNA isolated from formalin-fixed and paraffin-embedded samples of 202 gastric adenocarcinomas and 22 normal gastric mucosae was subjected to real-time methylation-specific PCR (Q-MSP). Two regions within the MAL promoter (M1 and M2) were analysed. In addition, 17 frozen gastric carcinomas and two gastric cancer cell lines were analysed both by Q-MSP and real-time RT-PCR. Methylation of M1 and M2 occurred in 71 and 80% of the gastric cancers, respectively, but not in normal gastric mucosa tissue. Hypermethylation of M2, but not M1, correlated with significantly better disease-free survival in a univariate (P=0.03) and multivariate analysis (P=0.03) and with downregulation of expression (P=0.01). These results indicate that MAL has a putative tumour-suppressor gene function in gastric cancer, and detection of promoter hypermethylation may be useful as a prognostic marker.

Mammaglobin and lipophilin B expression in breast tumors and their lack of effect on breast cancer cell proliferation.

BACKGROUND: Mammaglobin (SCGB2A2) and lipophilin B (SCGB1D2) are members of the secretoglobin polypeptide family. Mammaglobin has been shown to be overexpressed in breast tumor tissue, indicating that mammaglobin might confer a growth advantage to mammaglobin-expressing tumor cells. MATERIALS AND METHODS: The mammaglobin and lipophilin B mRNA expression levels were investigated in seven breast tumors and matched nonneoplastic tissues from the same patients using quantitative real-time RT-PCR. The effect of mammaglobin and lipophilin B expression on breast cancer cell proliferation rates was investigated by analyzing retrovirally transduced Hs578T cell clones. Cell proliferation rates were determined during the exponential growth phase by analyzing the change in lactate dehydrogenase activity over time. RESULTS: All analyzed breast cancer tumors had lower expression levels of mammaglobin and lipophilin B than the respective mean level of the nonneoplastic breast tissues; no prominent overexpression was evident. There was high variability in the expression of mammaglobin and lipophilin B among the non-neoplastic samples, showing that caution should be taken when evaluating their over- and underexpression in tumors. The expression levels of mammaglobin and lipophilin B correlated with each other in the analyzed samples (p = 0.001). Ectopic overexpression of mammaglobin and lipophilin B did not affect the cell proliferation rate of Hs578T breast carcinoma cells in vitro. CONCLUSION: Our findings suggest that the overexpression of mammaglobin observed in certain breast tumors is an epiphenomenon not causally involved in breast carcinogenesis.

Sentinel lymph node molecular pathology in breast carcinoma.

OBJECTIVES: The prognosis of breast cancer patients depends on primary tumor resection and axillary lymph nodes examination. The purpose of this study was to analyze by molecular biology techniques the presence of mammaglobin A and B messenger RNA in breast sentinel lymph node (SLN) by reverse-transcription polymerase chain reaction (RT-PCR). METHODS: Sentinel lymph nodes from 50 patients with a diagnosis of breast cancer were prospectively studied between June 2004 and August 2006. Lymph nodes were all examined every 2 mm by intraoperative cytology. Hematoxylin-eosin (HE), immunohistochemistry (IHC) with cytokeratin (clone AE1-AE3, DAKO, dilution 1:100), and molecular biology techniques were used in all cases. RESULTS: Deferred study with routine techniques showed subcapsular metastasis in 3/50 cases. Out of 50 cases, 5 were detected with IHC, and 2 of them were negative for HE. Multiplex RT-PCR allowed the detection of 18/50 positive SLN, which included the 5 above-mentioned cases. The other SLN studied (32/50) showed no metastases with the methods herein implemented. CONCLUSIONS: The epidemiologic impact of incomplete SLN study has been observed, as the HE technique fails to identify all SLN with micrometastases. In our opinion, SLN should be studied with IHC and molecular biology techniques. The multiplex RT-PCR technique for A and B mammaglobin proves to be specific and sensitive. This study will serve to formulate hypotheses. Further research, including a larger population and a longer-term follow-up period, will be required to confirm these hypotheses. Should our findings be confirmed in the future, molecular biology determinations could modify patients' staging and treatment.

Expression of mammaglobins A and B in nasal polyps is similar in patients with and without allergic rhinitis.

BACKGROUND: The causes of nasal polyposis remain unclear. Mammaglobins have been implicated in its pathogenesis. However, their association with the occurrence of nasal polyps in the presence of allergic rhinitis (AR) has not been explored. The aim of this study was to compare the expression levels of mammaglobins A and B with the nasal polyps of patients with and without AR. METHODS: Thirty-one patients with bilateral nasal polyposis underwent skin-prick tests to specific aeroallergens. Nasal polyp tissues were obtained from all patients and divided into two groups as nasal polyps with and without AR depending on clinical history and the skin-prick test results. All polyp tissues were analyzed for the levels of mammaglobin A and mammaglobin B by using real-time quantitative polymerase chain reaction technique. RESULTS: Of the 16 samples from patients having nasal polyps with AR, only 1 sample expressed a detectable level of mammaglobin A (1/16). There was no detectable expression of mammaglobin A in tissues from the group of nasal polyps without AR (0/15). Expression of mammaglobin B was detected in all nasal polyp tissues from both groups. The expression of mammaglobin B was not significantly different between nasal polyps with AR (median, 25th-75th percentiles; 0.023, 0.013-0.046) and nasal polyps without AR (0.032, 0.007-0.16). CONCLUSION: Expression levels of mammaglobins A and B in nasal polyps are not different between patients with and without AR. Our findings suggest that mammaglobins' implication in the pathogenesis of nasal polyps is independent of an underlying AR.

Liposomes-mediated delivery of pro-apoptotic therapeutic membrane proteins.

The delivery of functional therapeutic proteins by lipid vesicles into targeted living cells is one of the most promising strategies for treatment of different diseases and cancer. The use of this system in the delivery of membrane proteins directly into cells remains to be tested because the methods for producing membrane proteins are difficult to perform. Here we describe the effect of proteoliposomes containing the voltage-dependent anion channel (VDAC) and pro-apoptotic Bak, both produced with an optimized cell-free expression system. For the first time, recombinant VDAC and Bak proteins are synthesized and directly integrated into the lipidic bilayer of natural liposomes in a one-step reaction. VDAC has been shown to play an essential role in apoptosis in mammalian cells by regulating cytochrome c release from mitochondria and Bak modulates mitochondrial membrane permeability upon activation. Internalization of recombinant proteoliposomes into mammalian cells induces apoptosis by release of cytochrome c and caspases activation. These results highlight that membrane proteins integrated in natural liposomes can represent an excellent candidate for cancer protein therapy.

Characterization of Japanese flounder (Paralichthys olivaceus) NK-lysin, an antimicrobial peptide.

The NK-lysin cDNA of Japanese flounder, Paralichthys olivaceus, consists of 657bp, containing an open reading frame (ORF) of 444bp, which encodes 147 amino acid residues. The amino acid sequence of Japanese flounder NK-lysin has 21% identity to porcine NK-lysin and bovine NK-lysin, 23% to equine NK-lysin, and 46% to zebrafish NK-lysin-like protein. Multiple alignments of Japanese flounder NK-lysin and other known saposin-like proteins revealed that the six cysteine residues important for structural folding are completely conserved. The Japanese flounder NK-lysin gene is approximately 2kb and consists of five exons and four introns. Japanese flounder NK-lysin mRNA constitutive expression was mainly detected in gills, heart, head kidney, intestines, peripheral blood leukocytes (PBLs), spleen and trunk kidney, and was detected at low levels in liver, muscle and ovary. However, expression was not detected in brain, skin and stomach of apparently healthy Japanese flounder. Gene expression of Japanese flounder NK-lysin was not inducible by lipopolysaccharide (LPS) treatment. A synthesized NK-lysin peptide, consisting of 27 amino acid residues, showed antimicrobial activity against Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Photobacterium damselae subsp. piscicida.