Identification, expression analysis, and antibacterial activity of NK-lysin from common carp Cyprinus carpio.

Natural killer lysin (NK-lysin), produced by cytotoxic T lymphocytes and natural killer cells, is a cationic antimicrobial peptide that has a broad antimicrobial spectrum, including bacteria, viruses, and parasites. Nevertheless, the implication of NK-lysin in the protection against bacterial infection is not aware in common carp. In this study, six different NK-lysin genes (nkl1, nkl2, nkl3, nkl4, nkl5 and nkl6) were identified in the common carp genome. Each of the mature peptides of common carp NK-lysin has six well-conserved cysteine residues, and shares a Saposin B domain, characteristic of saposin-like protein (SALIP) family. The gene nkl1 contains 5 extrons and 4 introns, and nkl2, nkl3, nkl4 or nkl5 contains 4 extrons and 3 introns, however, the nkl6 has 3 extrons and 2 introns. By quantitative real-time PCR, nkl2 transcripts were predominantly expressed in spleen of healthy common carp, while elevated mainly in gill and spleen upon Aeromonas hydrophila infection. The recombinant NK-lysin-2 purified from Pichia pastoris shows antibacterial activity against Staphylococcus aureus (Gram-positive), and Escherichia coli M15, Aeromonas hydrophila, as well as Edwardsiella tarda (Gram-negative), the latter two are important pathogens of aquaculture. Our results indicate that NK-lysin in common carp might play an important role in fish immune response by enhancing antibacterial defense against bacterial pathogens.

Molecular cloning, biological effect, and tissue distribution of interleukin-8 protein in mandarin fish (Siniperca chuasti) upon Flavobacterium columnare infection.

Interleukin-8 (IL-8), a CXC-type chemokine, plays a key role in acute inflammation by recruiting neutrophils in mammals. In the present study, the open reading frame (ORF) of IL-8, encoding 99 amino acids was cloned in mandarin fish, and its function in inflammation was investigated. The IL-8 contains four conserved cysteine residues, with the first two forming the CXC signature motif. The genomic sequence of mandarin fish IL-8 has four exons and three introns, a typical gene organization of the CXC chemokine. Bioactive recombinant IL-8 (rIL-8) exhibited a chemotactic effect on head kidney leukocytes in vitro, and activates the transcription of the inflammatory genes, IL-8 and IL-1ß. When mandarin fish was challenged intraperitoneally with the pathogenic bacterium Flavobacterium columnare G4, the steady-state protein level of IL-8 was up-regulated in trunk kidney and head kidney. These results suggest that IL-8 is a functional CXC chemokine in mandarin fish, and plays a key role in the inflammatory responses towards bacterial infection.

Edwardsiella tarda EscE (Orf13 Protein) Is a Type III Secretion System-Secreted Protein That Is Required for the Injection of Effectors, Secretion of Translocators, and Pathogenesis in Fish.

The type III secretion system (T3SS) of Edwardsiella tarda is crucial for its intracellular survival and pathogenesis in fish. The orf13 gene (escE) of E. tarda is located 84 nucleotides (nt) upstream of esrC in the T3SS gene cluster. We found that EscE is secreted and translocated in a T3SS-dependent manner and that amino acids 2 to 15 in the N terminus were required for a completely functional T3SS in E. tarda. Deletion of escE abolished the secretion of T3SS translocators, as well as the secretion and translocation of T3SS effectors, but did not influence their intracellular protein levels in E. tarda. Complementation of the escE mutant with a secretion-incompetent EscE derivative restored the secretion of translocators and effectors. Interestingly, the effectors that were secreted and translocated were positively correlated with the EscE protein level in E. tarda. The escE mutant was attenuated in the blue gourami fish infection model, as its 50% lethal dose (LD50) increased to 4 times that of the wild type. The survival rate of the escE mutant-strain-infected fish was 69%, which was much higher than that of the fish infected with the wild-type bacteria (6%). Overall, EscE represents a secreted T3SS regulator that controls effector injection and translocator secretion, thus contributing to E. tarda pathogenesis in fish. The homology of EscE within the T3SSs of other bacterial species suggests that the mechanism of secretion and translocation control used by E. tarda may be commonly used by other bacterial pathogens.

[Biocompatibility of new bone tissue engineering scaffolds in vivo].

OBJECTIVE: To investigate the biocompatibility of new bone tissue engineering scaffolds, A:D, L-polylactic acid (PDLLA)/polylactic acid-polyethylene glycol-polylactic acid-polylactic acid (PLA-PEG-PLA)/Tricalcium phosphate and B: PDLLA/PLA-PEG-PLA in vivo, compared with PDLLA in repair of a rabbit mandibular body defect. METHODS: 24 New Zealand adult rabbits were divided into 4 groups randomly. 15 mm x 6 mm defects were made surgically in the bilateral mandibular bodies and each hemi-mandible was assigned as an experimental unit. The defects were randomly repaired with scaffold materials in each group. Specimens obtained were evaluated with general observation, X-ray, histomorphology and computerized graphical analysis at 2, 4 , 8, 12 weeks after surgery. RESULTS: Compared with PDLLA, the new scaffold materials B showed biocompatibility. At the same time the quantity of new bone produced was much more than that in control group (P<0.05). The new scaffold materials A showed the clear chronic granulomatous inflammation. CONCLUSION: New scaffold material B had sound biocompatibility. It was much better than PDLLA. So it may be an ideal bone tissue engineering scaffold material. A is not adapted to be used as scaffold material.

Syntheses of R-beta-rutinosides by rutin-degrading reaction.

Rutinose and five R-beta-rutinosides were obtained by means of rutin-degrading reaction in water or aqueous alcohol (ROH, R = methyl, ethyl, propyl, isopropyl, and benzyl) with rutin-degrading enzyme as catalyst and rutin as starting material in 84-94% yields, of which methyl-beta-rutinoside, propyl-beta-rutinoside, isopropyl-beta-rutinoside, and benzyl-beta-rutinoside are firstly reported in this paper. Based on spectral analysis, the structures of all products were elucidated.

[Effects of granulocyte colony-stimulating factor on peripheral endothelial progenitor cells in cholesterol-fed rabbits].

OBJECTIVE: To investigate the effects of granulocyte colony-stimulating factor (G-CSF) on peripheral endothelial progenitor cells (EPC) and atherosclerosis (AS) in cholesterol-fed rabbits. METHODS: Male New Zealand white rabbits were randomly divided into control group, G group (Recombinant Human Granulocyte Colony Stimulating Factor Injection rhG-CSF 50 microg/d), AS group (high cholesterol diet) and G + AS group (rhG-CSF 50 microg/d plus high cholesterol diet, n = 8 per group). Peripheral blood was collected at baseline and at 1, 4, 8 and 12 weeks, total mononuclear cells (MNCs) were isolated from peripheral blood by Ficoll density gradient centrifugation, and then the cells were plated on fibronectin-coated culture dishes. After being cultured for 7 days, EPCs were characterized as adherent cells double positive for DiLDL uptake and lectin binding by direct fluorescent staining under a laser scanning confocal microscope. After being cultured for 3 days, the number of EPC (PE-CD34/FITC-CD133 double-stained positive cells) was quantified by flow cytometric analysis. Serum NO was measured and aortic plaque area analyzed at 12 weeks. RESULTS: EPC number was low in control and AS groups and EPC number was significantly increased ( approximately 13-fold, P < 0.001) compared to baseline at 1 week in G and G + AS groups and remained at this level throughout the study period in G group while decreased gradually in G + AS group and returned to baseline level at 12 weeks. Aortic atherosclerotic plaque was visible in both AS and G + AS groups, however, the aortic atherosclerotic plaque area was smaller in G + AS group than that of in As group (59.8 mm(2) +/- 26.9 mm(2) vs. 251.5 mm(2) +/- 83.4 mm(2), P < 0.01). Serum NO was similar between AS and G + AS groups and significantly higher than that in control and G groups. CONCLUSION: CSF could attenuate atherosclerosis in cholesterol-fed rabbits by increasing circulating EPC.

[Time course of G-CSF, estrogen and various doses of atorvastatin on endothelial progenitor cells mobilization].

OBJECTIVE: To evaluate the time course of granulocyte-colony-stimulating-factor (G-CSF), estrogen and various doses of atorvastatin on endothelial progenitor cells (EPCs) mobilization. METHOD: A total of 48 male New Zealand White rabbits were treated with placebo, estrogen (0.25 mg.k(-1).d(-1)), Atorvastatin (2.5, 5, or 10 mg) and G-CSF (50 microg/rabbit/d), respectively. Peripheral EPCs number was surveyed weekly for 4 weeks by FACS analysis (double-positive for PE-CD34/FITC-CD133) and under fluorescent microscope (double-positive for FITC-UEA-1/Dil-acLDL). Serum nitric oxide (NO) and lipids were also measured at the third week. RESULTS: Peripheral EPCs was significantly increased in G-CSF treated animals and remained constant for 4 weeks compared to placebo treated animals. Atorvastatin increased peripheral EPCs dose-dependently from 2.5 to 5 mg and peaked at the third week while peripheral EPCs number was not affected by 10 mg.k(-1).d(-1) atorvastatin during the first 3 weeks and was significantly higher only in the fourth week compared to placebo group. Estrogen also significantly increased peripheral EPCs at the third and fourth week compared to placebo group. At the third week, serum NO was similar in G-CSF group, significantly higher in atorvastatin 5 mg.k(-1).d(-1) and estrogen groups while significantly lower in atorvastatin 10 mg.k(-1).d(-1) group compared to placebo group. Serum lipids were similar among various groups. CONCLUSION: Atorvastatin, estrogen and G-CSF could mobilize EPCs. The mobilization efficacy is as follows: G-CSF > atorvastatin 5 mg.k(-1).d(-1) > estrogen > atorvastatin 2.5 mg.k(-1).d(-1) > atorvastatin 10 mg.k(-1).d(-1). NO might partly contribute to the mobilizing effect of estrogen and atorvastatin.