Fish natural killer enhancing factor-A (NKEF-A) enhance cytotoxicity of nonspecific cytotoxic cells against bacterial infection.

Natural killer enhancing factor (NKEF)-A/B is a member of Peroxiredoxin (Prxs) family, which is named for the function of enhancing NK cells activity. NKEF also plays essential roles in multiple physiology/pathology processes including inflammation regulation, cancer development and redox reactions. However, the regulatory effects of fish NKEF on immune cells remain largely unknown. In this study, the full-length cDNA of NKEF-A (Accession No. MK584553, designated as On-NKEF-A) was identified from Nile tilapia (Oreochromis niloticus). On-NKEF-A encoded a 198 amino acid peptide with molecular mass of 22.085 kDa. On-NKEF-A protein contained a typical 2-Cys family domain, two active sites (51aa and 172aa) that were conserved in mammals, birds, amphibians and fish. Phylogenetic analysis showed that On-NKEF-A had the closest relationship with Zebra mbuna (Maylandia zebra) NKEF. The On-NKEF-A transcription was present in all examined tissues or organs. And the relative high expression levels of On-NKEF-A was found in head kidney leucocytes and nonspecific cytotoxic cells (NCC). After Streptococcus agalactiae stimulation, On-NKEF-A was significantly up-regulated in head kidney, spleen, gill and skin. Also, On-NKEF-A was markedly induced post S. agalactiae, LPS and poly I:C stimulation in head kidney-derived NCC. Moreover, On-NKEF-A was mainly distributed in cytoplasm of fathead minnow cells (FHM cells). The further in vitro analysis found that the recombinant protein of On-NKEF-A (rOn-NKEF-A) could induce the expression of various molecular markers of B cells, macrophages and NCC, enhanced the cytotoxicity of NCC via increasing the effectors expression. The present data collectively indicate that On-NKEF-A participates in anti-bacterial immune response via regulating NCC activity, which will provide new ideas to further explore the defense mechanism of fish against bacteria.

A Flounder Fish Peptide Shows Anti-Hypertensive Effects by Suppressing the Renin-Angiotensin-Aldosterone System and Endothelin-1.

BACKGROUND: Many fishes have been known for their good nutritional effects especially in the cardiovascular aspect. Some specific fish peptides have anti-hypertensive effects. OBJECTIVE: In the present study, we hypothesized that the hexapeptide (MEVFVP) from flounder fish muscle can be a potent antihypertensive peptide, therefore, decided to perform this experiment. METHODS: The peptide MEVFVP from flounder fish muscle (40 mg/kg) and vehicle were administered per os to spontaneously hypertensive rats (SHRs) (SHR-M and SHR-C, respectively). Additionally, plasma MEVFVP was measured serially before and after its oral administration to Sprague Dawley rats. RESULTS: Blood pressures (BPs), especially systolic BP, in SHR rats were decreased around 3-6 hours after MEVFVP administration. Compared with SHR-C rats, endothelin-1 (ET-1) mRNA expression in multiple tissues, and plasma levels of ET-1, angiotensin II, and aldosterone were lower in SHR-M rats, whereas the phosphorylation of AMP-activated protein kinase (AMPK) was increased in the kidney of SHR-M rats. The administered peptide was not detected in rat plasma, while ex vivo incubation of the peptide in rat plasma caused its rapid degradation within minutes. CONCLUSION: Our results show that the MEVFVP has an antihypertensive effect by regulating renin- angiotensin-aldosterone system, ET-1 and AMPK despite its limited bioavailability.

Epinecidin-1 Protects against Methicillin Resistant Staphylococcus aureus Infection and Sepsis in Pyemia Pigs.

Methicillin resistant Staphylococcus aureus (MRSA) may be found on the skin, nose, and throats of long-term hospitalized patients. While MRSA infections are usually minor, serious infections and death may occur in immunocompromised or diabetic patients, or after exposure of MRSA to blood. This report demonstrates that the antimicrobial peptide (AMP) epinecidin-1 (Epi-1) efficiently protects against MRSA infection in a pyemia pig model. We first found that Epi-1 exhibits bactericidal activity against MRSA. Next, pharmacokinetic analysis revealed that Epi-1 was stable in serum for 4 h after injection, followed by a gradual decrease. This pharmacokinetic profile suggested Epi-1 may bind serum albumin, which was confirmed in vitro. Harmful effects were not observed for doses up to 100 mg/kg body weight in pigs. When Epi-1 was supplied as a curative agent 30 min post-infection, MRSA-induced abnormalities in blood uric acid (UA), blood urea nitrogen (BUN), creatine (CRE), GOT, and GPT levels were restored to normal levels. We further showed that the bactericidal activity of Epi-1 was higher than that of the antibiotic drug vancomycin. Epi-1 significantly decreased MRSA counts in the blood, liver, kidney, heart, and lungs of infected pigs. Elevated levels of serum C reactive protein (CRP), proinflammatory cytokine IL6, IL1ß, and TNFα were also attenuated by Epi-1 treatment. Moreover, the MRSA genes, enterotoxin (et)-A, et-B, intrinsic methicillin resistance A (mecA), and methicillin resistance factor A (femA), were significantly reduced or abolished in MRSA-infected pigs after treatment with Epi-1. Hematoxylin and eosin staining of heart, liver, lung, and kidney sections indicated that Epi-1 attenuated MRSA toxicity in infected pigs. A survival study showed that the pyemia pigs infected with MRSA alone died within a week, whereas the pigs post-treated with 2.5 mg/kg Epi-1 were completely protected against death. The present investigation, thus, demonstrates that Epi-1 effectively protects pyemia pigs against pathogenic MRSA without major toxic side effects.

Pleurocidin congeners demonstrate activity against Streptococcus and low toxicity on gingival fibroblasts.

OBJECTIVES: Fish epidermal antimicrobial peptides, such as pleurocidin, are cathelicidins with broad-spectrum antimicrobial activity against gram negative and gram-positive bacteria, as well as fungi. In the current study, we attempted to optimize peptide bioactivity by sequence modification and assess the antimicrobial activities. METHODS: Fifteen pleurocidin analogues were designed, and the efficacy of pleurocidin congeners against common cariogenic microorganisms was tested; furthermore, we performed a preliminary study of the antimicrobial mechanism. We assayed the minimal inhibitory concentration (MIC), minimal bactericide concentration (MBC) and bactericidal kinetics to determine the cell killing activity. Scanning electron microscopy (SEM) was used to observe the bacterial membrane after treatment with congeners' peptides. Human gingival fibroblasts (HGFs) were also used in toxicity studies. RESULTS: The MIC and MBC results indicated that peptide congeners had different antimicrobial activities against the tested oral strains. Toxicity studies indicated that several congener peptides had little effect on human gingival fibroblasts (HGFs) with 5min of in vitro treatment. CONCLUSION: Our findings suggested that several pleurocidin congeners had the antimicrobial effect against Streptococcus mutans, Streptococcus sanguinis and Streptococcus sobrinus.

A novel antimicrobial peptide derived from fish goose type lysozyme disrupts the membrane of Salmonella enterica.

In aquaculture, accumulation of antibiotics resulted in development of resistance among bacterial pathogens. Consequently, it became mandatory to find alternative to synthetic antibiotics. Antimicrobial peptides (AMPs) which are described as evolutionary ancient weapons have been considered as promising alternates in recent years. In this study, a novel antimicrobial peptide had been derived from goose type lysozyme (LyzG) which was identified from the cDNA library of freshwater fish Channa striatus (Cs). The identified lysozyme cDNA contains 585 nucleotides which encodes a protein of 194 amino acids. CsLyzG was closely related to Siniperca chuatsi with 92.8% homology. The depicted protein sequence contained a GEWL domain with conserved GLMQ motif, 7 active residues and 2 catalytic residues. Gene expression analysis revealed that CsLyzG was distributed in major immune organs with highest expression in head kidney. Results of temporal expression analysis after bacterial (Aeromonas hydrophila) and fungal (Aphanomyces invadans) challenges indicated a stimulant-dependent expression pattern of CsLyzG. Two antimicrobial peptides IK12 and TS10 were identified from CsLyzG and synthesized. Antibiogram showed that IK12 was active against Salmonella enterica, a major multi-drug resistant (MDR) bacterial pathogen which produces beta lactamase. The IK12 induced loss of cell viability in the bacterial pathogen. Flow cytometry assay revealed that IK12 disrupt the membrane of S. enterica which is confirmed by scanning electron microscope (SEM) analysis that reveals blebs around the bacterial cell membrane. Conclusively, CsLyzG is a potential innate immune component and the identified antimicrobial peptide has great caliber to be used as an ecofriendly antibacterial substance in aquaculture.

Bioactive protein-based nanofibers interact with intestinal biological components resulting in transepithelial permeation of a therapeutic protein.

Proteins originating from natural sources may constitute a novel type of material for use in drug delivery. However, thorough understanding of the behavior and effects of such a material when processed into a matrix together with a drug is crucial prior to further development into a drug product. In the present study the potential of using bioactive electrospun fish sarcoplasmic proteins (FSP) as a carrier matrix for small therapeutic proteins was demonstrated in relation to the interactions with biological components of the intestinal tract. The inherent structural and chemical properties of FSP as a biomaterial facilitated interactions with cells and enzymes found in the gastrointestinal tract and displayed excellent biocompatibility. More specifically, insulin was efficiently encapsulated into FSP fibers maintaining its conformation, and subsequent controlled release was obtained in simulated intestinal fluid. The encapsulation of insulin into FSP fibers provided protection against chymotrypsin degradation, and resulted in an increase in insulin transport to around 12% without compromising the cellular viability. This increased transport was driven by interactions upon contact between the nanofibers and the Caco-2 cell monolayer leading to the opening of the tight junction proteins. Overall, electrospun FSP may constitute a novel material for oral delivery of biopharmaceuticals.

Evaluation of HPßCD-PEG microparticles for salmon calcitonin administration via pulmonary delivery.

For therapeutic peptides, the lung represents an attractive, noninvasive route into the bloodstream. To achieve optimal bioavailability and control their fast rate of absorption, peptides can be protected by coprocessing with polymers such as polyethylene glycol (PEG). Here, we formulated and characterized salmon calcitonin (sCT)-loaded microparticles using linear or branched PEG (L-PEG or B-PEG) and hydroxypropyl-beta-cyclodextrin (HPßCD) for pulmonary administration. Mixtures of sCT, L-PEG or B-PEG and HPßCD were co-spray dried. Based on the particle properties, the best PEG:HPßCD ratio was 1:1 w:w for both PEGs. In the sCT-loaded particles, the L-PEG was more crystalline than B-PEG. Thus, L-PEG-based particles had lower surface free energy and better aerodynamic behavior than B-PEG-based particles. However, B-PEG-based particles provided better protection against chemical degradation of sCT. A decrease in sCT permeability, measured across Calu-3 bronchial epithelial monolayers, occurred when the PEG and HPßCD concentrations were both 1.6 wt %. This was attributed to an increase in buffer viscosity, caused by the two excipients. sCT pharmacokinetic profiles in Wistar rats were evaluated using a 2-compartment model after iv injection or lung insufflation. The maximal sCT plasma concentration was reached within 3 min following nebulization of sCT solution. L-PEG and B-PEG-based microparticles were able to increase T(max) to 20 ± 1 min and 18 ± 8 min, respectively. Furthermore, sCT absolute bioavailability after L-PEG-based microparticle aerosolization at 100 µg/kg was 2.3 times greater than for the nebulized sCT solution.