Bovine Lactoferricin Research Articles

Design of bovine lactoferricin-derived peptide and its expression and activity in Pichia pastoris

Bovine lactoferrin peptide has been shown to be a broad-spectrum antimicrobial peptide. Based on the relationship between the structure and function of antimicrobial peptides, the antimicrobial peptide databases and protein analysis software were used to optimize the design of bovine lactoferricin peptide (LfcinB). The designed bovine lactoferricin-derived peptide (LfcinBD) gene fragment was inserted into a pPIC9K-His plasmid to construct a recombinant expression vector. After linearization of the Recombinant plasmid, Pichia pastoris GS115 cells were transfected with linearized recombinant plasmid by using electroporation and LfcinBD gene expression was induced with methanol. After the fermentation, supernatant was separated by low-temperature high-speed centrifugation. Ultrafiltration and freeze drying of the fermentation supernatant were performed, purified. Experimental results showed that the LfcinBD had stronger bacteriostatic activity against Staphylococcus aureus than the natural bovine lactoferrin peptide (LfcinB) produced under the same fermentation conditions. The effective expression of the optimized bovine lactoferricin-derived peptide was detected using SDS-PAGE electrophoresis. This study lays the foundation for further exploration to improve the biological activities of antimicrobial peptides.

Secondary Structural Transformation of Bovine Lactoferricin Affects Its Antibacterial Activity.

Lactoferricin (Lfcin) is a potent antibacterial peptide derived from lactoferrin by pepsin hydrolysis. It was hypothesized that structural transformation of Lfcin could affect its antibacterial function through forming and breaking of intramolecular disulfide bond. To prove this hypothesis, bovine Lfcin (bLfcin) and its two derivatives, bLfcin with a disulfide bond (bLfcin DB) and bLfcin with a mutation C36G (bLfcin C36G), were synthesized, purified, and identified. The circular dichroism (CD) spectra of the peptides were detected in solutions with different ionic and hydrophobic strength. Then, the secondary structure contents of the peptides were calculated on the basis of the CD spectra. The antibacterial activity of the peptides against Escherichia coli ATCC 25922, Salmonella typhimurium ATCC 14028, Shigella flexneri ATCC 12022, and Staphylococcus aureus ATCC 25923 was evaluated. The results showed that bLfcin and bLfcin C36G had similar percentages of secondary structure in water, while bLfcin and bLfcin DB had similar ratios of secondary structure under less hydrophobic conditions. The synthetic peptides exhibited antibacterial activity against all the tested bacteria, except for S. aureus ATCC 25923. bLfcin demonstrated higher antibacterial activity compared with its derivatives. The results suggested that bLfcin could transform its structure under alterative ionic strengths and hydrophobic conditions, and the transformation of structures was beneficial to enhancing the antibacterial function.

Effect of intramolecular disulfide bond of bovine lactoferricin on its molecular structure and antibacterial activity against Trueperella pyogenes separated from cow milk with mastitis

BackgroundLactoferricin (Lfcin) is an antimicrobial activity center of lactoferrin, produced by hydrolysis from the N-terminal of lactoferrin. It was hypothesized that the intramolecular disulfide bond in Lfcin could affect its antibacterial function through influencing its molecular structure. To prove this hypothesis, bovine Lfcin (bLfcin) and its two derivatives, bLfcin with an intramolecular disulfate bond (bLfcin DB) and bLfcin with a mutation C36G (bLfcin C36G), were synthesized, purified, and identified. The circular dichroism spectra of the peptides were detected in solutions with different ionic and hydrophobic strength. The antibacterial activity of the peptides against Trueperella pyogenes, separated from cow milk with mastitis, were determined.ResultsThe secondary structure of bLfcin DB showed more β-turn and less random coil than the other peptides in H2O, similar ratios of secondary structures with bLfcin and bLfcin C36G under ionic conditions, and close percentages of secondary structure with bLfcin under hydrophobic conditions. The synthetic peptides exhibited strong antimicrobial activity against T. pyogenes isolates, T. pyogenes ATCC 19,411, and E. coli ATCC 25,922. The antimicrobial activities of the three peptides were greater against T. pyogenes than against E. coli, and bLfcin DB exhibited higher antibacterial activity compared with its derivatives.ConclusionsThe intramolecular disulfide bond could change the molecular structure of bLfcin under alternative ionic strengths and hydrophobic effects, and the formation of the disulfide bond is beneficial to executing the antibacterial function of bLfcin.

Antibiofilm activity of lactoferrin-derived synthetic peptides against Pseudomonas aeruginosa PAO1.

Many pathogenic bacteria can protect themselves from the effects of antibiotics and the host immune response system by forming biofilms. Biofilms are polymer-entrapped bacterial cells, which adhere to each other and are often attached to a surface. Eradication of bacterial biofilms typically requires much higher concentrations of antibiotics than are normally needed to kill cultured planktonic cells, raising serious clinical concerns. In an attempt to prevent the formation of biofilms or to break up existing biofilms of pathogenic bacteria, herein we have used the standard crystal violet assay as well as the Calgary biofilm device to test several lactoferrin- and lactoferricin-derived antimicrobial peptides for their antibiofilm activity against Pseudomonas aeruginosa PAO1. Our results revealed that the short bovine lactoferricin-derived RRWQWR-NH2 (20-25) hexapeptide has no activity against P. aeruginosa PAO1. Moreover, the longer human lactoferricin-derived peptide GRRRRSVQWCA (1-11) and the bovine lactoferrampin (268-284) peptide were also almost devoid of activity. However, several different "mix-and-match" dimeric versions of the two lactoferricin-derived peptides proved quite effective in preventing the formation of biofilms at low concentrations, and in some cases, could even eradicate an existing biofilm. Moreover, the full-length bovine lactoferricinB (17-41) peptide also displayed considerable antimicrobial activity. Some of the longer lactoferricin-derived dimeric peptides acted through a bactericidal mechanism, whereas others seemed to interfere in cell-signalling processes. Taken together, our results indicate that synthetic dimeric peptides comprising short naturally occurring human and bovine lactoferricin constructs could be further developed as antibiofilm agents.

Evaluation of Antibacterial Properties of Chimeric Bovine Lactoferrin Peptide for Inhibition of Food and Plant Pathogens

: The resistance of microorganisms to conventional antibiotics has prompted researchers to produce new antimicrobial compounds. Antimicrobial peptides can be alternatives to chemical antibiotics. Antimicrobial peptides are produced approximately by all living organisms to fight infection. Lactoferrin is an iron glycoprotein that plays an important role in the immune system. Lactoferricin and lactoferrampine have stronger antimicrobial activities than lactoferrin. In this study, we investigated the inhibitory effects of a combination of chimeric bovine lactoferricin and lactoferrampine on microorganisms including Escherichia coli, Staphylococcus aureus, Salmonella typhimurium, Enterococcus faecalis, and Listeria monocytogenes, as well as plant pathogens including Pseudomonas syringae pv. syringae, Pseudomonas viridiflava, Xanthomonas translucens, Xanthomonas perforans, Erwinia amylovora, Pectobacterium carotovorum, and Agrobacterium tumefaciens. The results showed that chimeric bovine lactoferrin had a good inhibitory effect against pathogenic microorganisms and plant pathogens. The minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC) for pathogenic microorganisms Salmonella typhimurium and Listeria monocytogenes were 7.562 µg/mL and 15.125 µg/mL, respectively, which showed the highest sensitivity to chimeric bovine lactoferrin. The MIC and MBC for plant pathogens were 0.497 µg/mL and 0.997 µg/mL, respectively, which were related to Pseudomonas syringae pv. syringae and it showed the highest sensitivity to chimeric bovine lactoferrin.

Selective cytotoxic effect against the MDA-MB-468 breast cancer cell line of the antibacterial palindromic peptide derived from bovine lactoferricin.

The cytotoxic effect against the breast cancer cell line MDA-MB-468 of the palindromic peptide LfcinB (21–25)Pal: 1RWQWRWQWR9 and its analogous peptides, obtained via alanine scanning, was evaluated. The results indicate that the palindromic peptide exhibited a concentration-dependent cytotoxic effect against this cell line. The cytotoxic effect of the palindromic peptide was fast and selective and was sustained for up to 48 h of treatment. MDA-MB-468 cells treated with the palindromic peptide exhibited severe cellular damage, acquiring rounded forms and shrinkage, a behavior typical of apoptotic events. The analogous peptides exhibited fewer cytotoxic effects than the original palindromic peptide, suggesting that the substitution of any amino acid with alanine diminishes the cytotoxic effect. The Arg and Trp residues proved to be the most relevant for the cytotoxic effect; the analogous peptides with substitutions of Trp with Ala did not induce a change in cellular morphology, while analogous peptides with substitutions of Arg or Gln with Ala induced cellular damage. Also, neither the palindromic peptide nor its analogues exerted a significant cytotoxic effect on normal fibroblasts, indicating that the peptides had a selective cytotoxic effect on cancerous cells. The peptide LfcinB (21–25)Pal, and its analogues exhibited antibacterial activity against E. coli and S. aureus strains and a selective cytotoxic effect against the breast cancer cell line MDA-MB-468.

The Bovine Antimicrobial Peptide Lactoferricin Interacts with Polysialic Acid without Loss of Its Antimicrobial Activity against Escherichia coli.

Simple SummaryBovine milk contains a high concentration of the protein lactoferrin. It is an important antimicrobial biomolecule, which is also present in other bodily fluids like blood and semen. However, not only the intact protein but also its cleavage products have antimicrobial activity. Perhaps, the best-known cleavage product of lactoferrin is the peptide lactoferricin that has significant antimicrobial capacity against a broad range of pathogens such as enterohemorrhagic Escherichia coli (EHEC). Interestingly, lactoferricin can interact with the sugar polymer polysialic acid, which is also present in milk, blood, and semen. In the present study, we tested if the binding to polysialic acid influences the biological activity of bovine lactoferricin. Remarkably, neither different amounts of polysialic acid nor different chain lengths of this sugar polymer influenced the antimicrobial activity of lactoferricin. The ability of polysialic acid to bind and not inactivate lactoferricin may allow the development of novel endogenous and biodegradable polysialylated surfaces and/or hydrogels, which can be loaded with the antimicrobial peptide lactoferricin for biomedical applications in veterinary and human medicine.The lactoferrin-derived peptide lactoferricin (LFcin) belongs to the family of antimicrobial peptides, and its bovine form has already been successfully applied to counteract enterohemorrhagic Escherichia coli (EHEC) infection. Recently, it was described that LFcin interacts with the sugar polymer polysialic acid (polySia) and that the binding of lactoferrin to polySia is mediated by LFcin, included in the N-terminal domain of lactoferrin. For this reason, the impact of polySia on the antimicrobial activity of bovine LFcin was investigated. Initially, the interaction of LFcin was characterized in more detail by native agarose gel electrophoresis, demonstrating that a chain length of 10 sialic acid residues was necessary to bind LFcin, whereas approximately twice-as-long chains were needed to detect binding of lactoferrin. Remarkably, the binding of polySia showed, independently of the chain length, no impact on the antimicrobial effects of LFcin. Thus, LFcin binds polySia without loss of its protective activity as an antimicrobial peptide.

PH and Thermal Dual-Sensitive Nanoparticle-Mediated Synergistic Antitumor Effect of Immunotherapy and Microwave Thermotherapy.

Cationic anticancer peptides, which can induce tumor cell immunogenic death and further promote systemic tumor-specific immune responses, have offered a promising solution to relieve the tumor immunosuppressive microenvironment. However, peptide drugs are easily degraded and lack of targeting ability when administered systemically, leading to limitations in their applications. Herein, we report a pH and thermal dual-sensitive bovine lactoferricin-loaded (one of the most widely studied cationic anticancer peptides) nanoparticles, which simultaneously exhibited antitumor and immune cell activated effects when applied with microwave thermotherapy, an auxiliary method of immunotherapy. The bovine lactoferricin could be delivered to the tumor site by nanoparticles, be immediately released from nanoparticles in the acidic environment of lysosomes and the thermal condition caused by microwave radiation, and ultimately induce tumor apoptosis with the release of damage-associated molecular patterns (DAMPs). It is worth noting that the strategy of bovine lactoferricin-loaded nanoparticles intravenous injection combined with local microwave thermotherapy not only showed excellent efficacy in relieving tumor growth but also resulted in strong antitumor immunities, which was due to the released bovine lactoferricin under stimulating conditions, and the pool of tumor-associated antigens generated by tumor destruction. In conclusion, this work presents a strategy for tumor treatment based on dual-sensitive bovine lactoferricin-loaded nanoparticles combined with microwave thermotherapy, which may provide a solution for cationic anticancer peptides delivery and improving antitumor immune responses.

Polyhistidine facilitates direct membrane translocation of cell-penetrating peptides into cells

The bovine lactoferricin L6 (RRWQWR) has been previously identified as a novel cell-penetrating peptide (CPP) that is able to efficiently internalize into human cells. L6 interacts with quantum dots (QDs) noncovalently to generate stable L6/QD complexes that enter cells by endocytosis. In this study, we demonstrate a modified L6 (HL6; CHHHHHRRWQWRHHHHHC), in which short polyhistidine peptides are introduced into both flanks of L6, has enhanced cell-penetrating ability in human bronchoalveolar carcinoma A549 cells. The mechanism of cellular uptake of HL6/QD complexes is primarily direct membrane translocation rather than endocytosis. Dimethyl sulfoxide (DMSO), but not pyrenebutyrate (PB), ethanol, oleic acid, or 1,2-benzisothiazol-3(2 H)-one (BIT), slightly enhances HL6-mediated protein transduction efficiency. Neither HL6 nor HL6/QD complexes are cytotoxic to A549 or HeLa cells. These results indicate that HL6 could be a more efficient drug carrier than L6 for biomedical as well as biotechnological applications, and that the function of polyhistidine peptides is critical to CPP-mediated protein transduction.

Effects of Lactococcus lactis MG1363 producing fusion proteins of bovine lactoferricin-lactoferrampin on growth, intestinal morphology and immune function in weaned piglet.

We developed a strategy for localized delivery of the LFCA (lactoferricinlactoferrampin), which is actively synthesized in situ by Lactococcus lactis (pAMJ399-LFCA/LLMG1363), and explored the possibility of using pAMJ399-LFCA/LLMG1363 as an alternative additive diet to antibiotics. The antimicrobial activities of the LFCA derived from pAMJ399-LFCA/LLMG1363 were tested in vitro. The results showed that LFCA had an inhibitory effect on Staphylococcus aureus, Escherichia coli and Salmonella enteritidis. Then, the pAMJ399-LFCA/LLMG1363 was used as an additive diet for piglets. Our data demonstrated that oral administration of pAMJ399-LFCA/LLMG1363 significantly improved the average daily gain, feed-to-gain ratio, intestinal mucosal integrity and decreased the serum endotoxin and d-lactic acid levels. The mRNA expression levels of intestinal tight junction proteins (including occludin, Claudin-1 and ZO-1) were significantly upregulated by pAMJ399-LFCA/LLMG1363 administration. The serum immunoglobulin G (IgG) levels, intestinal secretory immunoglobulin A (sIgA) levels, IL-2, IL-10 and TGF-β levels were significantly increased by pAMJ399-LFCA/LLMG1363. Furthermore, our data revealed that oral administration of pAMJ399-LFCA/LLMG1363 significantly increased the number of general Lactobacillus, and decreased the total viable E. coli counts in the ileum and cecum contents. We developed a novel pAMJ399-LFCA/LLMG1363 secreting LFCA, which had probiotic effects on the growth performance, intestinal morphology, intestinal barrier function and immunological indices of weaned piglets. pAMJ399-LFCA/LLMG1363, with probiotic effects on the health of weaned piglets, may be a promising feed additive for weaned piglets.

Oral delivery of a Lactococcus lactis strain secreting bovine lactoferricin-lactoferrampin alleviates the development of acute colitis in mice.

Ulcerative colitis (UC) is a chronic relapsing disease. Treatment of UC would benefit from specific targeting of therapeutics to the intestine. Previous studies have demonstrated that bovine lactoferricin and lactoferrampin have bactericidal, anti-inflammatory, and immunomodulatory effects. Here, we investigated whether oral administration of a bovine lactoferricin-lactoferrampin (LFCA)-encoding Lactococcus lactis (LL-LFCA) strain could alleviate experimental colitis. LFCA derived from LL-LFCA inhibited the growth of Escherichia coli and Staphylococcus aureus in vitro. In mice, administration of LL-LFCA decreased the disease activity index and attenuated dextran sulfate sodium (DSS)-induced body weight loss and colon shortening. LL-LFCA treatment also ameliorated DSS-induced colon damage, inhibited inflammatory cell infiltration, significantly decreased myeloperoxidase activity, and ameliorated DSS-induced disruption of intestinal permeability and tight junctions. In addition, 16S rDNA sequencing showed that LL-LFCA reversed DSS-induced gut dysbiosis. The production of proinflammatory mediators in serum and the colon was also reduced by administration of LL-LFCA. In vitro, LFCA derived from LL-LFCA decreased the messenger RNA expression of proinflammatory factors. The underlying mechanisms may involve inhibition of the nuclear factor kappa B (NF-κB) pathway. The results demonstrate that LL-LFCA ameliorates DSS-induced intestinal injury in mice, suggesting that LL-LFCA might be an effective drug for the treatment of inflammatory bowel diseases.

Peptide conjugates of lactoferricin analogues and antimicrobials—Design, chemical synthesis, and evaluation of antimicrobial activity and mammalian cytotoxicity

Eight new peptide conjugates composed of modified bovine lactoferricin truncated analogues (LFcinB) and one of the three antimicrobials — ciprofloxacin (CIP), levofloxacin (LVX), and fluconazole (FLC) — were synthesized. Four different linkers were applied to connect a peptide and an antimicrobial agent. The FLC-containing peptidic conjugates were synthesized using the “click chemistry” method. This novel approach is reported here for the first time. Unlike their components, CIP- and LVX-based conjugates exerted activity against Candida yeast. Similarly to the constituent peptides, synthesized conjugates showed activity against Gram-positive bacteria, especially S. epidermidis. The most active were the conjugates containing CIP linked to the peptide by the redox-sensitive disulfide bridge. Our results show a significant role of a linker between antimicrobial agent and a peptide. This was also confirmed by the lack of synergistic effects on the antimicrobial activity of the constituent compounds. Moreover, cytotoxicity assays revealed that the proposed conjugates cause a comparatively low cytotoxic effect in reference to antibiotics widely used in therapies. Therefore, they can be deliberated as attractive leading structures for the development of drugs.

Intracellular Delivery of Nanoparticles Mediated by Lactoferricin Cell-Penetrating Peptides in an Endocytic Pathway.

Cell-penetrating peptides (CPPs) containing a preponderance of basic amino acids are able to deliver biologically active macromolecules and nanomaterials into live cells. Quantum dots (QDs) are nanoparticles with unique fluorescence properties that have found wide application in biomedical imaging. In this study, we demonstrate transduction of an L6 CPP (RRWQWR) derived from bovine lactoferricin (LFcin) into human lung cancer cells. L6 noncovalently interacts with QDs to form stable complexes. L6/QD complexes enter cells most efficiently when prepared at a nitrogen/phosphate ratio of 60. Mechanistic studies indicate that L6/QD complexes enter cells by endocytosis. Treatment with 1,2-benzisothiazol-3(2H)-one (BIT), an industrial preservative that enhances uptake of certain CPPs, does not affect L6 CPP-mediated protein transduction efficiency. L6 and L6/QD complexes are not cytotoxic. These results indicate that L6 LFcin might be an efficient and safe nanoshuttle for nanoparticles or nanomedicines in biomedical applications.

The tetrameric peptide LfcinB (20-25)4 derived from bovine lactoferricin induces apoptosis in the MCF-7 breast cancer cell line.

The cytotoxic effect of the tetrameric peptide LfcinB (20–25)4 against breast cancer cell line ATCC® HTB-22™ (MCF-7) was evaluated. The tetrameric peptide exhibited a concentration-dependent cytotoxic effect against MCF-7 cancer cells. The peptide at 22 µM had the maximum cytotoxic effect against MCF-7 cancer cells, reducing their cell viability to ∼20%. The cytotoxic effect of the tetrameric peptide against MCF-7 cells was sustained for 24 hours. Furthermore, the tetrameric peptide did not exhibit a significant cytotoxic effect against the non-tumorogenic trophoblastic cell line, which confirms their selectivity for breast cancer cell lines. The MCF-7 cells treated at 12.2 µM for 1 h exhibited morphological changes characteristic of apoptosis, such as rounded forms and cellular shrinkage. Furthermore, this peptide induces severe cellular damage to MCF-7 cells, mitochondrial membrane depolarization, and increase of cytoplasmic calcium concentration. Our results suggest that it has a significant selective cytotoxic effect against MCF-7 cells, which may be mainly associated with the apoptotic pathway. This peptide, which contains the RRWQWR motif, could be considered to be a promising candidate for developing therapeutic agents for the treatment of breast cancer.

Enzymatic On-Resin Peptide Cleavage and in Situ Cyclization One-Pot Strategy for the Synthesis of Cyclopeptide and Cyclotide.

A one-pot strategy combining sortase A mediated on-resin peptide cleavage and in situ cyclization was developed for the synthesis of cyclic peptides. This strategy was applied to synthesize head-to-tail cyclic antibacterial bovine lactoferricin peptide LFcinB20-35 in a yield of 67%. The one-pot strategy was compatible with an oxidative folding reaction, and complex cyclotides containing one or two disulfide bonds, such as sunflower trypsin inhibitors-1 and α-conotoxin MII, were successfully synthesized in one pot in a yield of 77% and 61%, respectively.

Antibacterial Activity and Mechanism of Action of Bovine Lactoferricin Derivatives with Symmetrical Amino Acid Sequences.

In recent years, the overuse of antibiotics has become very serious. Many pathogenic bacteria have become resistant to them, with serious potential health consequences. Thus, it is urgent that we develop new antibiotic drugs. Antimicrobial peptides (AMPs) are important endogenous antibacterial molecules that contribute to immunity. Most have spectral antibacterial properties and do not confer drug resistance. In this paper, an 11-residue peptide (LFcinB18–28) with a sequence of KCRRWQWRMKK was modified by amino acid substitution to form a symmetrical amino acid sequence. The antibacterial activities and mechanisms of action of engineered peptides including KW-WK (KWRRWQWRRWK), FP-PF (FPRRWQWRRPF), FW-WF (FWRRWQWRRWF), and KK-KK (KKRRWQWRRKK) were investigated. The four engineered peptides could more effectively inhibit bacteria than the original peptide, LFcinB18–28. This suggested that a symmetrical amino acid sequence might enhance the antibacterial activity of AMPs. However, only peptides KW-WK, FP-PF, and KK-KK were safe; FW-WF displayed hemolytic activity. The engineered peptides shared cationic and amphipathic characteristics that facilitated interactions with the anionic microbial membranes, leading to disruption of membrane integrity and permeabilizing microbial membranes, resulting in cell death. Therefore, a symmetrical amino acid sequence and related structural parameters offer an alternative approach to the design of AMPs. This will provide a scientific basis for the design and synthesis of new AMPs.

LFchimera protects HeLa cells from invasion by Yersinia spp. in vitro

Yersinia pestis is the causative agent of plague. As adequate antibiotic treatment falls short and currently no effective vaccine is available, alternative therapeutic strategies are needed. In order to contribute to solving this problem we investigated the therapeutic potential of the peptide construct LFchimera against the safer-to-handle Y. pestis simulants Yersinia enterocolitica and Yersinia pseudotuberculosis in vitro. LFchimera is a heterodimeric peptide construct mimicking two antimicrobial domains of bovine lactoferrin, i.e. lactoferrampin and lactoferricin. LFchimera has been shown to be a potent antimicrobial peptide against a variety of bacteria in vitro and in vivo. Also Y. enterocolitica and Y. pseudotuberculosis have been shown to be susceptible for LFchimera in vitro. As Yersiniae spp. adhere to and invade host cells upon infection, we here investigated the effects of LFchimera on these processes. It was found that LFchimera has the capacity to inhibit host-cell invasion by Yersiniae spp. in vitro. This effect appeared to be host-cell mediated, not bacteria-mediated. Furthermore it was found that exposure of human HeLa epithelial cells to both LFchimera and the bacterial strains evoked a pro-inflammatory cytokine release from the cells in vitro.

Molecular Therapeutic Cancer Peptides: A Closer Look at Bovine Lactoferricin

Background: In spite of the progress that was recorded in the development of anticancer drugs, challenges continue to rises particularly due to resistance of the cancer chemotherapies and low sensitivity of the commercially available anticancer drugs.
 Methodology: collection and review of available literatures.
 Results: Bovine lactoferricin (LfcinB) is a cationic peptide with broad spectrum antimicrobial and anticancer activity. It also shows promising activity against many varieties of cancer cells including colon carcinoma cells, melanoma, fibrosarcoma, monocytic leukemic cells and neuroblastoma. It is unaffected by multidrug resistance mechanisms seen with conventional chemotherapeutic drugs, and displays a higher specificity for cancer cells versus normal cells in comparison to conventional chemotherapy. 
 Conclusion: LfcinB exhibit strong antitumor activity effectively penetrating cell membrane, activating caspases and induce apoptosis to cancer cells.

Construction and characterization of thymidine auxotrophic (\u0394thyA) recombinant Lactobacillus casei expressing bovine lactoferricin

BackgroundLactobacillus casei (L. casei) is well known for its probiotic property in human and animals. Lactoferricin (Lfcin) polypeptide can effectively modulate host immune responses and have antimicrobial activity in vivo and in vitro. In order to develop a food-grade L. casei system constitutively expressing bovine Lfcin, this study constructed a thymidine auxotrophy (ΔthyA) recombinant L. casei.ResultsBased on the thymidylate synthase gene (thyA) insert site, LFEC(Lfcin expression cassette)was inserted into L. casei genome through homologous recombination, successfully expressed and could be stably inherited. The recombinant L. casei, ΔthyA L. casei-LFEC, is sensitive to chloramphenicol and limited when cultured without thymine. Meanwhile, ΔthyA L. casei-LFEC has both good antibacterial activity against Escherichia coli and Staphylococcus aureus and antiviral activity against porcine epidemic diarrhea virus (PEDV).ConclusionsWe successfully constructed a recombinant L. casei strain expressing Lfcin, ΔthyA L. casei-LFEC, which could only survive in the presence of thymine, and had excellent antimicrobial and antiviral activity with good genetic stability and sensitivity. This research provides a cost-effective alternative to the antibiotics with additional biological functions and wider applicability prospect. Using ΔthyA as the selectable mark instead of antibiotic to construct genetic engineering L.casei provides a safe and effective approach of feed additives in livestock raising.

Effect of Polyvalence on the Antibacterial Activity of a Synthetic Peptide Derived from Bovine Lactoferricin against Healthcare-Associated Infectious Pathogens.

Antimicrobial peptides (AMPs) are gaining interest as potential therapeutic agents. Peptides derived from bovine lactoferricin B (LfcinB) have been reported to exhibit antimicrobial activity, and the LfcinB RRWQWR sequence is the smallest known motif that exhibits antibacterial and cytotoxic activity. Our goal was to examine the effect of multicopy arrangements of the RRWQWR motif, on its antibacterial activity against healthcare-associated infections (HCAIs). Linear and branched peptides containing the RRWQWR motif were generated using solid phase peptide synthesis-Fmoc/tBu methodology, purified, and characterized using reverse phase-high performance liquid chromatography and matrix-assisted laser desorption/ionization time of flight mass spectrometry. For each peptide, the antibacterial activity against Staphylococcus aureus (ATCC 25923 and 33591 strains) and Klebsiella pneumoniae (ATCC 13883 and 700603 strains) was assessed by measuring the minimum inhibitory and the minimum bactericidal concentrations, in the exponential phase. Cells were observed by scanning electron microscopy, and the hemolytic activity of the peptides was assessed. The overall results demonstrate that, compared to linear analogues, polyvalent presentation of the RRWQWR motif enhances its antibacterial activity against both Gram-negative and Gram-positive bacteria even on resistant strain.