Cecropin P1 Research Articles

Heterologous expression and activity of α-helical antimicrobial peptide SW in Bacillus subtilis

The antimicrobial peptide Cecropin P1 (CP1) was isolated from the pig-intestinal parasitic nematode Ascaris lumbricoides and had potent antimicrobial activity. However, its cytotoxicity is high. Herein, Using CP1 as the parent peptide, we designed a derivative peptide SW by replacing amino acids based on the structure and function relationship of antimicrobial peptides (AMPs) to reduce the toxicity of CP1. Subsequently, the gene sequence encoding SacB-6 ×His-SUMO-SW was constructed into the recombinant expression vector PHY-P43 of Bacillus subtilis WB800N. The fusion proteins with concentrations of 26.97 mg/L were obtained after purification by a Ni–NTA column. A total of 5.37 mg of SW was obtained from 1 L fermentation cultures. Recombinant SW (rSW) exhibits potent antimicrobial activity against gram-negative bacteria by inducing membrane instabilities. In addition, rSW showed low hemolytic activity and cytotoxicity against human red blood cells and IPEC-J2, respectively. In the mice model of peritonitis infection, rSW could reduce Escherichia coli-loading in the liver, spleen, lung, and kidney of mice. These results indicate that rSW production by Bacillus. subtilis is a safe and reliable strategy, and rSW has the potential to be a novel antimicrobial agent against gram-negative bacterial infections.

Potential of antimicrobial peptide-fused endolysin LysC02 as therapeutics for infections and disinfectants for food contact surfaces to control Cronobacter sakazakii

Although endolysin (lysin), a muralytic enzyme of bacteriophages, has emerged as a promising antibacterial agent, its use against Gram-negative bacteria is challenging due to the presence of an outer membrane (OM) that acts as a physical barrier. To overcome this limitation, it is crucial to provide lysins with the ability to penetrate or disrupt the OM to access the target peptidoglycan. Here, we fused a lysin LysC02 from phage ΦC02 with nine different amphipathic antimicrobial peptides (AMPs) to enhance its intrinsic bactericidal activity against the notorious opportunistic foodborne Gram-negative pathogen Cronobacter sakazakii. Even without OM permeabilizers, the engineered LysC02 fused at the C-terminus with Cecropin P1, Lactoferricin B, or Thanatin exhibited the most enhanced bactericidal activity against C. sakazakii with ∼3-log reduction within 2 h. The action of LysC02-AMPs, the physical destruction of bacterial cells facilitated by the destabilization of the OM by fused AMPs and the intracellular turgor pressure of the cell, occurred over a wide range of temperatures up to 55 °C and pH from 4 to 10. Not only C. sakazakii but also other Gram-negative pathogens were lysed by LysC02-AMPs. Accordingly, LysC02-AMPs were able to significantly rescue Galleria mellonella larvae infected with C. sakazakii in the in vivo treatment model and significantly reduce C. sakazakii cells in food and food contact surface models. Taken together, LysC02-AMPs could be developed as effective alternative disinfectants and therapeutics to control C. sakazakii.

Cecropin P1 antimicrobial peptide modulates differential expression of immune relevant genes in rainbow trout (Oncorhynchus mykiss) gill cell line, RTgill-W1

Accumulated evidence indicates that antimicrobial peptides modulate immune activities in fish. In this study, we profiled the differential expression patterns of representative immune relevant genes in an epithelial-like cell line of rainbow trout gill, RTgill-W1, in response to exposure of cecropin P1 antimicrobial peptide. RTgill-W1 cells were treated with synthetic cecropin P1 over time (0, 2, 4 and 24 h) with or without the present of lipopolysaccharide (LPS) or polyinosinic polycytidylic acid (PolyI:C). The relative abundances of each mRNA were measured by real-time quantitative PCR. The dose-response study revealed significant perturbations of mRNA levels of genes related to pro-inflammation, acute phase, surface proteins and transcription factors at 30 μM of cecropin P1. In addition, cecropin P1 altered the differential expression patterns that were induced by LPS or PolyI:C, at different time points in RTgill-W1. Overall, our results indicate that cecropin P1 exhibits pro-inflammation activity, modulate cell-cell interaction and cytokine signal transduction in rainbow trout gill cell, and may suggest a potential application of this peptide as an immune adjuvant for disease control in aquaculture.

Three-Dimensional Structure of the Antimicrobial Peptide Cecropin P1 in Dodecylphosphocholine Micelles and the Role of the C-Terminal Residues.

Cecropin P1 (CP1) isolated from a large roundworm Ascaris suum, which is found in pig intestines, has been extensively studied as a model antimicrobial peptide (AMP). However, despite being a model AMP, its antibacterial mechanism is not well understood, particularly the function of its C-terminus. By using an Escherichia coli overexpression system with calmodulin as a fusion partner, we succeeded in the mass expression of recombinant peptides, avoiding toxicity to the host and degradation of CP1. The structure of the recombinant 15N- and 13C-labeled CP1 and its C-terminus truncated analogue in dodecylphosphocholine (DPC) micelles was determined by NMR. In this membrane-mimetic environment, CP1 formed an α-helix for almost its entire length, except for a short region at the C-terminus, and there was no evidence of a hinge, which is considered important for the expression of activity in other cecropins. Several NMR analyses showed that the entire length of CP1 was protected from water by micelles. Since the loss of the C-terminus of the analogue had little effect on the NMR structure or its interaction with the micelle, we investigated another role of the C-terminus of CP1 in its antimicrobial activity. The results showed that the C-terminal region affected the DNA-binding capacity of CP1, and this mechanism of action was also newly suggested that it contributed to the antimicrobial activity of CP1.

Analysis of the influence of transgenic kalanchoe extract with cecropin P1 on the state of a non-infected wound in rats

In this article data on the analysis of the effect of transgenic Kalanchoe pinnate extract with cecropin P1 on the state of uninfected wounds in rats are presented. The study used Wistar rats weighing 220 ± 20 g. A wound of 20 × 20 mm was modeled under anesthesia in sterile conditions. The wound was treated during 14 days with the 3% hydrogen peroxide solution in all experimental groups. Additional treatment with the 10% cefazolin solution was used in group No. 2; with Kalanchoe juice it was used in group No. 3, and with transgenic Kalanchoe extract cecropin P1 it was used in group No. 4. The effectiveness of the treatment of the wound process was evaluated by the external state of the wound and planimetry indicators. It was found that all analyzed parameters of the experimental groups were better than in the control group of animals. However, the effect was most pronounced in the group treated by transgenic Kalanchoe extract with cecropin P1, The results of the study allow us to recommend further preclinical study of the substance for the possible treatment of trophic ulcers in patients with diabetes mellitus.

The Brassica napus L. plants expressing antimicrobial peptide cecropin P1 are safe for colonization by beneficial associative microorganisms

ABSTRACT This work was aimed to study the opportunity of transgenic oilseed rape (Brassica napus L.), expressing the gene of antimicrobial peptide cecropin P1 (cecP1) to be inhabited with associative microorganisms Methylobacterium mesophilicum and Pseudomonas aureofaciens. Previously, resistance was demonstrated to the microbial pathogens:Erwinia carotovora B15, Pseudomonas syringae, Fusarium oxysporum, and Botrytis cinerea. Analysis of the fatty acid composition of transgenic plant seeds showed an increase in the proportion of unsaturated fatty acids. Plants studied showed increased photosynthetic activity in oxidative stress induced by paraquat instead of in oxidative stress and UV radiation compared to control ones. A study of the interaction of transgenic plants with the associative microorganisms showed that bacteria were located in all initially colonized plants, as well as in plants obtained after several passages of microproliferation. Accordingly, transgenic plants, containing the cecP1 gene were actively colonized by associative bacteria, indicating their safety for associative bacteria, indicating their safety for beneficial microorganisms.

Expression of antimicrobial peptide Cecropin P1 in Saccharomyces cerevisiae and its antibacterial and antiviral activity in vitro

Cecropin P1, acting as an antimicrobial, has a broad-spectrum antibacterial activity with some antiviral and antifungal properties. It is a promising natural alternative to antibiotics which is originally isolated from the pig intestinal parasitic nematode Ascaris suum. Many studies have shown that Cecropin P1 is helpful for the prevention or treatment of clinical diseases. Therefore, it is very necessary to establish a safe, nontoxic, and efficient expression method of Cecropin P1. The results indicated that the recombinant protein was about 5.5 kDa showed by Tricine–SDS–PAGE and Western blot. And Cecropin P1 was efficiently secreted and expressed after 12 h of induction, with an increasing yield over the course of the induction. Its maximum concentration was 7.83 mg/L after concentration and purification. In addition, in vitro experiments demonstrated that Cecropin P1 not only exerted a strong inhibitory effect on Escherichia coli, Salmonella sp., Shigella sp., and Pasteurella sp., but also displayed an antiviral activity against PRRSV NADC30-Like strain. Collectively, the strategy of expressing Cecropin P1 in Saccharomyces cerevisiae is harmless, efficient, and safe for cells. In addition, the expressed Cecropin P1 has antiviral and antibacterial properties concurrently. How to cite: Jiang R, Zhang P, Wu X, et al., Expression of antimicrobial peptide Cecropin P1 in Saccharomyces cerevisiae and its antibacterial and antiviral activity in vitro. Electron J Biotechnol 2021;50. https://doi.org/10.1016/j.ejbt.2020.12.006

Conjugation of Carboxylated Graphene Quantum Dots with Cecropin P1 for Bacterial Biosensing Applications.

Biosensors that can accurately and rapidly detect bacterial concentrations in solution are important for potential applications such as assessing drinking water safety. Meanwhile, quantum dots have proven to be strong candidates for biosensing applications in recent years because of their strong light emission properties and their ability to be modified with a variety of functional groups for the detection of different analytes. Here, we investigate the use of conjugated carboxylated graphene quantum dots (CGQDs) for the detection of Escherichia coli using a biosensing assay that focuses on measuring changes in fluorescence intensity. We have further developed this assay into a novel, compact, field-deployable biosensor focused on rapidly measuring changes in absorbance to determine E. coli concentrations. Our CGQDs were conjugated with cecropin P1, a naturally produced antibacterial peptide that facilitates the attachment of CGQDs to E. coli cells; to our knowledge, this is the first instance of cecropin P1 being used as a biorecognition element for quantum dot biosensors. As such, we confirm the structural modification of these conjugated CGQDs in addition to analyzing their optical characteristics. Our findings have the potential to be used in situations where rapid, reliable detection of bacteria in liquids, such as drinking water, is required, especially given the low range of E. coli concentrations (103 to 106 CFU/mL) within which our two biosensing assays have collectively been shown to function.

Sandwich immunoassay based on antimicrobial peptide-mediated nanocomposite pair for determination of Escherichia coli O157:H7 using personal glucose meter as readout.

A sandwich immunoassay was developed for determination of E. coli O157:H7. This is based on an antimicrobial peptide-mediated nanocomposite pair and uses a personal glucose meter as signal readout. The antimicrobial peptides, magainins I, and cecropin P1 were employed as recognition molecules for the nanocomposite pair, respectively. With a one-step process, copper phosphate nanocomposites embedded by magainins I and Fe3O4 were used as "capturing" probes for bacterial magnetic isolation, and calcium phosphate nanocomplexes composed of cecropin P1 and invertase were used as signal tags. After magnetic separation, the invertase of the signal tags hydrolyzed sucrose to glucose, thereby converting E. coli O157:H7 levels to glucose levels. This lattercan be quantified by a personal glucose meter. Under optimal conditions, the concentration of E. coli O157:H7 can be determined in a linear range of 10 to 107CFU·mL-1 with a detection limit of 10CFU·mL-1. The method was successfully applied to the determination of E. coli O157:H7 in milk samples. Graphical abstract Schematic representation of sandwich immunoassay for E. coli O157:H7. One-pot synthetic of Fe3O4-magainins I nanocomposites (MMP) were used for magnetic capture. Cecropin P1-invertase nanocomposites (PIP) were used as signal tags. A personal glucose meter was used as readout to determine the target.

Obtainment and Analysis of Marker-Free Oil Plants Camelina sativa (L.) Expressing of Antimicrobial Peptide Cecropin P1 Gene

Marker-free transgenic Camelina sativa (L.) plants carrying a synthetic gene for cecropin P1, an antimicrobial peptide, under the control of the cauliflower mosaic virus 35S RNA promoter have been obtained and analyzed. The plants were transformed with an agrobacterial binary vector free of selective genes of antibiotic and herbicide resistance. The marker-free transformants were screened via measurement of the antibacterial activity of cecropin P1 and enzyme immunoassay. The obtained plants exhibited an increased resistance to infection with the bacteria Erwinia carotovora, the fungi Fusarium graminearum, and oxidative stress during infection. Analysis of the fatty acid composition of seed oil showed an increased amount of α-linolenic acid in the transgenic Camelina lines as compared to unmodified plants. The results indicate that the cecropin P1 gene can be included in an integral antistress plant-protective system.

Integrated evolutionary analysis reveals antimicrobial peptides with limited resistance

Antimicrobial peptides (AMPs) are promising antimicrobials, however, the potential of bacterial resistance is a major concern. Here we systematically study the evolution of resistance to 14 chemically diverse AMPs and 12 antibiotics in Escherichia coli. Our work indicates that evolution of resistance against certain AMPs, such as tachyplesin II and cecropin P1, is limited. Resistance level provided by point mutations and gene amplification is very low and antibiotic-resistant bacteria display no cross-resistance to these AMPs. Moreover, genomic fragments derived from a wide range of soil bacteria confer no detectable resistance against these AMPs when introduced into native host bacteria on plasmids. We have found that simple physicochemical features dictate bacterial propensity to evolve resistance against AMPs. Our work could serve as a promising source for the development of new AMP-based therapeutics less prone to resistance, a feature necessary to avoid any possible interference with our innate immune system.

A pathway-focused RT-qPCR array study on immune relevant genes in rainbow trout (Oncorhynchus mykiss) harboring cecropin P1 transgene

Recently, our laboratory had produced five families of transgenic rainbow trout harboring cecropin P1 transgene, and via repeated challenge studies these fish exhibited a significant elevation of resistance to infection by microbial pathogens. By cDNA microarray and mRNA deep sequencing (mRNA-seq) analyses on two of the five families of cecropin P1 transgenic fish, differentially expressed genes (DEGs) relevant to the innate and adaptive immune pathways in three different immune-related tissues, (i.e. spleen, kidney and liver) were profiled. These results supported our hypothesis that in addition to its direct microbicidal activity, the transgene product of cecropin P1 induces immunomodulatory activity in the transgenic host. Here, we have adapted the technique of quantitative reverse transcription real time PCR (RT-qPCR) array to analyze the expression of genes relevant to the innate and adaptive immune pathways in the rest three families. A RT-qPCR array was constructed with oligonucleotide primers of fifty-two innate/adaptive immune relevant DEGs shown to be the most perturbed by cecropin P1 transgene product in previous studies. Messenger RNA isolated from the spleen, kidney and liver of transgenic fish and non-transgenic fish control were studied on this array. Results of RT-qPCR array revealed that statistically significant perturbations of gene expression were detected in pathways of cytokine/chemokine signaling, Toll-like receptor signaling, complement cascade, antigen processing/presentation, lysosomal phagocytosis and leukocyte trans-endothelial migration in the transgenic spleen; extracellular matrix (ECM) organization and leukocyte trans-endothelial migration pathways in the transgenic kidney; lysosomal activity pathway in the transgenic liver. Furthermore, genes related to the pathways of the peroxisome proliferator-activated receptors (PPAR) signaling, lipid metabolism process and arachidonic acid metabolism were also impacted in the transgenic liver. Findings of the current study are in good agreement with those discoveries in previous two transgenic families by cDNA microarray and mRNA-seq analyses.

Получение и анализ безмаркерных масличных растений Camelina sativa (L.), экспрессирующих ген антимикробного пептида цекропина Р1

The marker-free transgenic Camelina sativa (L.) plants carrying a synthetic gene for cecropin P1, an antimicrobial peptide, under the control of the cauliflower mosaic virus 35S RNA promoter have been obtained and analyzed. The plants were transformed by the agrobacterial binary vector free of selection genes of antibiotic and herbicide resistance. Screening of the marker-free transformants was performed by measuring the antibacterial activity of cecropin P1 and by enzyme immunoassay. The obtained plants exhibited increased resistance to the infection by the Erwinia carotovora bacteria, Fusarium graminearum fungi, and to oxidative stress during infection. The analysis of the fatty acid composition of seed oil showed an increased amount of α-linolenic acid in the transgenic camelina lines as compared to unmodified plants. The obtained results indicate that the gene for cecropin P1 can be included in the integral antistress plant protective system. Camelina sativa, cecropin Р1, agrobacterium-mediated transformation, marker-free transgenic plants, fatty acid compositions, microbial phytopathogenes, oxidative stress, resistance, cecropin P1. The work was financially supported by State job № 0101-2014-0046, № РК 01201352439 and with the partial financial support the Russian Fund for Basic Investigation, Grants № 18-08-00752, № 16-04-00623.

Detergent-assisted Enhancement of the Translation Rate during Cell-free Synthesis of Peptides in an Escherichia coli Extract

The open nature of cell-free synthesis allows customization of the reaction conditions for given target molecules using diverse biological and non-biological substances. This study demonstrates that non-ionic detergents can be used to enhance translation during the synthesis of peptides in a cell-free system derived from an Escherichia coli extract. The yield of the antimicrobial peptide Cecropin P1 was markedly increased in the presence of detergents. The stimulatory effect of detergents was not limited to the Cecropin P1 peptide, but the detergent also enhanced the translation of other antimicrobial peptides. Furthermore, the enhanced translation rate by detergents was maintained for extended periods by a continuous exchange cell-free synthesis reaction, leading to production of antimicrobial peptides with markedly improved yields.

RNA-Seq analysis of differentially expressed genes relevant to innate and adaptive immunity in cecropin P1 transgenic rainbow trout (Oncorhynchus mykiss)

BackgroundIn the past years, our laboratory successfully generated transgenic rainbow trout bearing cecropin P1 transgene. These fish exhibited resistant characteristic to infection by Aeromonas salmonicida, Infectious Hematopoietic Necrosis Virus (IHNV) and Ceratomyxa shasta (a parasitic pathogen). Previously, treating rainbow trout macrophage cells (RTS-11) with cecropin B, pleurocidin and CF17, respectively, resulted in elevated expression of two pro-inflammatory genes, e.g. cyclooxygenase-2 (cox-2) and interleukin-1β (il-1β). In addition, a profiling of global gene expression by 44 k salmonid microarray analysis was conducted, and the results showed that immune relevant processes have been perturbed in cecopin P1 transgenic rainbow trout. Therefore, we hypothesized that cecropin P1 may not only eliminate pathogens directly, but also modulate the host immune systems, leading to increased resistance against pathogen infections. To confirm this hypothesis, we performed de novo mRNA deep sequencing (RNA-Seq) to analyze the transcriptomic expression profiles in three immune competent tissues of cecropin P1 transgenic rainbow trout.ResultsDe novo sequencing of mRNA of the rainbow trout spleen, liver and kidney tissues were conducted by second-generation Illumina system, followed by Trinity assembly. Tissue specific unigenes were obtained, and annotated according to the Gene Ontology (GO) and the Nucleotide Basic Local Alignment Search Tool (BLAST). Over 2000 differentially expressed genes (DEGs) were determined by normalized ratio of Reads Per Kilobase of transcript per million mapped reads (RPKM) among the transgenic and non-transgenic fish in a tissue specific manner, and there were 82 DEGs in common among the three tissues. In addition, the enrichment analysis according to Gene Ontology Biological Process (GO:BP), and Kyoto Encyclopedia of Genes and Genomes (KEGG) based pathway analysis associated with innate/adaptive immunity of fish were also performed to illustrate the altered immune-related functions in each tissue.ConclusionsAccording to the RNA-Seq data, the correlations between alteration of gene expression profiles and the functional perturbations of the host immune processes were revealed. In comparison with the results of cDNA microarray analysis conducted by Lo et al., the overall results supported our hypothesis that the gene product of cecropin P1 transgene may not only directly eliminate pathogens, but also modulate the host immune system. Results of this study present valuable genetic information for Oncorhynchus mykiss, and will benefit future studies on the immunology of this fish species.

Nucleation and growth of pores in 1,2-Dimyristoyl-sn-glycero-3-phosphocholine (DMPC) / cholesterol bilayer by antimicrobial peptides melittin, its mutants and cecropin P1.

Antimicrobial peptides are one of the most promising alternatives to antibiotics for targeting pathogens without developing resistance. In this study, pore formation in 1,2-Dimyristoyl-snglycero-3-phosphocholine (DMPC) / cholesterol liposome induced by native melittin, its two mutant variants (G1I and I17 K), and cecropin P1 was investigated by monitoring the dynamics of fluorescence dye leakage. A critical peptide concentration was required for dye leakage with the rate of leakage being dependent on peptide concentration above a critical value. A lag time was required for dye leakage for low peptide concentrations that are above the critical value, which decreased at higher peptide concentrations eventually approaching zero. Lag time was found to be in the order I17 K mutant with lower hydrophobicity and higher net charge > G1I with higher hydrophobicity > melittin > cecropin P1. Cecropin P1 exhibited the highest rate of dye leakage followed by melittin, G1I, and I17 K. Size distribution and transmission electron microscopy (TEM) of liposomes exposed to peptides of different concentrations indicated pore formation with accompanied stretching of liposomes at low peptide concentrations for both melittin and cecropin P1. At much higher concentrations, however, size distribution indicated three peaks for both peptides. In both cases, TEM images show that the middle and small peaks are shown to be due to stretched liposome and broken stretched liposome respectively. For melittin, the large peak is due to peptide aggregates as well as aggregates of liposome. For cecropin P1, however, the large peak indicates cecropin P1 aggregates with solubilized lipids thus suggesting carpet mechanism.

The Role of Outer Membrane Proteins and Lipopolysaccharides for the Sensitivity of Escherichia coli to Antimicrobial Peptides

Bacterial resistance to classical antibiotics is emerging worldwide. The number of infections caused by multidrug resistant bacteria is increasing and becoming a serious threat for human health globally. In particular, Gram-negative pathogens including multidrug resistant Escherichia coli are of serious concern being resistant to the currently available antibiotics. All Gram-negative bacteria are enclosed by an outer membrane which acts as an additional protection barrier preventing the entry of toxic compounds including antibiotics and antimicrobial peptides (AMPs). In this study we report that the outer membrane component lipopolysaccharide (LPS) plays a crucial role for the antimicrobial susceptibility of E. coli BW25113 against the cationic AMPs Cap18, Cap11, Cap11-1-18m2, melittin, indolicidin, cecropin P1, cecropin B, and the polypeptide antibiotic colistin, whereas the outer membrane protease OmpT and the lipoprotein Lpp only play a minor role for the susceptibility against cationic AMPs. Increased susceptibility toward cationic AMPs was found for LPS deficient mutants of E. coli BW25113 harboring deletions in any of the genes required for the inner part of core-oligosaccharide of the LPS, waaC, waaE, waaF, waaG, and gmhA. In addition, our study demonstrates that the antimicrobial activity of Cap18, Cap11, Cap11-1-18m2, cecropin B, and cecropin P1 is not only dependent on the inner part of the core oligosaccharide, but also on the outer part and its sugar composition. Finally, we demonstrated that the antimicrobial activity of selected Cap18 derivatives harboring amino acid substitutions in the hydrophobic interface, are non-active against wild-type E. coli ATCC29522. By deleting waaC, waaE, waaF, or waaG the antimicrobial activity of the non-active derivatives can be partially or fully restored, suggesting a very close interplay between the LPS core oligosaccharide and the specific Cap18 derivative. Summarizing, this study implicates that the nature of the outer membrane component LPS has a big impact on the antimicrobial activity of cationic AMPs against E. coli. In particular, the inner as well as the outer part of the core oligosaccharide are important elements determining the antimicrobial susceptibility of E. coli against cationic AMPs.

Synergistic effect of low power ultrasonication on antimicrobial activity of cecropin P1 against E. coli in food systems

Recent studies have shown that both low frequency (20–100 kHz) ultrasonication and antimicrobial peptides (AMPs) treatment processes have a significant advantage in inactivating bacterial cells than the conventional heat treatment due to higher food texture quality of the final product. However, the effect of the combined process has not been fully investigated in complex matrices such as food. In this study, deactivation of Escherichia coli in different concentrations of milk and orange juice were performed using three different treatments: low frequency ultrasonication (20 kHz) at different power levels, antimicrobial peptide Cecropin P1 at different concentrations, and combination of both. The results of all samples showed that the combined treatment is more efficient, reducing the cell density of E. coli up to four orders of magnitude, compared to individual treatments. However, the milk concentration results in lower synergistic effect. This is believed to be due to complexation of milk proteins with Cecropin P1 thus resulting in less availability of the latter for antimicrobial action. This dependence was not observed in orange juice samples. Ultrasonication resulted in insignificant decrease in viscosity, color and vitamin C for both milk and orange juice except at higher power level of 160 W at longer exposure time.

Antibacterial activities and molecular mechanism of amino-terminal fragments from pig nematode antimicrobial peptide CP-1.

High manufacturing costs and weak cell selectivity have limited the clinical application of naturally occurring peptides when faced with an outbreak of drug resistance. To overcome these limitations, a set of antimicrobial peptides was synthesized with the general sequence of (WL)n, where n=1, 2, 3, and WL was truncated from the N-terminus of Cecropin P1 without initial serine residues. The antimicrobial peptide WL3 exhibited stronger antimicrobial activity against both Gram-negative and Gram-positive microbes than the parental peptide CP-1. WL3 showed no hemolysis even at the highest test concentrations compared to the parental peptide CP-1. The condition sensitivity assays (salts, serum, and trypsin) demonstrated that WL3 had high stability in vitro. Fluorescence spectroscopy and electron microscopy indicated that WL3 killed microbes by means of penetrating the membrane and causing cell lysis. In a mouse model, WL3 was able to significantly reduce the bacteria load in major organs and cytokines (TNF-α, IL-6, and IL-1β) levels in serum. In summary, these findings suggest that WL3, which was modified from a natural antimicrobial peptide, has enormous potential for application as a novel antibacterial agent.

Biological Activity of Leaf Extracts from Cecropin P1-Synthesizing Kalanchoe Plants: Pharmacological Prospects

The method of producing of water extract from transgenic Kalanchoe pinnata L. plants expressing cecropin P1 gene has been developed and adapted for pharmaceutical production. Chlorophorm has been shown to be an efficient preservative for this extract. The antibiotic activity of the extract has kept well even after 1-h boiling. In addition, the prolonged antimicrobial activity of the extract has been confirmed by the “accelerated aging” method. Being added to culture medium, the leaf extract from transgenic K. pinnata plants has provided more intensive callus growth and improved rhizogenesis in ice plant (Mesembryanthemum crystallinum). The obtained results demonstrate an increased biological activity of the extract from kalanchoe plants producing antimicrobial cecropin P1 peptide and good prospects for its use in pharmacology.