"Green" Synthesis of Cytotoxic Silver Nanoparticles Based on Secondary Metabolites of Lavandula Angustifolia Mill.

In this study, we used "green" synthesis to prepare silver nanoparticles (NPs) from aqueous plant and callus extracts of the narrow-leaved lavender Lavandula angustifolia Mill. 35.4 ± 1.6 nm and 56.4 ± 2.4 nm nanoparticles, colloidally stable in phosphate-buffered saline, were synthesized using the plant extract and the callus extract, respectively. NPs were characterized by spectrophotometry, dynamic light scattering, and scanning electron microscopy. We studied the dynamics of the nanoparticle synthesis and evaluated the cytotoxic properties of the plant extract-based NPs. Modification of NPs with bovine serum albumin demonstrated that blockage of the nanoparticle surface completely suppressed NP cytotoxic activity in vitro. The synthesized NPs possess localized surface plasmon resonance properties and are of small sizes, and their surface can be modified with protein molecules, which makes them promising agents for cancer theranostics.

Diversity of Harpin-Like Defense Peptides from Barnyard Grass (Echinochloa crusgalli L.) Seeds.

The results of the study of the structure and function of harpin-like peptides (alpha-harpinins) of the EcAMP group from the barnyard grass (E. crusgalli) seeds and the possibility of their involvement in the innate immunity to biotic stresses are presented.

A Low-Molecular-Weight Compound Derived from Human Leukocytes Determines a Bactericidal Activity of the Interferon Preparation.

The aim of this study was to characterize the structure and mode of action of antimicrobials derived from a commercial preparation of alfa-interferon. By combination of semi-preparative/analytical reversed-phase high-performance liquid chromatography, we isolated and purified a novel active substance based on carbohydrate with a complex of amino acids, which determines antimicrobial property of commercial preparation of interferon. A size-exclusion chromatography was performed and LC/ESI-MS revealed molecular masses of active substance were in the range of 180-249 Da. Edman sequencing identified phenylthiohydantoin (PTH) derivatives which consisted a set of preliminary (Asp, Glu, Gly, and Ala) and minor amino acids (Leu and Thr) at equimolar ratio. Thus, the purified active substance is a compound containing the complex of amino acids connected with carbohydrate background and called leucidin. Leucidin demonstrated antimicrobial activity against the model Escherichia coli (E. coli) K12 strain at a minimal inhibitory concentration of 20 µg mL-1. The revealed antimicrobial mechanism of action is associated with violation of the bacterial cell wall leading to a SOS response and bacterial autolysis. Despite the preliminary nature of the results, obtained data allowed us to discover the previously unknown leukocyte-derived antimicrobial molecules.

A Novel High-Molecular-Mass Bacteriocin Produced by Enterococcus faecium: Biochemical Features and Mode of Action.

Discovery of a novel bacteriocin is always an event in sciences, since cultivation of most bacterial species is a general problem in microbiology. This statement is reflected by the fact that number of bacteriocins is smaller for tenfold comparing to known antimicrobial peptides. We cultivated Enterococcus faecium on simplified medium to reduce amount of purification steps. This approach allows to purify the novel heavy weight bacteriocin produced by E. faecium ICIS 7. The novelty of this bacteriocin, named enterocin-7, was confirmed by N-terminal sequencing and by comparing the structural-functional properties with available data. Purified enterocin-7 is characterized by a sequence of amino acid residues having no homology in UniProt/SwissProt/TrEMBL databases: NH2 - Asp - Ala - His - Leu - Ser - Glu - Val - Ala - Glu - Arg - Phe - Glu - Asp - Leu - Gly. Isolated thermostable protein has a molecular mass of 65 kDa, which allows it to be classified into class III in bacteriocin classification schemes. Enterocin-7 displayed a broad spectrum of activity against some Gram-positive and Gram-negative microorganisms. Fluorescent microscopy and spectroscopy showed the permeabilizing mechanism of the action of enterocin-7, which is realized within a few minutes.

Antimicrobial activity of the indolicidin-derived novel synthetic peptide In-58.

Natural peptides with antimicrobial activity are extremely diverse, and peptide synthesis technologies make it possible to significantly improve their properties for specific tasks. Here, we investigate the biological properties of the natural peptide indolicidin and the indolicidin-derived novel synthetic peptide In-58. In-58 was generated by replacing all tryptophan residues on phenylalanine in D-configuration; the α-amino group in the main chain also was modified by unsaturated fatty acid. Compared with indolicidin, In-58 is more bactericidal, more resistant to proteinase K, and less toxic to mammalian cells. Using molecular physics approaches, we characterized the action of In-58 on bacterial cells at the cellular level. Also, we have found that studied peptides damage bacterial membranes. Using the Escherichia coli luminescent biosensor strain MG1655 (pcolD'::lux), we investigated the action of indolicidin and In-58 at the subcellular level. At subinhibitory concentrations, indolicidin and In-58 induced an SOS response. Our data suggest that indolicidin damages the DNA, but bacterial membrane perturbation is its principal mode of action. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.

Derivatization of Aminoglycoside Antibiotics with Tris(2,6-dimethoxyphenyl)carbenium Ion.

Detection of aminoglycoside antibiotics by MS or HPLC is complicated, because a) carbohydrate molecules have low ionization ability in comparison with other organic molecules (particularly in MALDI-MS), and b) the lack of aromatics and/or amide bonds in the molecules makes common HPLC UV-detectors useless. Here, we report on the application of a previously developed method for amine derivatization with tris(2,6- dimethoxyphenyl)carbenium ion to selective modification of aminoglycoside antibiotics. Only amino groups bound to primary carbons get modified. The attached aromatic residue carries a permanent positive charge. This makes it easy to detect aminoglycoside antibiotics by MS-methods and HPLC, both as individual compounds and in mixtures.

Novel haemoglobin-derived antimicrobial peptides from chicken (Gallus gallus) blood: purification, structural aspects and biological activity.

AIM: To purify and characterize antimicrobial peptides derived from the acid extract of Gallus gallus blood cells. METHODS AND RESULTS: Two polypeptides (i.e. CHb-1 and CHb-2) with antibacterial activity were detected in the acidic extract of blood cells from chicken (G. gallus). The isolated peptides that possessed a potent antibacterial activity were purified using a two-step chromatography procedure that involved solid-phase extraction of a total protein/peptide extract followed by thin fractionation by reversed-phase high performance liquid chromatography (RP-HPLC). The molecular masses of the purified peptides were similar and were 4824·4 and 4825·2 Da, which have been measured by matrix-assisted laser desorption/ionization mass spectrometry (MALDI TOF MS). Their amino acid sequences were determined by Edman degradation and showed that the peptides were fully identical to the two fragments of G. gallus α-haemoglobin localized into different subunits (A and D respectively). The peptides were active in micromolar concentrations against Gram-negative Escherichia coli K12 TG1. Using the 1-N-phenylnaphthylamine, the FITC-dextran labelled probes and the live/dead staining allowed to show the hemocidin mode of action and estimate the pore size. CONCLUSION: In this study, for the first time, α-haemoglobin from chicken (G. gallus) has been investigated as a donor of the two high homologous native peptide fragments that possess potent antibacterial activity in vitro. These are membrane-active peptides and their mechanism of action against E. coli involves a toroidal pore formation. SIGNIFICANCE AND IMPACT OF THE STUDY: The obtained results expand the perception of the role of haemoglobin in a living system, describing it as a source of multifunction substances. Additionally, the data presented in this paper may contribute to the development of new, cost-effective, antimicrobial agents.

[BIOLOGICAL ACTIVITY OF ANTIMICROBIAL PEPTIDES FROM CHICKENS THROMBOCYTES].

AIM: Isolation and study of biological activity of antimicrobial peptides from chickens thrombocytes. MATERIALS AND METHODS: Peptides from chickens thrombocytes, obtained by reverse-phase high-performance liquid chromatography method with stepped and linear gradients of concentration increase of the organic solvent were used in the study. Their antimicrobial activity was determined by microtitration method in broth; mechanism of biological effect--by using fluorescent spectroscopy method with DNA-tropic dyes. RESULTS: Individual fractions of peptides were isolated from chickens thrombocytes, that possess antimicrobial activity against Staphylococcus aureus P209 and Escherichia coli K12. A disruption of integrity of barrier structures of microorganisms under the effect of thrombocyte antimicrobial peptides and predominance of cells with damaged membrane in the population of E. coli was established. CONCLUSION: The data obtained on antimicrobial activity and mechanism of bactericidal effect of the peptide fractions from chickens thrombocytes isolated for the first time expand the understanding of functional properties of chickens thrombocytes and open a perspective for their further study with the aim of use as antimicrobial means.

Investigation of Thionins from Black Cumin (Nigella sativa L.) Seeds Showing Cytotoxic, Regulatory and Antifungal Activity.

Thionins (NsW1 and NsW2), earlier isolated from the seeds of endemic Middle-Asian black cumin (Aligella sativa L.), showing signilicant inhibitory action on some bacterial and yeast pathogens were investigated for cytotoxic properties against several tumor cell lines (AsPC-1, Colo357, RD and Jukart) in vitro within nano- and micromolar ranges of the active concentrations and as modulators of expression of the genes controlling conversion of normal cells to malignant ones. Suppression of the expression of the genes from MMP, RhoA, miR21 families in human rhabdomyosarcoma (RD) cells was observed, whereas the influence of the molecules on the genes in normal blood cells was not identified. It was shown that the thionins from black cumin induced almost 90% of the cell death in RD and Jukart lines. Moreover, the polypeptides inhibited clinical isolates of Aspergillus ochraceus and A.fimigatus at the level comparable with that of amphotericin B. The data demonstrated that the peptides could be considered as perspective antitumor and antimycotic agents.

Antibiotic Activity of Probiotic Strain Bacillus subtilis 534 Against Clinical Isolates of Acinetobacter baumannii.

Probiotic strain Bacillus subtilis 534 is the base of sporobacterin, a pharmaceutical. In submerged culture it showed antibiotic activity against many of gram-positive and gram-negative bacteria and fungi. The spectrum of the antimicrobial activity of the culture fluid depended on the.cultivation time and aeration intensity. It was shown that component No. 1 of the antibiotic complex was effective against clinical isolates of Acinetobacter baumannii: 20 out of 24 isolates were susceptible to component No. 1, including 15 strains out of 16 panresistant isolates.

[Genes encoding hevein-like antimicrobial peptides WAMPs: Expression in response to phytohormones and environmental factors].

We investigated the role of genes of hevein-like antimicrobial peptides of the WAMP family in the protection of wheat plants against biotic and abiotic stress. The semiquantitative RT-PCR method was used to examine the expression of wamp genes in wheat seedlings in response to infection by pathogens and exposure to phytohormones and ions of a heavy metal ion­cadmium. We discovered that wheat germ contamination by harmful fungi significantly increases expression of genes of the wamp family, and the primary transcript is wamp-2. We determined that salicylic acid, rather than methyl jasmonate, induces expression of genes of the wamp family. We showed that abiotic stress induced by cadmium ions inhibits expression of wamp genes in the roots with no effect on their expression in shoots. The results support the protective role of wamp genes in the response of wheat plants to infections by pathogens. In turn, the resistance to abiotic stress induced by cadmium ions does not appear to be associated with expression of genes of the wamp family.

Characterization of Extracellular Yeast Peptide Factors and Their Stress-Protective Effect on Probiotic Lactic Acid Bacteria.

Protective effect of the extracellular peptide fraction (reactivating factors, RF) produced by yeasts of various taxonomic groups (Saccharomyces cerevisiae, Kluyveromyces lactis, Candida utilis, and Yarrowia li- polytica) on probiotic lactic acid bacteria (LAB) Lactobacillus casei, L. acidophilus,'and L. reuteri under bile salt (BS)-induced stress was shown. RF of all yeasts were shown to be of peptide nature; the active component of the S. cerevisiae RF was identified as a combination of low-molecular polypeptides with molecular masses of 0.6 to 1.5 kDa. The protective and reactivating effects of the yeast factors were not species-specific and were similar to those of the Luteococcusjaponicus subsp. casei R. In BS-treated cells of the tester bacteria, a pro- tective effect was observed after 10-min preincubation of the LAB cell suspension with yeast RE: the number of surviving cells (CFU) was 2 to 4.5 times higher than in the control. The reactivating effect was observed when RF was added to LAB cell suspensions not later than 15 min after stress treatment. It was less pro- nounced than the protector effect, with the CFU number I to 3 times that of the control. Both the protector and the reactivating effects were most pronounced in the S. cerevisiae and decreased in the row: C. utilis > K. lactis > Y lipolytica. The efficiency of protective action of yeast RF was found to depend on the properties of recepient LAB cells, with the L. casei strain being most sensitive to BS treatment. In both variants, the highest protective effect of RF (increase in the CFU number) was observed for L. acidophilus, while the least pronounced one, for L. casei. The reasons for application of the LAB strains combining high stress resistance and high response to stress-protecting metabolites, including RF factors, as probiotics, is discussed.

[BIOLOGICAL ACTIVITY OF ANTIMICROBIAL PEPTIDES OF ENTEROCOCCUS FAECIUM].

AIM: Isolate bacteriocins from Enterococcus faecium metabolites and characterize their effect on cells of Gram positive (Listeria monocytogenes) and Gram negative (Escherichia coli) bacteria. MATERIALS AND METHODS: Methods of solid-phase extraction, ion-exchange and reversed phase chromatography were applied for isolation of bacteriocins from cultural medium of bacteria MALDI time-of-flight mass-spectrometry was used for characterization of the obtained preparations. The mechanism of biological effect of peptides was evaluated using DNA-tropic dyes (SYTO 9 and PI) with subsequent registration of fluorescence spectra: Atomic-force microscopy (AFM) was used for characterization of morpho-functional reaction of target cells. RESULTS: Peptide fractions with mass of 1.0 - 3.0 kDa were isolated from enterococci metabolites, that inhibit the growth of indicator microorganisms. E. faecium strain exoproducts were shown to increase membrane permeability during interaction with L. monocytogenes, that results in subsequent detectable disturbance of normal cell morphology of listeria. Alterations of E. coli surface during the effect of purified peptide fraction was detected using AFM. CONCLUSION: The studies carried out have revealed the effect of bacteriocins of enterococci on microorganisms with various types of cell wall composition and have confirmed the importance of bacterial barrier structure permeability disturbance in the mechanism of antimicrobial effect of enterocins.

[Antimicrobial Activity of Fungi Strains of Trichoderma from Middle Siberia].

The antibiotic activity in 42 strains of 8 species of the Trichoderma genus (T. asperellum, T. viride, T. hamatum, T. koningii, T. atroviride, T. harzianum, T. Citrinoviride, and T. longibrachiatum) isolated from different Siberian ecotops was studied. It was shown that these species differ in the degree of their antibacterial and antifungal activity. The chosen strain, T. citrinoviride TV4-1, exhibited high activity and a wide range of actions against the opportunistic and pathogenic fungi of the Aspergillus and Candida albicans genus; bacteria, including methicillin-resistant Staphylococcus aureus; and cancer cells. According to mass and I R spectrometry data and the spectrum of biological action, peptaibols are probably the most active compounds in the strain culture extracts.

[Comparative analysis of natural and synthetic antimutagens as regulators of gene expression in human cells under exposure to ionizing radiation].

This paper studies the effect of plant peptides of thionine Ns-W2 extracted from seeds of fennel flower (Nigella sativa) and ß-purothionine from wheat germs (Triticum kiharae), as well as a synthetic antimutagen (crown-compound), on the expression of several genes involved in the.control of cellular homeostasis, processes of carcinogenesis, and radiation response in human rhabdomyosarcoma cells (RD cells), T-lymphoblastoid cell line Jurkat, and blood cells. All of these agents acted as antimutagens-anticarcinogens, reducing the expression of genes involved in carcinogenesis (genes of families MMP, TIMP, and IAP and G-protein genes) in a tumor cell. A pronounced reduction in the mRNA level of these genes was caused by thionine Ns-W2, and the least effect was demonstrated by ß-purothionine. Antimutagens had very little effect on the mRNA levels of the several studied genes in normal blood cells.

[Reactivating factor of Luteococcus japonicus subsp. casei: isolation and characterization].

It has been shown that a producer strain of reactivating factor (RF) is identical to a typical strain of Luteococcus japonicus DSM 10546 from the Propionibacteriaceae family according to the physiological and biochemical properties and the sequencing of 16S rRNA fragments. A number of phenotypical differences from the model strain allowed the producer strain to be considered a subspecies of Luteococcus japonicus, and it was named Luteococcus japonicus subsp. casei. At cultivation of the producer, RF is secreted into the medium and plays the role of a signaling molecule. RF antioxidant activities towards various organic radicals may be a possible mechanism of its protective and reactivating effects. Metabolites secreted by the L. casei producer strain into the culture medium were separated by a combination of liquid chromatographies. Four components possessing biological activities were found. The most active one was studied by MALDI-TOF mass spectrometry, which revealed that it is a polypeptide. Primary identification of some amino acid residues was performed. Sugar residues were found in the structure.

[Antimicrobial Activity of Substances Produced by Trichoderma citrinoviride Strain VKPM F-1228: Optimization of Cultivation and Assessment of Spectrum of Individual Peptaibols].

The Trichoderma citrinoviride VKPM F-1228 strain produces a complex of peptide-based antibiotics with antibacterial and antimycotic action. Synthesis of peptaibols is closely related to the conidiogenesis in the culture. The optimal procedure of the strain cultivation for production of peptaibols is stationary growing for 14 days at a temperature of 28 degrees C and pH 7.5 followed by formation of a dense mycelium film on the modified Saburo medium containing 30 gr/l of glucose and 12.5 gr/l of peptone. Eight individual peptaibols were extracted. The spectrum of their activity was estimated with the use of opportunistic bacteria and micromycetes as well as pathogenic clinical aspergilli. Compounds 9, 13, 14, 15 and 16 were shown active against opportunistic fungi and bacteria including methicillin resistant S. aureus, whereas compounds 9, 13 and 14 in addition showed antimycotic activity against clinical aspergilli.

Defense peptides from barnyard grass (Echinochloa crusgalli L.) seeds.

A number of defense polypeptides from latent seeds of weed cereal barnyard grass (Echinochloa crusgalli L.) has been isolated and characterized using an acidic extraction and high performance liquid chromatography methods in combination with MALDI-TOF mass spectrometry and Edman sequencing. Members of three antimicrobial peptide families and two protease inhibitor families were found to be localized in barnyard grass seeds. Their biological activity concerning to Gram-Positive and Gram-Negative phytopathogenic bacteria, as well as oomycete Phytophthora infestans, has been investigated. Diversity of barnyard grass defense peptides is a significant factor that provides a resistance of E. crusgalli seeds to germination and latent phases.