Новый экспресс-метод определения активности противоопухолевого фермента L-лизин-α-оксидазы из <i>Trichoderma harzianum</i> Rifai F-180

For the first time, a new express method for the determination of the antitumor activity of L-lysine-α-oxidase obtained by cultivating the fungus Trichoderma harzianum Rifai F-180. The carcinogenic reagent, ortho-dianisidine-hydrochloride, which was present in the reaction medium, was replaced by environmentally friendly reagents of the chromogenic mixture, which included tetramethylbenzidine (TMB). This method is more accurate in determining the enzyme and has a 40-fold increased sensitivity. In addition, it provides the ability to determine enzyme activity in producer strains not only with pronounced L-lysine-α-oxidase activity, but also in those where this activity has not been previously determined.Представлен новый экспресс-метод определения активности противоопухолевого фермента L-лизин-α-оксидазы, полученного при культивировании гриба Trichoderma harzianum Rifai F-180. Содержащийся в реакционной среде канцерогенный реактив орто-дианизидингидрохлорид был заменён на экологически чистые реагенты хромогенной смеси, в которую входил тетраметилбензидин. Метод повышает точность определения фермента в 10 раз, обладает повышенной в 40 раз чувствительностью и обеспечивает возможность определения активности фермента не только у штаммов-продуцентов с выраженной активностью L-лизин-a-оксидазы, но и с неопределённой активностью

A Novel Express Method to Determine Activity of Antitumor Enzyme L-Lysine-α-Oxidase of Trichoderma harzianum Rifai F-180

A novel express method is developed to determine activity of antitumor enzyme L-lysine-α-oxidase obtained by culturing Trichoderma harzianum Rifai F-180 fungus. The carcinogenic reagent ortho-dianisidine-hydrochloride was replaced in the reaction medium with environmentally friendly reagents of the chromogenic mixture that included tetramethylbenzidine. This method improved precision and sensitivity of ELISA by 10 and 40 times, respectively. In addition, it could detect activity of L-lysine-α-oxidase not only in the producer strains with a pronounced activity of this enzyme, but also in the strains where this activity has not been previously determined. © 2020, Springer Science+Business Media, LLC, part of Springer Nature

Modulating the Antifungal Activity of Antimycotic Drugs with Farnesol

Introduction. Clinical strains of microorganisms, including opportunistic yeast-like fungi (YLF) of the genus Candida, are resistant to currently used antifungal drugs. In this regard, the search for alternative ways to potentiate the activity of antimicrobial agents in relation to the infectious agent is an important and relevant area of research. The study of combinations of existing antimycotic drugs and a medicinal extract of plant origin – farnesol – is one of the promising approaches in the fight against resistant strains of YLF genus Candida. In our previous studies, farnesol has been shown to exhibit relative activity against YLF Candida albicans biofilms. In this study, we used 6 clinical isolates and one museum strain YLF C. albicans to study the effect of farnesol on the antifungal activity of antimycotic drugs. Aim. To prove that farnesol can increase the antifungal activity of certain antimycotics. Materials and methods. To determine the sensitivity of 7 strains of YLF C. albicans to the antimycotic drugs "Nystatin" (NYS 50 µg), "Ketoconazole" (KET 10 µg), "Clotrimazole" (CTR 10 µg), "Amphotericin B" (AMB 10 µg), "Voriconazole" (VRC 10 µg) disk diffusion test was used. A solution of farnesol in concentrations of 100, 50 and 25 µM in a volume of 25 µl was applied to the disk with the antimycotic drug. Sterile physiological (PhS) solution was used as a control (pH 7.0; V = 25 µl). Results and discussion. In 34.3 % of of experiments we can talk about the modulating effect of farnesol solutions on the antifungal activity of antimycotic drugs. In all these cases, the sensitivity of YLF C. albicans to the antimycotic drug increases. Conclusion. The results of this study provide useful information for understanding the mechanism of QS-molecules action with antifungal activity, as well as they are the basis for the practical application of some QS-molecules in the treatment of infectious diseases caused by YLF of the genus Candida. The study demonstrates that farnesol can be recommended as an active substance that improves the sensitivity of YLF Candida to antimycotic drugs, especially in the case of multi-resistant strains Candida. © 2021, Center of Pharmaceutical Analytics. All rights reserved

Evaluation of Changes Induced in the Probiotic Escherichia coli M17 Following Recurrent Exposure to Antimicrobials

Introduction: It is already well known that the exposure of certain bacteria, pathogenic or not, to antimicrobials is likely to increase their virulence and induce the development of direct or cross resistance to antimicrobials, but there is almost no information available regarding probiotics. Aim: To assess the changes induced in susceptibility to antibiotics, biofilm formation, growth rate and relative pathogenicity in the probiotic Escherichia coli M17 (EC-M17) after long exposure to antimicrobials namely ampicillin, kanamycin, cefazolin and silver nanoparticles (AgNPs). Methods: After determining the minimum inhibitory concentrations (MIC) and minimum bactericidal concentrations (MBC) of the 4 antimicrobials above-mentioned by the microdilution method, EC-M17 was exposed to increasing subinhibitory doses ranging from MIC/8 to MIC for 8 days. The susceptibility to antibiotics of the mutants obtained was assessed by the Kirby Bauer disc diffusion method, biofilm formation by the Congo red agar method and with crystal violet bacterial attachment assay, and relative pathogenicity was assessed using a Galleria melonella waxworm model. Results: Exposure to antimicrobials induces noticeable changes in EC-M17. The highest adaptation to antimicrobials was observed on AgNPs with 8-fold increase in MIC and 16-fold increase in MBC of AgNPs. EC-M17 exposed to ampicillin, kanamycin and silver nanoparticles became resistant to ampicillin, ceftazidime, ceftazidime/clavulanate and tetracycline while exposure to cefazolin induced a significant decrease in sensitivity to tetracycline and ampicillin and resistance to ceftazidime/clavulanate and ceftazidime. The strain exposed to ampicillin was the only one to produce more biofilm than the control strain and except the EC-M17 exposed to cefazolin, all other EC-M17 strains were more pathogenic on G. melonella model than the control. Conclusion: Data in this investigation suggest that repeated exposure of the probiotic EC-M17 to antimicrobials may induce changes in antimicrobials susceptibility, biofilm formation, growth rate, and relative pathogenicity. Therefore, as far as possible, the probiotic E. coli M17 should not be used in combination with antibiotics and further investigations are required to expand similar work on more probiotics in order to avoid resistance build-up which might be transmitted by horizontal transfer

Prevotella rara sp. nov., isolated from human faeces

A strain of obligately anaerobic, Gram-stain-negative rods was isolated from human faeces and characterized both phenotypically and genotypically. Phylogenetic analysis based on 16S rRNA gene and whole-genome sequences revealed the strain to represent a member of the genus Prevotella, distant from the species with validly published names, with the closest relationship to Prevotella oryzae. The strain was moderately saccharolytic and proteolytic. The predominant menaquinones were MK-13 and MK-12. The major cellular long-chain fatty acids were anteiso-C15 : 0 and iso-C15 : 0. The genomic DNA G+C content was 45.7 mol%. On the basis of chemotaxonomic and genotypic properties, it was concluded that the strain represent a novel species within the genus Prevotella, for which the name Prevotellarara sp. nov. is proposed. The type strain of Prevotellarara is 109T (=VKM B-2992T=DSM 105141T)

Definitions of antibodies to the antitumor enzyme l-lysine-α-oxidase, the study of its immunogenic properties and allergenic activity [Definiciones de anticuerpos contra la enzima antitumoral l-lisina-α-oxidasa, estudio de sus propiedades inmunogénicas y actividad alergénica]

he determination of antibodies to the antitumor enzyme L-lysine-α-oxidase Trichoderma har-zianum Rifai F-180 was studied using enzyme-linked immunosorbent assay. It was shown that when testing the enzyme in mice and guinea pigs at a dose of 35 U / kg, the antitumor enzyme L-lysine-α-oxidase has low immunogenicity. Antibodies not only reduce catalytic activity, but also affect the affinity of the enzyme with the substrate. Guinea pigs showed that native and modified L-lysine-α-oxidase at a dose of 35 U / kg in vivo and in vitro has a weak allergenic activity. © 2021, Venezuelan Society of Pharmacology and Clinical and Therapeutic Pharmacology. All rights reserved

Characterization of L-Lysine-Α-Oxidase, A New Antitumor And Antiviral Drug Substance Synthesized By Trichoderma

The trigger to the studies of L-lysine-α-oxidase is of great interest for the scientists across the world. Regarding Russian Federation, the enzyme is under examination by several laboratories. Attracted by the polyfunctionality of the enzyme, the likelihood of utilizing this substance in the creation of multiple dosage forms of different therapeutic orientation. Thus, the main aim of the study is to analyze the new characteristics of L-lysine-α-oxidase as a new antitumor and antiviral drug substance synthesized by Trichoderma. To meet the study's aim, a biological experiments were carried out on guinea pigs weighing 200-250 g and CBA mice (CBA x C57BI) weighing 18-20 g, C57 BI, SHR.Native L-lysine-α-oxidase was injected into C57BI mice five times intravenously at a dose of 35 U/kg. Blood was sampled every week for four weeks from the beginning of immunization (n=7), and the obtained sera of mice were analyzed by enzyme-linked immunosorbent assay according to a previously developed method. The sera were titrated in increments of 2. Tests of L-lysine-α-oxidase in mice and guinea pigs found low immunogenicity of L-lysine-α-oxidase when administered in mice at a dose of 35 U/kg, or 0.8 ml of protein/kg. Based on the results obtained, antibodies not only reduce its catalytic activity but also affect the affinity of the enzyme with the substrate. Experiments on guinea pigs have shown that native and modified L-lysine-α-oxidase at a dose of 35 U/kg in vivo and in vitro has a low allergenic activity. The allergenic activity of L-lysine-α-oxidase was investigated in comparison with other drugs. The results od this article, can be considered as one of a brand-new and effective methods for assessing the body sensitization caused by the use of enzymes and can contribute to the development of the respective field

New Trichoderma harzianum Rifai F-180 - L-lysine-α-oxidase antitumor enzyme producer - culture liquid-based substance biotechnology

The biosynthesis of L-lysine-α-oxidase from Trichoderma harzianum Rifai F-180, an antitumor enzyme producer, was carried out on a wheat bran medium. Hydrophilic bases of lightly crosslinked acrylic polymers and methylcellulose derivatives were selected, and model samples were prepared with a 1% concentration of the culture liquid of the fungus. The wound healing activity of the obtained gel samples was investigated on an experimental model of guinea pig wounds. The results of experimental studies showed a high wound healing activity of the gel with a 1% concentration of the culture liquid of the fungus and can serve as a basis for further preclinical trials of this dosage form of wound healing action. © 2020 EManuscript Technologies. All rights reserved