A staphylolytic fusion protein (chimeric enzyme K-L) was created, harboring three unique lytic activities composed of the LysK CHAP endopeptidase, and amidase domains, and the lysostaphin glycyl-glycine endopeptidase domain. To assess the potential of possible therapeutic applications, the kinetic behavior of chimeric enzyme K-L was investigated. As a protein antimicrobial, with potential antigenic properties, the biophysical effect of including chimeric enzyme K-L in anionic polymer matrices that might help reduce the immunogenicity of the enzyme was tested. Chimeric enzyme K-L reveals a high lytic activity under the following optimal () conditions: pH 6.0-10.0, t 20-30 °C, NaCl 400-800 mM. At the working temperature of 37 °C, chimeric enzyme K-L is inactivated by a monomolecular mechanism and possesses a high half-inactivation time of 12.7 ± 3.0 h. At storage temperatures of 22 and 4 °C, a complex mechanism (combination of monomolecular and bimolecular mechanisms) is involved in the chimeric enzyme K-L inactivation. The optimal storage conditions under which the enzyme retains 100 % activity after 140 days of incubation (4 °C, the enzyme concentration of 0.8 mg/mL, pH 6.0 or 7.5) were established. Chimeric enzyme K-L is included in complexes with block-copolymers of poly-L-glutamic acid and polyethylene glycol, while the enzyme activity and stability are retained, thus suggesting methods to improve the application of this fusion as an effective antimicrobial agent.
Location of phosphodiester bonds essential for ammoacylation of bovine tRNATn' was identified using a randomly cleaved transcript synthesized in vitro. It was found that cleavage of phosphodiester bonds after nucleotides in positions 21. 22, 36-38. 57-59, 62 and 64 were critical for aminoacylation capacity of tRNA'IP -transcript. These cleavage sites were located in the regions of tRNA molecule protected by the cognate synthetase against chemical modification and m the regions presumably outside the contact area as well. These results indicate that for mamtenance of aminoacylation ability the intactness of the certain regions of the tRNA backbone structure is necessary. Random splitting of non-modified RNA with alkali followed by separation of active and inactive molecules and identification of cleavage sites developed m this work may become a general approach for studying the role of RNA covalent structure in its interaction with proteins.
The problems of bacterial infections in medicine and veterinary medicine require careful study and rapid solution. Due to continuous and, in some cases, irrational use of antibiotics, the efficiency of their effect on host has been noticeably decreasing; moreover, resistance to antibacterial drugs is steadily growing, antibiotic-resistant strains emerge, which are not amenable to conventional medical treatment. The unprecedented rise of pathogenic bacteria resistance to antibiotics requires generation of new drugs to combat them. One of the ways to increase the effectiveness of antibiotic therapy is to use combination drugs. Combination dosage forms provide an increased therapeutic effect and should not be toxic to the body. To overcome the microbial resistance reducing host burden of antibiotics, we proposed a combination preparation based on antibiotic, cluster silver and specific bacteriophage for treatment of infectious diseases caused by S. aureus, including MRSA strains. Each component has already proven in the treatment of pathogen-caused infectious diseases. But while using this combination agent, it became possible to reduce the amount of antibiotic and get rid of antibiotic-resistant and phage-resistant forms of bacteria. The study showed the effectiveness of the combination preparation on S.aureus MRSA bacteria, while reducing the amount of antibiotics in proposed composition by 2-4 times compared to use of antibiotic alone. The efficacy of the preparation containing was as follows: 10mcg/ml gentamicin, 7mcg/ml cluster silver and 106 bov/ml bacteriophage, on S.acureus MRSA bacteria in suspension is comparable to the effectiveness of gentamicin with a concentration of 40mcg/ml.
For S. aureus 209 and S. aureus ssp. bacteria with reduced resistance to antibiotics, the use of two-component compositions (antibiotic + cluster silver; antibiotic + bacteriophage) also allowed to reduce the antibiotic concentration by 2-3 times. It should be noted that with a constant amount of antibiotic, increasing the amount of cluster silver by 4-5 times, there is a rise in bactericidal properties of the resulting preparations. Repeated introduction of drug dose allows to achieve a marked decrease in level of pathogenic microorganisms in the suspension studied. This drug has no limitations depending on the state and degree of microbial resistance.
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