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The study of antimicrobial compounds of animal origin, particularly antimicrobial peptides (AMPs), is a current research topic. However, extracting endogenous AMPs is a challenging process and requires the application of targeted enzymatic processing principles based on knowledge of the structure of prepropeptide molecules — precursors of AMPs. In this study, a search was conducted for antimicrobial peptides present in Sus scrofa and Bos taurus organisms, as well as their precursors, using The Antimicrobial Peptide Database and UniProtKB databases. In the amino acid sequences of prepropeptides, the sequences of the mature peptides were found, and cleavage sites for trypsin, bacterial collagenase (type I), and neutrophil elastase were determined. As a result of the search for antimicrobial compounds in The Antimicrobial Peptide Database, 18 antimicrobial peptides from Sus scrofa and 40 antimicrobial peptides from Bos taurus were identified. Based on the results of determining cleavage sites in AMP precursors, enzymes were ranked from less preferred to more preferred for AMP release as follows: bacterial collagenase (type I) ≤ trypsin < neutrophil elastase. This order is justified not only by the number of suitable cleavage sites and their accuracy but also by the action of enzymes within mature AMPs: it is important to consider that enzymes can “cut” the peptides themselves, thereby reducing their antimicrobial activity. The bioinformatics analysis conducted is applicable for both primary screening of raw material potential and determining of suitable enzymes for extracting antimicrobial compounds from Sus scrofa and Bos taurus organisms.

Introduction. The manufacturing and application of enzyme preparations in the semisolid topical products has features associated with their lability. In previous studies, on the set of specifications the gel form was selected, containing aerosil and olive oil, in which the stabilization of collagenase from Paralithodes camtschaticus was ensured by lipophilic base. In the pharmaceutical development process, the drug critical quality attributes must be defined. Taking into account the lability of Paralithodes camtschaticus collagenase, the identification and minimizing risks аre relevant.Aim. The aim was the risk factors assessment and minimization during the pharmaceutical development of the gel, containing Paralithodes camtschaticus collagenase within the QbD concept.Materials and methods. The laboratory samples of gels, containing Paralithodes camtschaticus collagenase, were tested, using the analysis methods recommended by the SP RF 14th ed., as well as GOST 29188.3-91.Results and discussion. Based on the potential critical characteristics of the material, the pharmaceutical substance Paralithodes camtschaticus collagenase (particle size) and excipients (concentration and sorption properties of aerosil, oxidation of olive oil) risk factors were assessed, and potential measures to reduce these risks are considered. It was revealed that the substance particle size in its origin exceeds the norm and requires preliminary grinding. The aerosil content factor was reveal as critical. The sorption properties of aerosil were not affected the proteolytic and collagenolytic activity of the collgenase gel. It was proved that chemical instability of oil refers to critical factors and the antioxidants (alpha-tocopherol or butylhydroxytoluene) content was justified.Conclusion. In the course of the study, the Paralithodes camtschaticus collagenase gel the possible risks of pharmaceutical substances and excipients were theoretically justified in accordance with the QbD concept. An experimental assessment of their possible impact on the gel quality was given, which can improve the finished product quality during manufacturing manufacturing by eliminating the identified risks.

Purpose of the study. Testing of new chemotherapeutic agents in translational and biology medicine needs studies on immortalized cell lines. However, such models do not always have the biological properties of a tumor in situ, in contrast to primary cell cultures. Primary cultures of lung cancer cells have biological, morphological and molecular characteristics close or identical to tumor cells in vivo. Obtaining collections of primary lung cancer cell lines is an important task in creating various models for preclinical studies.Materials and methods. The materials are represented by postoperative tumor samples obtained from 25 patients with newly diagnosed lung cancer without prior treatment. The following methods were used to obtain primary cultures: enzymatic dissociation in Hanks' solution with the addition of 300 units/ml collagenase I (Thermo Fisher Scientific, USA), enzymatic dissociation using the Brain Tumor Dissoсiation Kit (Miltenyi Biotec, Germany) and 150 units/ml. ml of collagenase I, as well as the method of explants. The following methods were used to remove fibroblasts: the use of the FibrOut™ system (CHI Scientific, USA), magnetic separation of fibroblasts using Anti-Fibroblast MicroBeads (Miltenyi Biotec, Germany), and cold trypsinization.Results. We have obtained 15 primary lung cancer cell cultures that have passed the zero order passage. In this work, the method of enzymatic dissociation turned out to be the most effective. Incubation of lung tumor samples with collagenase for 1 hour preserves the viability and adhesiveness of the cells. The explant method did not show its effectiveness for long-term cultivation, there was no migration of tumor cells to plastic. Magnetic separation, as a method of removing stromal components of fibroblasts, showed the greatest efficiency, while maintaining the viability of tumor cells.Conclusion. The obtained primary cell cultures of lung cancer can be used for many tasks of experimental oncology: studies of the biological characteristics of lung cancer, development of preclinical models for the studies on new chemotherapeutic drugs.

The success of pancreatic islet allotransplantation in the treatment of patients with a difficult-to-manage type 1 diabetes depends mainly on the quantity and quality of islets isolated from the pancreas of deceased donors using enzyme preparations, primarily collagenase. Numerous studies on improvement and standardization of islet isolation techniques have reached their limits in the last decade. This has made it impossible to further boost the number and quality of clinical transplants. Taking into account the negative impact of collagenase technique on the morphofunctional properties of isolated islets, this work has studied the possibility of enzyme-free isolation of islet tissue purified of exocrine ballast. Experiments using the pancreas of newborn and young rabbits showed that developing methodological approaches to obtaining islet-like cultures without the use of exogenous enzymes is feasible.

Domestic and international experience confirm the expediency of enzymes in meat industry. Enzymes are used for biomodification proposes when it goes about raw materials with the high-content of connective tissue, e.g. chopped semi-finished products, delicatessen, sausages, etc. Ostrich meat is an advantageous source of functional food products due to its high content of native protein, selenium, magnesium, phosphorus, and group B vitamins and low contents of cholesterol. However, a significant amount of connective tissue makes it rigid. Application of collagenase improves the functional and technological properties of minced meat and product yield. The purpose of the present research was to optimize the technological parameters of fermented ostrich minced meat by the method of multifactorial experiment planning. The ostrich thighs parts were produced on the territory of the Leningrad Region. The authors used the fractional factorial experiment to study the influence of three factors on response functions: the mass fraction of collagenase (C, the coded variable X1), the holding time (τ, the coded variable X2), and the holding temperature (t, the coded variable X3). The values of moisture-holding ability (Y1) and the content of amino nitrogen (Y2) were chosen as response functions. The study had the following parameters of fermentation at the main level and the interval of variation: C0 = 0,04%, ΔC = 0,02%; τ0 = 4 hr, Δτ = 2 hr; t0 = 12o C, Δt = 7oC. The experiment resulted in a matrix of experiment planning and several regression equations, which describe the process in question. The study revealed the optimal technological parameters of minced meat fermentation with collagenase: mass fraction of collagenase was 0.05% while the duration of exposure of minced meat was 4.5 hours at t = 13°C. These technological parameters allowed the authors to obtain minced meat with better organoleptic characteristics and functional and technological properties in comparison with those of the control sample. The obtained fermented minced meat and the selected regimes are recommended for chopped semi-finished products, cooked sausages, and meat-vegetable canned foods.

Biotechnological stage in technology of fodder products of starfish is substantiated. The starfishes Evasterias echinosoma and Patiria pectinifera caught in the Severnaya Bay (Primorye coast of the Japan Sea) are used in the experiments on enzymic hydrolysis for production of the fodder additive with high biological value. The hydrolysis provides the more accessible forms of nutrients for animals and birds. Impacts of the enzyme preparation concentration, temperature, pH, time of hydrolysis, and water duty on efficiency of enzymic hydrolysis are tested in the experiments; these parameters influence on the amount of accumulated amino nitrogen and on the hydrolysis degree is evaluated. The following rational values are determined for the parameters in the process of protein-mineral fodder additive production: concentration of enzyme collagenase preparation - 0.25-1.00 %; time of treatment - 2-4 hours; temperature - 45-55oC; pH - 6.0; water duty - 0.2.

Purpose. To study the stability of a crosslinking modified donor cornea to collagenase preserved in the Borzenok-Moroz solution.Material and methods. Donor cornea (n=24) divided into 4 groups were the target of the research. All cornea were preserved in the Borzenok-Moroz solution for 24 hours according to standard procedure (2) before the experiment start. The first group contained only the donor cornea preserved in the BorzenokMoroz solution (n=6). The second group included preserved donor corneas with the added riboflavin treated according to the crosslinking method (n=6). The third group was formed by preserved donor corneas treated with collagenasa: result was evaluated in dynamics 24 hours (n=3) as well as 72 hours (n=3) later. The forth group consisted of preserved donor corneas with the added riboflavin after cross-linking and treated with collagenasa: result was evaluated in dynamics 24 hours (n=3) as well as 72 hours (n=3) later. Scanning electron microscopy (SEM) was carried outResults. The first group of corneas was used for reference, and according to the SEM results the corneal stroma was presented by plates of collagen protein fibers strictly oriented. In the second corneal stroma group a thickening of collagen fibrils was observed as well as a reduction of distance between the collagen plates and a more compact stacking of plates under influence of riboflavin and ultraviolet compared to the reference group. In the third group of corneas, upon collagenase impact a destruction of collagen fibrils, a breaking of the longitudinal collagen plates orientation, a disorder of protein structure were also noted after 24 hours, the remaining coagulated protein fractions were visualized. Corneal collagen fibrils in the fourth group treated with riboflavin, ultraviolet and collagenase after 24 hours retained their structure and orientation and a more compact stacking of collagen plates was noticed compared to the third group corneas. Dynamic evaluation of collagenase impact on the third and fourth groups after 72 hours showed that in the group 4 the impact of collagenase on the cornea was minimal, the structure and orientation of the collagen layers were preserved.Conclusions. Cross-linking procedure has a stabilizing physical-chemical effect on corneal collagen fibrils, enhances biochemical resistance to proteolytic tear enzymes and inflammatory cells what can be effective in treatment of corneal diseases.

The results of the experimental and theoretical researches of kinetics of erythrosine penetration into collagenase molecules have represented in this paper. The case with introduction of the compound (fluorescent probe) which has dimers to enzyme solution as an injection has been considered. It was shown that monomers and dimers can penetrate into enzyme molecules with formation complexes monomer - enzyme, dimer- enzyme. Moreover, transformation of probe fluorescence spectra is at each time moment. Spectrum maximum shift, and its form change. At a time, the immobilized dye dimers greatly impact to formation of end fluorescence spectrum. Well correlation between experimental and theoretical results confirms reality of the obtained data.

Objective: To study the effect of type II collagen peptide (CP) on the decomposition of type II collagen and the differentiation of embryonic chondrocytes of various morphotypes, isolated from the bovine growth plate. Material and methods. Bovine growth plate chondrocytes were separated into morphotypes on a Percoll gradient in accordance with different differentiation stages and then cultured in the presence or absence of 10 ßM of CP. Cartilage explants from patients with osteoarthrosis (OA) were also cultured in the presence of 10 μM of CP. Type II collagen decomposition activity was evaluated by ELISA. Gene expressions were determined by reverse transcription polymerase chain reaction (RT-PCR). Results. The cells having the highest buoyant density on a Percoll gradient (subpopulation A) were represented as early proliferative zone chondrocytes in the growth plate. In their presence of CP, there was increased expression of the genes of transforming growth factor (TGF) β2, parathyroid hormone-related peptide (PTHrP), fibroblast growth factor (FGF) 2, and cyclin B2, which were expressed in the proliferative zone of the growth plate, as well as metalloproteinase matrix (MMP) 13 and collagen decomposition activity. The large cells with the lowest buoyant density on a Percoll gradient (subpopulation B) were represented as hypertrophic chondrocytes. Cultivation of these cells in the presence of CP increased the expression of the genes associated with the terminal chondrocyte differentiation of type X collagen (COL10A1), Indian hedgehog (Ihh), core-binding factor (CBFA) 1 and TGF β1; and the expression of MMP 13 was suppressed. At the same time, collagen decomposition activity in cultured chondrocytes remained unchanged in the presence of CP. Although type II collagen was able to induce the collagen decomposition in the healthy articular cartilage explants, which was attended by the increased expression of the genes associated with the hypertrophic zone of an embryonic growth plate, it did not affect collagen decomposition rate in the cartilage of patients with OA. Conclusion. The effect of CP on gene expression and collagen decomposition activity depends on the morphotype of embryonic chondrocytes. Lack of effect of CP on collagen decomposition activity in both the embryonic hypertrophic chondrocytes and the cartilage explants from OA patients supports the hypothesis that the hypertrophic morphotype is a dominant morphotype of articular chondrocytes in OA. Moreover, collagen decomposition products can be involved in the resorption of matrix in OA and in the maintenance of chronic nature of the pathology.

Objective: To study the effect of type II collagen peptide (CB12-2) that is able to induce the collagen decomposition in the knee articular cartilage explants, which activates matrix metalloproteinase (MMP) and articular chondrocyte differentiation. Material and methods. Human cartilage explants were cultured in the presence of CB12-2 (amino acid residues 195-218) at a concentration of 10 μM. Type II collagen decomposition was evaluated by enzyme immunoassay. Immunohistochemical assay of type X collagen (C0L10A1) used frozen cartilage sections. Apoptotic activity was measured by terminal deoxynucleotidyl transferase-mediated dUPTnick end-labeling method (TUNEL). Gene expressions were determined by semiquantitative reverse transcription polymerase chain reaction (RT-PCR). Results. CB12-2 at high, but naturally occurring concentrations (10 μM) induced type II collagen decomposition by collagenase in the cartilage explants from a healthy individual. The peptide caused an increase in the expression of MMP 13, 1, 9, MT1-MMP, and the genes associated with the differentiation of embryo chondrocytes in the growth plate - transforming growth factor (TGF) β1/2, sex-determining region Y-box 9 (Sox9), Indian hedgehog (Ihh), parathyroid hormone-related peptide (PTHrP), fibroblast growth factor (FGF-2); a marker for proliferation of cyclin B2 and cytokines of tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1Я). At the same time the increased expression of the chondrocyte hypertrophy marker C0L10A1 was accompanied by enhanced staining for type X collagen in the frozen cartilage sections whereas the number of TUNEL-positive cells increased in the cartilage surface area as the expression of the apoptotic marker caspase 3 became higher. Conclusion. This study has shown that the induction of collagenase activity by CB12-2 in the human articular cartilage chondrocytes is attended by terminal differentiation/hypertrophy of these cells. The terminal differentiation of chondrocytes may be one of the mechanisms of chondrolysis in osteoarthrosis since it naturally occurs not only in endochondrial ossification, but also in the development of pathology.

The studies aimed at modeling the process of fish collagen raw material proteolysis by using enzyme preparations of collagenase and bromelin are given in the paper. The data for determining reasonable ranges of the proteolysis parameters, in particular, the temperature and duration of the proteolysis, pH and concentration of enzyme preparations, are given. The obtained set of data indicates the high efficiency of collagenase in relation to connective-tissue proteins. The mathematical model of the process of fish collagen raw materials proteolysis was developed and significant technological factors that provide functional and technological properties of the final product were determined. The proteolysis parameters, namely duration of (9...11)×60 s, temperature of 40±2 ℃, type and concentration of the enzyme preparation ?collagenase= 0,050±0,005 % to the mass of fish raw materials, value of the pH medium 7,0±0,2 were scientifically justified. The obtained results can be used for solving the problems of control and management of the process of the fish collagen proteolysis and controlled accumulation of water-soluble fractions of connective tissue of the salmon skin, depending on the composition and type of the raw materials. [ABSTRACT FROM AUTHOR]

Aging of extracellular proteins colloidal systems is one of major synchronizing mechanism in mammal`s «biological clock». We hypothesized that induced controllable modification of connective tissue composition could reverse aging. In murine experimental models collagenase was used for selective destruction of old collagen. Oxygen consumption, urine hydroxyproline excretion, density and distribution of mature and old collagen and elastine fibers in dermal biopsies were determined. Collagenase injections significantly increased hydroxyproline excretion. We observed reduced density of mature and old collagen fibers and increased oxygen consumption in dermal biopsies after course of collagenase injections. Collagenase treatment intensified the destruction of mature and old collagen matrix and enhanced synthesis of new collagen and elastine fibers. Furthermore oxygen consumption increased. Our findings can be considered as indicator of collagenase systemic anti-aging (rejuvenation) activity.