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1.
Dokl Biol Sci ; 513(Suppl 1): S41-S44, 2023 Dec.
Article in English | MEDLINE | ID: mdl-38472685

ABSTRACT

The effect of recombinant spidroin (RS) hydrogel (HG) on anterior epithelial cells and keratocytes of the human cornea was studied in vitro. Corneal injuries are highly prevalent in developing countries according to the World Health Organization. Various technologies have recently been proposed to restore the damaged surface of the cornea. Use of biodegradable silk-based materials, including recombinant analogs of the spider silk protein spidroin, is an important avenue of research in the field of wound healing and corneal regeneration. Spidroins are well known for their optimal balance of strength and elasticity. Given their biological compatibility, lack of immunogenicity, and biodegradability, spidroins provide a biomaterial for tissue engineering and regenerative medicine. HGs based on RS rS2/12-RGDS were therefore tested for cytotoxicity toward isolated corneal epithelial cells and keratocytes with regard to possible changes in cell phenotype and migratory activity. A promising outlook and therapeutic potential were demonstrated for RS-based HGs.


Subject(s)
Fibroins , Humans , Fibroins/pharmacology , Fibroins/genetics , Silk/genetics , Cornea , Biocompatible Materials , Cell Proliferation
2.
Biochemistry (Mosc) ; 85(7): 808-819, 2020 Jul.
Article in English | MEDLINE | ID: mdl-33040725

ABSTRACT

The effect of bioresorbable materials on aging in cultured mouse NIH 3T3 fibroblasts treated with elevated glucose concentration was investigated. The cells were grown on films produced from the silkworm fibroin and rS1/9, a recombinant analog of Nephila clavipes spidroin 1. Exposure to 50 mM glucose of the cells grown on uncoated glass support resulted in the cell growth retardation. The average areas of the cells and nuclei and the percentage of apoptotic cells increased, whereas the amount of soluble collagen decreased. In contrast, on the fibroin and spidroin films, the cell density and the percentage of 5-bromo-2'-deoxyuridine (BrdU)-positive cells were higher vs. the cells grown on the glass support. The films protected NIH 3T3 fibroblasts from the glucose-induced death. The most prominent effects on the cell density, BrdU incorporation, and apoptosis prevention were observed in the cells cultured on spidroin films. Unlike the cells grown on glass support (decrease in the soluble collagen production) or fibroin (no effect), production of soluble collagen by the cells grown on spidroin films increased after cell exposure to 50 mM glucose. Molecular analysis demonstrated that 50 mM glucose upregulated phosphorylation of the NFκB heterodimer p65 subunit in the cells grown on the glass support. The treatment of cells grown on fibroin films with 5.5 mM or 50 mM glucose had no effect on p65 phosphorylation. The same treatment decreased p65 phosphorylation in the cells on the spidroin films. These results demonstrate the anti-aging efficacy of biomaterials derived from the silk proteins and suggest that spidroin is more advantageous for tissue engineering and therapy than fibroin.


Subject(s)
Aging/drug effects , Aging/metabolism , Fibroins/pharmacology , Aging/genetics , Animals , Cell Proliferation/drug effects , Fibroblasts/metabolism , Fibroins/genetics , Fibroins/metabolism , Glucose/metabolism , Mice , NIH 3T3 Cells/drug effects , Tissue Engineering/methods
3.
Dokl Biochem Biophys ; 466: 9-12, 2016.
Article in English | MEDLINE | ID: mdl-27025477

ABSTRACT

The study of the stimulating effect of the microgels (MGs) based on recombinant 1F9 spidroin on the regeneration of the deep skin wound in mice was carried out. The use of spidroin MGs was shown to increase significantly the quality of healing compared to the control. The introduction of the MG in the wound edges led to recovery of all the structural elements of the skin: the epidermis, the dermis, including vascular and nervous network, in the periphery of the wound underlying muscles, and skin appendages (sebaceous and sweat glands and hair follicles) was revealed.


Subject(s)
Fibroins/pharmacology , Hydrogels/pharmacology , Wound Healing/drug effects , Animals , Female , Fibroins/genetics , Hydrogels/chemistry , Mice , Mice, Inbred C57BL , Recombinant Proteins/genetics , Recombinant Proteins/pharmacology
4.
Genetika ; 52(10): 1210-4, 2016 Oct.
Article in Russian | MEDLINE | ID: mdl-29369593

ABSTRACT

Escherichia coli open reading frames ydiO and ydiQRST were identified as genes encoding components of the acyl-CoA dehydrogenase complex of anaerobic fatty acid ß-oxidation. Individual or concomitant inactivation of fadE gene, encoding known aerobic acyl-CoA dehydrogenase, and ydiO and/or ydiQRST genes did not affect cellular growth on glucose as a sole carbon source. Aerobic growth on sodium oleate was observed only for the cells with intact fadE gene. With an alternative electron acceptor, the cells possessing intact fadE gene demonstrated anaerobic growth on sodium oleate irrespective of the presence or absence of ydiO and ydiQRST genes. For the fadE-deficient mutants, anaerobic growth on sodium oleate was observed only for cells with intact ydiO and ydiQRST genes, while the fadE/ydiO and fadE/ydiQRST mutants failed to grow under the similar conditions.


Subject(s)
Acyl-CoA Dehydrogenase/genetics , Escherichia coli Proteins/genetics , Escherichia coli/genetics , Fatty Acids/genetics , Open Reading Frames , Acyl-CoA Dehydrogenase/metabolism , Anaerobiosis/physiology , Escherichia coli/enzymology , Escherichia coli Proteins/metabolism , Fatty Acids/metabolism , Oxidation-Reduction
5.
Dokl Biochem Biophys ; 464: 325-8, 2015.
Article in English | MEDLINE | ID: mdl-26518560

ABSTRACT

Electrochemical parameters of bacterial cells Shewanella oneidensis MR-1 were investigated. For registration of the direct electron transfer between S. oneidensis MR-1 and electrode, bacterial cells were pretreated with didodecyldimethylammonium bromide (DDAB), a synthetic membrane-like substance of polycationic nature that exhibits membrane-loosening properties. Such pretreatment of S. oneidensis MR-1 allowed increasing the efficiency of extracellular electron transfer by the proteobacterium due to better availability of electroactive proteins for registration of electron transfer processes. The electroanalysis of bacterial cells S. oneidensis MR-1 under anaerobic conditions allows registering redox-active proteins and biomolecules in the range of potentials of-0.40,-0.16, and-0 V, which corresponds to flavohemoproteins, quinone derivatives, and c-type cytochromes of the external membrane of S. oneidensis MR-1 cells.


Subject(s)
Electrochemical Techniques , Shewanella/chemistry , Electrodes , Electrons , Extracellular Space/chemistry , Extracellular Space/drug effects , Graphite/chemistry , Quaternary Ammonium Compounds/pharmacology , Shewanella/drug effects , Water/chemistry
6.
Dokl Biochem Biophys ; 463: 232-5, 2015.
Article in English | MEDLINE | ID: mdl-26335819

ABSTRACT

Microcarriers generated from recombinant spidroin 1F9 are suitable for use as an injection material. The microcarriers were a heterogeneous mixture of microgel particles ranging from 50 to 300 µm in size with the predominance of particles of 50-150 µm. The surface of these microparticles had a complex topography and ensured efficient cultivation of primary and immortalized fibroblasts. Intradermal injections of microgel suspensions into the area of full-thickness skin wounds did not lead to the development of acute inflammation in mice; instead, they accelerated the recovery of skin tissue and stimulated neurogenesis and angiogenesis.


Subject(s)
Drug Carriers/chemistry , Fibroins/chemistry , Microspheres , Recombinant Proteins/chemistry , Regenerative Medicine/methods , 3T3 Cells , Animals , Female , Mice , Particle Size , Skin Physiological Phenomena , Wound Healing/drug effects
7.
Genetika ; 51(4): 443-55, 2015 Apr.
Article in Russian | MEDLINE | ID: mdl-26087620

ABSTRACT

Microorganism producer strains are the basis of industrial biotechnology. Their properties determine the economical parameters of the production. Methods of rational design (metabolic engineering) and combinatorial methods of mutagenesis and selection (laboratory evolution, adaptive evolution, protein and genomic shuffling) are used for the construction of microorganism strains. Combination of these methods is frequently used. Modern strains usually do not contain plasmids and markers of drug resistance. All changes are introduced into the chromosome by the methods of homologous and site-specific recombination. The sum of such approaches is called recombineering. Gene expression is carried out at the optimal level under the control of promoters of a certain power (frequently regulated). Knowledge of a complete genomic sequence is almost a mandatory condition for the use of methods of metabolic engineering. Bioinformatics significantly assists in the selection of enzymes and the search for necessary genes and metabolic reactions. Measurement of metabolic fluxes largely assists in the construction of strains. The current level of science makes it possible to construct metabolic pathways de novo in strains for the production of chemicals and biofuel. Carbon dioxide has potential as a raw material for microbiological industry; therefore, the study of CO2 fixation by acetogens and electrogens is a promising direction of studies.


Subject(s)
Industrial Microbiology/methods , Industrial Microbiology/trends , Gene Expression , Genetic Engineering/methods , Genetic Engineering/trends , Selection, Genetic , Transformation, Genetic
8.
Biofizika ; 59(3): 500-7, 2014.
Article in Russian | MEDLINE | ID: mdl-25715592

ABSTRACT

In the present work it was shown that biosynthesis of silver sulfide nanoparticles from silver nitrate and sodium thiosulfate solutions of millimolar concentration occurs efficiently by living Shewanella oneidensis MR-1 cells, as well as by ultrasonically-disrupted cells and by the membrane fraction of the cells. The size of nanoparticles synthesized in the presence of living cells was 7.8 ± 1.5 nm, while in the presence of ultrasonically-disrupted cells--it was 6.52 nm. The shape of nanoparticles in both cases was close to spherical. It was also shown, that synthesis of nanoparticles occurs in a cell-free solution of sodium thiosulfate that has been incubated with cells previously and to which then a silver nitrate solution was added. In this case the nanoparticles were of elongated shape and their size was (11 ± 4) x (24 ± 6) nm. In the control experiment, when only silver nitrate and sodium thiosulfate solutions not incubated with cells were used, the nanoparticles were not detected. It was shown that biosynthesis of nanoparticles occurs both in aerobic and anaerobic conditions. Nanoparticles are not formed by using thermally inactivated cells as it was shown by us previously. The results show the important role of the native structures of cells for the nanoparticles formation.


Subject(s)
Nanoparticles/chemistry , Shewanella/chemistry , Silver Compounds/chemistry , Cell-Free System/chemistry , Oxidation-Reduction , Thiosulfates/chemistry
9.
Prikl Biokhim Mikrobiol ; 49(2): 136-43, 2013.
Article in Russian | MEDLINE | ID: mdl-23795471

ABSTRACT

In this study, we constructed and characterized Escherichia coli strains deficient for mixed acid fermentation pathways, which are capable of rapid aerobic growth on glucose without pronounced bacterial Crabtree effect. The main pathways of production of acetic and lactic acids and ethanol in these strains were inactivated by a deletion of the ackA, pta, poxB, IdhA, and adhEgenes. The phosphoenolpyruvate-dependent phosphotransferase system of glucose transport and phosphorylation was inactivated in the strains by a deletion of the ptsG gene. The possibility of alternative transport and phosphorylation of the carbohydrate substrate was ensured in recombinants by constitutive expression of the galP and glk genes, which encode the low-affinity H+-symporter of D-galactose and glucokinase, respectively. SGMI.0DeltaptsG PtacgalP and SG M1.0DeltaptsG PIglk PtacgalP strains were capable of rapid aerobic growth in a minimal medium containing 2.0 and 10.0 g/l of glucose and secreted only small amounts of acetic acid and trace amounts of pyruvic acid.


Subject(s)
Escherichia coli Proteins/metabolism , Escherichia coli/metabolism , Glucokinase/metabolism , Glucose/metabolism , Metabolic Engineering , Phosphoenolpyruvate/metabolism , Aerobiosis , Biological Transport , Escherichia coli/genetics , Escherichia coli/growth & development , Escherichia coli Proteins/genetics , Fermentation , Galactose/metabolism , Gene Deletion , Glucokinase/genetics
12.
Prikl Biokhim Mikrobiol ; 48(4): 383-8, 2012.
Article in Russian | MEDLINE | ID: mdl-23035570

ABSTRACT

Anaerobic biosynthesis of 1-butanol from glucose is investigated in recombinant Escherichia coli strains which form butyryl-CoA using the heterologous enzyme complex of clostridia or as a result of a reversal in the action of native enzymes of the fatty acid beta-oxidation pathway. It was revealed that when the basic pathways of acetic and lactic acid formation are inactivated due to deletions in the ackA, pta, poxB, and ldhA genes, the efficiency of butyryl-CoA biosynthesis and its reduced product, i.e., 1-butanol, by two types of recombinant stains is comparable. The limiting factor for 1-butanol production by the obtained strains is the low substrate specificity of the basic CoA-dependent alcohol/aldehyde AdhE dehydrogenase from E. coli to butyryl-CoA. It was concluded that, in order to construct an efficient 1-butanol producer based on a model strain synthesizing butyryl-CoA as a result of a reversal in fatty acid beta-oxidation enzymes, it is necessary to provide intensive formation of acetyl-CoA and enhanced activity of alternative alcohol and aldehyde dehydrogenases in the cells of a strain.


Subject(s)
1-Butanol/metabolism , 3-Hydroxyacyl CoA Dehydrogenases/metabolism , Acyl Coenzyme A/metabolism , Bacterial Proteins/metabolism , Clostridium/enzymology , Escherichia coli/enzymology , 3-Hydroxyacyl CoA Dehydrogenases/genetics , Bacterial Proteins/genetics , Clostridium/genetics , Clostridium/metabolism , DNA Primers , Escherichia coli/genetics , Escherichia coli/metabolism , Fatty Acids/metabolism , Gene Deletion , Genetic Engineering , Oxidation-Reduction , Polymerase Chain Reaction , Substrate Specificity
14.
Aviakosm Ekolog Med ; 46(1): 62-7, 2012.
Article in Russian | MEDLINE | ID: mdl-22629587

ABSTRACT

Purpose of the work was designing and prototyping of microbial fuel cells (MFC) and comparative evaluation of the electrogenic activity of wastewater autochthonous microorganisms as well as bacterial monocultures. Objects were model electrogenic strain Shewanella oneidensis MR-1, and an Ochrobactrum sp. strain isolated from the active anode biofilm of MFC composed as an electricity generating system. The study employed the methods typically used for aerobic and anaerobic strains, current measurement, identification of new electrogenic strains in microbial association of wastewater sludge and species definition by rRNA 16-S. As a result, two MFCs prototypes were tried out. Besides, it was shown that electrogenic activity of S. oneidensis MR-1 and Ochrobactrum sp. monocultures is similar but differs from that of the microbial association of the anode biofilm.


Subject(s)
Bioelectric Energy Sources , Biofilms , Ecological Systems, Closed , Ochrobactrum/physiology , Shewanella/physiology , Space Flight , Electric Power Supplies , Electricity , Electrochemical Techniques , Electrodes , Humans , Life Support Systems , Microbial Consortia , Sewage/microbiology
15.
Prikl Biokhim Mikrobiol ; 47(4): 415-23, 2011.
Article in Russian | MEDLINE | ID: mdl-21950115

ABSTRACT

Effect of constitutive expression of the aceEF-lpdA operon genes coding for the enzymes of NAD+ reducing pyruvate dehydrogenase complex on the anaerobic production of succinic acids from glucose by recombinant Escherichia coli strains was studied. Basic producer strains were obtained by inactivation of the main pathways for synthesis of acetic and lactic acids by deletion of the genes ackA, pta, poxB, and ldhA (SGMO.1) in E. coli strain MG 1655 cells and additional introduction of the Bacillus subtilis pyruvate carboxylase (SG M0.1 [pPYC]). A constitutive expression of the genes aceEF-lpdA in derivatives of the basic strains SGM0.1 PL-aceEF-lpdA and SGM0.1 PL-aceEF-lpdA [pPYC] was provided by replacing the native regulatory region of the operon with the lambda phage PL promoter. Molar yields of succinic acid in anaerobic glucose fermentation by strains SGM0.1 P(L)-aceEF-lpdA and SGM0.1 PL-aceEF-lpdA [pPYC] exceeded the corresponding yields displayed by several control strains (exceeded considerably in the case of the strains with a pyruvate carboxylase activity). It is concluded that an increase in the succinic acid production by strain SGM0.1 PL-aceEF-lpdA [pPYC] as compared with the strains SGM0.1 and SGM0.1 [pPYC], which synthesize this substance in the reductive tricarboxylic acid cycle, is determined by activation of the glyoxylate shunt.


Subject(s)
Bacillus subtilis/enzymology , Bacterial Proteins/metabolism , Escherichia coli , Genetic Engineering/methods , Glucose/metabolism , NAD/metabolism , Pyruvate Carboxylase/metabolism , Pyruvate Dehydrogenase Complex/metabolism , Succinic Acid , Anaerobiosis , Bacillus subtilis/chemistry , Bacterial Proteins/genetics , Bacteriophage lambda/genetics , Bacteriophage lambda/metabolism , Citric Acid Cycle , Cloning, Molecular , Escherichia coli/enzymology , Escherichia coli/genetics , Fermentation , Gene Deletion , Gene Expression , Glyoxylates/metabolism , Operon , Plasmids , Promoter Regions, Genetic , Pyruvate Carboxylase/genetics , Pyruvate Dehydrogenase Complex/genetics , Succinic Acid/isolation & purification , Succinic Acid/metabolism , Transfection
17.
J Biomed Mater Res A ; 96(1): 125-31, 2011 Jan.
Article in English | MEDLINE | ID: mdl-21105160

ABSTRACT

The goal of this study was to generate porous scaffolds from the genetically engineered protein, an analogue of Nephila clavipes spidroin 1 (rS1/9) and to assess the properties of new rS1/9 scaffolds essential for bioengineering. The salt leaching technique was used to make the rS1/9 scaffolds of interconnected macroporous structure with spontaneously formed micropores. The tensile strength of scaffolds was 18 ± 5 N/cm(2). Scaffolds were relatively stable in a phosphate buffer but degraded in oxidizing environment after 11 weeks of incubation. Applicability of the recombinant spidroin 1 as a substrate for cell culture was demonstrated by successful 3T3 cells growth on the surface of rS1/9 films (270 ± 20 cells/mm(2) vs. 97 ± 8 cells/mm(2) on the glass surface, p < 0.01). The 3T3 fibroblasts readily proliferated within the rS1/9 scaffold (from initially plated 19 ± 2 cells/mm(3) to 3800 ± 304 cells/mm(3) after 2 weeks). By this time, cells were uniformly distributed between the surface and deeper layers (27% ± 8% and 33% ± 4%, respectively; p > 0.05), whereas the initial distribution was 58% ± 7% and 11% ± 8%, respectively; p < 0.05). The rS1/9 scaffolds implanted subcutaneously into Balb/c mice were well tolerated. Over a 2-month period, the scaffolds promoted an ingrowth of de novo formed vascularized connective tissue elements and nerve fibers. Thus, scaffolds made of the novel recombinant spidroin 1 analogue are potentially applicable in tissue engineering.


Subject(s)
Biocompatible Materials/chemistry , Fibroins/chemistry , Recombinant Proteins/chemistry , Tissue Scaffolds/chemistry , Animals , Cells, Cultured , Female , Fibroblasts/cytology , Fibroins/genetics , Materials Testing , Mice , Mice, Inbred BALB C , Porosity , Recombinant Proteins/genetics , Tissue Engineering/methods
19.
Mol Biol (Mosk) ; 44(1): 162-9, 2010.
Article in Russian | MEDLINE | ID: mdl-20198870

ABSTRACT

A study has been conducted on the morphology of artificial spider silk fibers, prepared from recombinant analogues of spiridons 1 and 2. It has been shown that by stretching out the "as spun" fiber, a reorganization of its spongy matrix occurs, which leads to the formation of microfibrills, followed by a reduction of the diameter of the fiber. The durability of an artificial fiber depends on the degree of stretching and on the substructure of the microfibrills. The model process of artificial fibers preparation reproduces to the great detail the natural process of spider web spinning. Future applications of this model include production of biomaterials with unique properties.


Subject(s)
Fibroins/chemistry , Recombinant Proteins/chemistry , Animals , Fibroins/genetics , Fibroins/ultrastructure , Protein Structure, Quaternary , Recombinant Proteins/genetics , Recombinant Proteins/ultrastructure , Spiders , Tensile Strength
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