A simplified «exon» method was developed for producing cDNA of low-copy and silent eukaryotic genes. It is based on assembly of the target gene from genomic DNA by direct synthesis of its exons, followed by their PCR-based joining without further purification of the amplicons. During the synthesis of exons, direct primers were used; these included about ~ 20 nucleotides of the 3`-terminal sequence previous (from the amplified) exon and ~ 20 nucleotides of the 5`-initial sequence of the amplified exon. Reverse primers included ~ 20 nucleotides complementary to the terminal sequence of the amplified exon. Forward and reverse primers flanking the gene to be assembled included the restriction sites necessary for insertion into the expression vector. Using this approach it is possible to assemble almost any eukaryotic gene with a known nucleotide sequence of genomic DNA available in the database.
{"title":"A Simplified Method for Obtaining cDNA of Low-Copy and Silent Eukaryotic Genes Using Human Renalase as an the Example","authors":"V. Fedchenko, A. Kaloshin","doi":"10.18097/BMCRM00101","DOIUrl":"https://doi.org/10.18097/BMCRM00101","url":null,"abstract":"A simplified «exon» method was developed for producing cDNA of low-copy and silent eukaryotic genes. It is based on assembly of the target gene from genomic DNA by direct synthesis of its exons, followed by their PCR-based joining without further purification of the amplicons. During the synthesis of exons, direct primers were used; these included about ~ 20 nucleotides of the 3`-terminal sequence previous (from the amplified) exon and ~ 20 nucleotides of the 5`-initial sequence of the amplified exon. Reverse primers included ~ 20 nucleotides complementary to the terminal sequence of the amplified exon. Forward and reverse primers flanking the gene to be assembled included the restriction sites necessary for insertion into the expression vector. Using this approach it is possible to assemble almost any eukaryotic gene with a known nucleotide sequence of genomic DNA available in the database.","PeriodicalId":286037,"journal":{"name":"Biomedical Chemistry: Research and Methods","volume":"1 1","pages":"0"},"PeriodicalIF":0.0,"publicationDate":"2019-06-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"133911765","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
A. Kopylov, O. Tikhonova, T. E. Farafonova, N. Petushkova, Yu. V. Miroshnichenko, V. Zgoda
LC-MS/MS allows identification of thousands of proteins in the complex proteomes. However, a significant part of a proteome remains inaccessible for identification due to the absence or poor quality of MS/MS spectra. The method described herein allows identifying the desired proteins of human blood plasma by comparing aligned chromatographic data of digested by trypsin sample and the same sample with spikedin synthetic peptides. Identification of human blood plasma proteins is archived by assigning tandem mass spectra of spiked-in peptides to the corresponding aligned chromatographic peaks of proteolytic peptides. Using the described approach we have identified 19 low abundant proteins in human blood plasma, which corresponded to 19 synthetic peptides used in the study. SRM verification of the identifications with isotopically labelled standards (SIS) confirmed the presence in the plasma of above 17 proteins.
{"title":"Identification of Human Blood Plasma Proteins Using Spike-In Peptides in Shotgun Proteomics","authors":"A. Kopylov, O. Tikhonova, T. E. Farafonova, N. Petushkova, Yu. V. Miroshnichenko, V. Zgoda","doi":"10.18097/BMCRM00093","DOIUrl":"https://doi.org/10.18097/BMCRM00093","url":null,"abstract":"LC-MS/MS allows identification of thousands of proteins in the complex proteomes. However, a significant part of a proteome remains inaccessible for identification due to the absence or poor quality of MS/MS spectra. The method described herein allows identifying the desired proteins of human blood plasma by comparing aligned chromatographic data of digested by trypsin sample and the same sample with spikedin synthetic peptides. Identification of human blood plasma proteins is archived by assigning tandem mass spectra of spiked-in peptides to the corresponding aligned chromatographic peaks of proteolytic peptides. Using the described approach we have identified 19 low abundant proteins in human blood plasma, which corresponded to 19 synthetic peptides used in the study. SRM verification of the identifications with isotopically labelled standards (SIS) confirmed the presence in the plasma of above 17 proteins.","PeriodicalId":286037,"journal":{"name":"Biomedical Chemistry: Research and Methods","volume":"48 1","pages":"0"},"PeriodicalIF":0.0,"publicationDate":"2019-05-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"116967613","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Infrared spectroscopy of saliva is an express and non-invasive method of analysis, applicable for diagnostics of various diseases and for studying metabolic processes and adaptive changes in the body. The goal of this study was to determine possibility of analyzing the products of lipid peroxidation (LPO) by using IR spectroscopy of saliva on the example of oncological diseases. The study involved 203 patients with lung cancer (n = 40), breast cancer (n = 50) and the control group (n = 113). Saliva samples were collected in the morning after overnight fast. The content of LPO products (conjugated dienes, and trienes, Schiff bases, malonic dialdehyde MDA) was determined in all samples and the IR absorption spectra were recorded in the range of 500–4000 cm–1. In the IR spectra, an increase in the intensity of the absorption bands of lipids was observed; it corresponded to an increase in the total lipid content and correlated with the content of MDA, and a decrease in the intensity of vibrations of oxygen-containing groups, which corresponded to a negative correlation with secondary LPO products. Apparently, on the IR spectra, we simultaneously register both primary, intermediate, and final LPO products. Statistically significant regression equations were obtained, allowing to estimate the content of intermediate LPO products - conjugated triene, and Schiff bases. The proposed method allows to monitor LPO processes, as well as to characterize the direction of the equilibrium shift in these processes.
{"title":"The Use of IR Fourier Spectroscopy of Saliva for Rapid Assessment of the Level of Lipid Peroxidation Products","authors":"L. Bel’skaya, E. Sarf","doi":"10.18097/bmcrm00094","DOIUrl":"https://doi.org/10.18097/bmcrm00094","url":null,"abstract":"Infrared spectroscopy of saliva is an express and non-invasive method of analysis, applicable for diagnostics of various diseases and for studying metabolic processes and adaptive changes in the body. The goal of this study was to determine possibility of analyzing the products of lipid peroxidation (LPO) by using IR spectroscopy of saliva on the example of oncological diseases. The study involved 203 patients with lung cancer (n = 40), breast cancer (n = 50) and the control group (n = 113). Saliva samples were collected in the morning after overnight fast. The content of LPO products (conjugated dienes, and trienes, Schiff bases, malonic dialdehyde MDA) was determined in all samples and the IR absorption spectra were recorded in the range of 500–4000 cm–1. In the IR spectra, an increase in the intensity of the absorption bands of lipids was observed; it corresponded to an increase in the total lipid content and correlated with the content of MDA, and a decrease in the intensity of vibrations of oxygen-containing groups, which corresponded to a negative correlation with secondary LPO products. Apparently, on the IR spectra, we simultaneously register both primary, intermediate, and final LPO products. Statistically significant regression equations were obtained, allowing to estimate the content of intermediate LPO products - conjugated triene, and Schiff bases. The proposed method allows to monitor LPO processes, as well as to characterize the direction of the equilibrium shift in these processes.","PeriodicalId":286037,"journal":{"name":"Biomedical Chemistry: Research and Methods","volume":"21 1","pages":"0"},"PeriodicalIF":0.0,"publicationDate":"2019-05-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"128003590","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
M. Senina, Y. Savochkina, L. V. Skvortsov, T. Popova, L. V. Dzhedzheia
The correct information about of the vaginal microflora plays an important role in preventing the occurrence of urinary tract infections and sexually transmitted infections among women. Disbalance of obligate and facultative microflora causes disbacteriosis, a risk factor for emergence of infectious diseases. It is known that the cause of bacterial vaginosis (BV) is not a single pathogen but a impairments in of the general balance of the vaginal microflora, which manifests a decrease of the normal microflora (Lactobacillus spp) and intense increase of pathogenic aerobic and anaerobic bacteria. The development of molecular genetic analysis methods, in particular, approaches based on the use of polymerase chain reaction (PCR), significantly expanded understanding of the diversity of microbial biotopes, including identification of the key and new «players» in the development of BV. The aim of our study was to evaluate the performance of real-time PCR kit «Femoscreen» («Lytech», Russia) for comprehensive BV diagnosis.
{"title":"The qPCR analysis of vaginal microflora for the diagnosis of bacterial vaginosis","authors":"M. Senina, Y. Savochkina, L. V. Skvortsov, T. Popova, L. V. Dzhedzheia","doi":"10.18097/BMCRM00084","DOIUrl":"https://doi.org/10.18097/BMCRM00084","url":null,"abstract":"The correct information about of the vaginal microflora plays an important role in preventing the occurrence of urinary tract infections and sexually transmitted infections among women. Disbalance of obligate and facultative microflora causes disbacteriosis, a risk factor for emergence of infectious diseases. It is known that the cause of bacterial vaginosis (BV) is not a single pathogen but a impairments in of the general balance of the vaginal microflora, which manifests a decrease of the normal microflora (Lactobacillus spp) and intense increase of pathogenic aerobic and anaerobic bacteria. The development of molecular genetic analysis methods, in particular, approaches based on the use of polymerase chain reaction (PCR), significantly expanded understanding of the diversity of microbial biotopes, including identification of the key and new «players» in the development of BV. The aim of our study was to evaluate the performance of real-time PCR kit «Femoscreen» («Lytech», Russia) for comprehensive BV diagnosis.","PeriodicalId":286037,"journal":{"name":"Biomedical Chemistry: Research and Methods","volume":"41 1","pages":"0"},"PeriodicalIF":0.0,"publicationDate":"2019-04-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"127638552","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
P. Ershov, Y. Mezentsev, E. Yablokov, L. Kaluzhskiy, I. Vakhrushev, O. Gnedenko, A. V. Florinskaya, A. Gilep, S. Usanov, K. Yarygin, A. Ivanov
The aim of this work was to test modifications of the standard protocol for the sample preparation of cell/tissue lysate before performing the affinity isolation of lysate protein partners for the target protein (bait protein) which is covalently immobilized on an inert sorbent (e.g. BrCN-, SH-Sepharose 4B) or a carrier (e.g. paramagnetic nanoparticles). The series of our previous works on applying the approach to direct molecular fishing procedure with combination of affinity chromatography and LC-MS/MS analysis using a number of proteins, encoded by the genes of human chromosome 18, have shown that there are at least two problems affecting the specificity and the effectiveness of this procedure. These include: (i) redundancy of the background proteins in the eluates from an affinity sorbent (carrier) due to isolation of multiprotein complexes “labeled” with a direct protein partner which binds with a bait protein immobilized on the sorbent; (ii) low enrichment of the eluates with appropriate protein partners due to the fact that some direct protein partners in the lysate exist in stable “wild type” complexes with the bait protein itself. This means that latter group of protein partners will not be sufficiently isolated from lysate. Therefore, in order to increase the specificity and efficiency of affinity isolation of protein partners for the bait protein, we modified the standard protocol of lysate preparation and the preliminary step on dissociation of lysate protein complexes was added. Several model experiments for the choice of regeneration solution, assessment of their efficiency in the dissociation of lysate protein complexes as well as the stability and binding capacity of proteins were performed under the control of surface plasmon resonance (SPR) biosensor Biacore 3000 using HepG2 cell lysate. It was shown that acid treatment and incubation of the cell lysate for one min on ice (final lysate dilution 20 times) and subsequent neutralization (pH shift from 2.0 to 7.4) resulted in maximal dissociation of the lysate protein complexes without significant negative effects on the protein-protein interactions tested.
{"title":"A Method of Lysate Preparation to Improve the Isolation Efficiency of Protein Partners for Target Proteins Encoded by the Genes of Human Chromosome 18","authors":"P. Ershov, Y. Mezentsev, E. Yablokov, L. Kaluzhskiy, I. Vakhrushev, O. Gnedenko, A. V. Florinskaya, A. Gilep, S. Usanov, K. Yarygin, A. Ivanov","doi":"10.18097/BMCRM00090","DOIUrl":"https://doi.org/10.18097/BMCRM00090","url":null,"abstract":"The aim of this work was to test modifications of the standard protocol for the sample preparation of cell/tissue lysate before performing the affinity isolation of lysate protein partners for the target protein (bait protein) which is covalently immobilized on an inert sorbent (e.g. BrCN-, SH-Sepharose 4B) or a carrier (e.g. paramagnetic nanoparticles). The series of our previous works on applying the approach to direct molecular fishing procedure with combination of affinity chromatography and LC-MS/MS analysis using a number of proteins, encoded by the genes of human chromosome 18, have shown that there are at least two problems affecting the specificity and the effectiveness of this procedure. These include: (i) redundancy of the background proteins in the eluates from an affinity sorbent (carrier) due to isolation of multiprotein complexes “labeled” with a direct protein partner which binds with a bait protein immobilized on the sorbent; (ii) low enrichment of the eluates with appropriate protein partners due to the fact that some direct protein partners in the lysate exist in stable “wild type” complexes with the bait protein itself. This means that latter group of protein partners will not be sufficiently isolated from lysate. Therefore, in order to increase the specificity and efficiency of affinity isolation of protein partners for the bait protein, we modified the standard protocol of lysate preparation and the preliminary step on dissociation of lysate protein complexes was added. Several model experiments for the choice of regeneration solution, assessment of their efficiency in the dissociation of lysate protein complexes as well as the stability and binding capacity of proteins were performed under the control of surface plasmon resonance (SPR) biosensor Biacore 3000 using HepG2 cell lysate. It was shown that acid treatment and incubation of the cell lysate for one min on ice (final lysate dilution 20 times) and subsequent neutralization (pH shift from 2.0 to 7.4) resulted in maximal dissociation of the lysate protein complexes without significant negative effects on the protein-protein interactions tested.","PeriodicalId":286037,"journal":{"name":"Biomedical Chemistry: Research and Methods","volume":"2 1","pages":"0"},"PeriodicalIF":0.0,"publicationDate":"2019-03-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"114991524","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
S. S. Boyko, Zherdev Vp, R. Shevchenko, O. G. Gribakina
Experimental pharmacokinetics of new pharmacologically active peptides, modified analogues of endogenous neuropeptides, has been investigated in rats and rabbits. The study icluded 3 new drugs: (i) the nootropic drug noopept (phenylacetyl-prolyl-glycine ethyl ester); (ii) dilept (N-caproyl-L-prolyl-L-tyrosine methyl ester) – the antipsychotic with positive mnemotropic action; (iii) compound GB-115 – selective anxiolytic (phenylhexanoyl-prolyl-tryptophan amide). Differences in pharmacokinetics and biotransformation of the studied drugs depended on their structural features. The ether derivatives noopept and dilept underwent intensive metabolism by rat gastrointestinal esterases and peptidases with the formation of active metabolites. Being an amide, the compound GB-115 was more resistant to the enzymatic effects of peptidases and was detected for a longer period in the blood of experimental animals. In rabbits the studied compounds were less exposed to the enzymatic action by gastrointestinal peptidases, and were detected plasma of rabbits for a longer period. The higher stability of the compounds studied in rabbits may be attributed not only to the structural features of the studied dipeptides, but also to differences in the activity of the enzymatic systems of the gastrointestinal tract participating in their metabolism, as well as differences in the rate of hepatic and renal blood flow in rats and rabbits.
{"title":"Comparative Analysis of Experimental Pharmacokinetics of New Neurotropic Peptides","authors":"S. S. Boyko, Zherdev Vp, R. Shevchenko, O. G. Gribakina","doi":"10.18097/BMCRM00092","DOIUrl":"https://doi.org/10.18097/BMCRM00092","url":null,"abstract":"Experimental pharmacokinetics of new pharmacologically active peptides, modified analogues of endogenous neuropeptides, has been investigated in rats and rabbits. The study icluded 3 new drugs: (i) the nootropic drug noopept (phenylacetyl-prolyl-glycine ethyl ester); (ii) dilept (N-caproyl-L-prolyl-L-tyrosine methyl ester) – the antipsychotic with positive mnemotropic action; (iii) compound GB-115 – selective anxiolytic (phenylhexanoyl-prolyl-tryptophan amide). Differences in pharmacokinetics and biotransformation of the studied drugs depended on their structural features. The ether derivatives noopept and dilept underwent intensive metabolism by rat gastrointestinal esterases and peptidases with the formation of active metabolites. Being an amide, the compound GB-115 was more resistant to the enzymatic effects of peptidases and was detected for a longer period in the blood of experimental animals. In rabbits the studied compounds were less exposed to the enzymatic action by gastrointestinal peptidases, and were detected plasma of rabbits for a longer period. The higher stability of the compounds studied in rabbits may be attributed not only to the structural features of the studied dipeptides, but also to differences in the activity of the enzymatic systems of the gastrointestinal tract participating in their metabolism, as well as differences in the rate of hepatic and renal blood flow in rats and rabbits.","PeriodicalId":286037,"journal":{"name":"Biomedical Chemistry: Research and Methods","volume":"1 1","pages":"0"},"PeriodicalIF":0.0,"publicationDate":"2019-03-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"129508883","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
N. D. Oltarzhevskaja, G. E. Krichevskij, M. Korovina, V. Shvets, A. A. Kubatiev
The review focuses on the analysis of various methods of obtaining and applying therapeutic materials used for targeted drug delivery to the lesion site of cancer patients. Special attention is paid to creation of targeted drugs by using nanotransporters, obtained by dispersing lipids in water and, in particular, liposomes; efficiencyof such nanotransporters depends the nature of drugs introduced into them (cytostatics). The review also describes methods of targeted transport of cytostatics to tumor tissues. The use of hydrogel therapeutic compositions based on biopolymers polysaccharides for the targeted delivery of chemotherapy drugs introduced into them, allows to control the mass transfer rate of drugs to tumor and to create therapeutic materials with predetermined properties in terms of drug concentration in the lesion site and time prolongation, which reduces toxicity of the treatment and increase its effectiveness.
{"title":"Methods of Delivery of Medications for the Treatment of Oncological Diseases","authors":"N. D. Oltarzhevskaja, G. E. Krichevskij, M. Korovina, V. Shvets, A. A. Kubatiev","doi":"10.18097/BMCRM00089","DOIUrl":"https://doi.org/10.18097/BMCRM00089","url":null,"abstract":"The review focuses on the analysis of various methods of obtaining and applying therapeutic materials used for targeted drug delivery to the lesion site of cancer patients. Special attention is paid to creation of targeted drugs by using nanotransporters, obtained by dispersing lipids in water and, in particular, liposomes; efficiencyof such nanotransporters depends the nature of drugs introduced into them (cytostatics). The review also describes methods of targeted transport of cytostatics to tumor tissues. The use of hydrogel therapeutic compositions based on biopolymers polysaccharides for the targeted delivery of chemotherapy drugs introduced into them, allows to control the mass transfer rate of drugs to tumor and to create therapeutic materials with predetermined properties in terms of drug concentration in the lesion site and time prolongation, which reduces toxicity of the treatment and increase its effectiveness.","PeriodicalId":286037,"journal":{"name":"Biomedical Chemistry: Research and Methods","volume":"9 1","pages":"0"},"PeriodicalIF":0.0,"publicationDate":"2019-02-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"123034644","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
M. Pokrovskaya, S. Aleksandrova, A. Veselovsky, D. D. Zdanov, V. Pokrovsky, M. Eldarov, D. V. Grishin, Y. Gladilina, I.Yu. Toropigin, N. Sokolov
Rru_A3730 protein is a bacterial Rhodospirillum rubrum L-asparaginase (RrA), which is known by its anticancer activity. RrA variants with point amino acid substitutions in the region of 150 amino acids residues: RrA17N, K149E, RrAE149R, V150P, F151T, RrА17N, E149R, V150P, RrAE149R, V150P, showed antiproliferative properties, and also by their ability to suppress telomerase activity. This work is devoted to comparison of physical-chemical and catalytic properties of these mutant forms of RrA. It is shown that pH optimum is in the alkaline zone (8.5 – 9.3); L-glutaminase and D-asparaginase activity is respectively not more than 0.1% and 1.6% of L-asparaginase for all studied variants of RrA. The presence of the N17-terminal amino acid sequence MASMTGGQMGRGSSRQ of the capsid protein of bacteriophage T7 in the RrA structure leads to an increase in the thermal stability of mutant RrA analogues (from 50°C to 56°C) and their resistance to denaturation in the presence of 3 – 4 M urea. It is of Metal ions exhibit multidirectional effects on L-asparaginase activity of RrA. K+, Ca2+, Zn2+, Cs+, Co2+ in significantly affect the activity of L-asparaginase, while Mn2+, Cu2+, Fe3+ ions inhibit it. There was no correlation between antitelomerase (antiproliferative) activity and kinetic properties of mutant forms of L-asparaginase RrA.
{"title":"Physical-Chemical Properties of L-asparaginase Mutants From Rhodospirillum Rubrum which Showed Antitelomerase Activity","authors":"M. Pokrovskaya, S. Aleksandrova, A. Veselovsky, D. D. Zdanov, V. Pokrovsky, M. Eldarov, D. V. Grishin, Y. Gladilina, I.Yu. Toropigin, N. Sokolov","doi":"10.18097/BMCRM00071","DOIUrl":"https://doi.org/10.18097/BMCRM00071","url":null,"abstract":"Rru_A3730 protein is a bacterial Rhodospirillum rubrum L-asparaginase (RrA), which is known by its anticancer activity. RrA variants with point amino acid substitutions in the region of 150 amino acids residues: RrA17N, K149E, RrAE149R, V150P, F151T, RrА17N, E149R, V150P, RrAE149R, V150P, showed antiproliferative properties, and also by their ability to suppress telomerase activity. This work is devoted to comparison of physical-chemical and catalytic properties of these mutant forms of RrA. It is shown that pH optimum is in the alkaline zone (8.5 – 9.3); L-glutaminase and D-asparaginase activity is respectively not more than 0.1% and 1.6% of L-asparaginase for all studied variants of RrA. The presence of the N17-terminal amino acid sequence MASMTGGQMGRGSSRQ of the capsid protein of bacteriophage T7 in the RrA structure leads to an increase in the thermal stability of mutant RrA analogues (from 50°C to 56°C) and their resistance to denaturation in the presence of 3 – 4 M urea. It is of Metal ions exhibit multidirectional effects on L-asparaginase activity of RrA. K+, Ca2+, Zn2+, Cs+, Co2+ in significantly affect the activity of L-asparaginase, while Mn2+, Cu2+, Fe3+ ions inhibit it. There was no correlation between antitelomerase (antiproliferative) activity and kinetic properties of mutant forms of L-asparaginase RrA.","PeriodicalId":286037,"journal":{"name":"Biomedical Chemistry: Research and Methods","volume":"70 5","pages":"0"},"PeriodicalIF":0.0,"publicationDate":"2019-02-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"113943487","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
The purpose of the study was to study parameters of the antioxidant protection system in saliva for non-small cell lung cancer. In the case-control study, included 683 volunteers, which were divided into 3 groups: primary (lung cancer patients, n = 290), comparison group (patients with nonmalignant pulmonary pathologies, n = 178) and control (conditionally healthy individuals, n = 215). Biochemical examination of saliva, histological verification of the diagnosis were carried out for all participants. The parameters of the antioxidant defense was determined spectrophotometrically. Intergroup differences were estimated by a nonparametric criterion. Saliva of lung cancer patients was characterized by imbalance in the antioxidant defense. It is shown that the activity of the enzymes of the first link of antioxidant protection (catalase, SOD) was significantly reduced (p ˂ 0.0001), whereas activity of salivary peroxidases increase (p = 0.0037). The parameters of non-enzymatic protection varied in opposite directions: the level of uric acid in lung pathologies decreases (p = 0.0399), whereas albumin concentration increased, under these conditions, it begins to exhibit prooxidant properties. Differences between adenocarcinoma and squamous cell lung cancer have been found in terms of the mode of the dynamics of antioxidant protection parameters. Probably, against the background of squamous cell lung cancer, an enzymatic link (catalase, SOD) contributes to the antioxidant protection system, whereas against adenocarcinoma - nonenzymatic (uric acid, albumin).
{"title":"Antioxidant Protection System in the Saliva of Patients with Non-Small Cell Lung Cancer","authors":"L. Bel’skaya, V. Kosenok, G. Massard","doi":"10.18097/BMCRM00061","DOIUrl":"https://doi.org/10.18097/BMCRM00061","url":null,"abstract":"The purpose of the study was to study parameters of the antioxidant protection system in saliva for non-small cell lung cancer. In the case-control study, included 683 volunteers, which were divided into 3 groups: primary (lung cancer patients, n = 290), comparison group (patients with nonmalignant pulmonary pathologies, n = 178) and control (conditionally healthy individuals, n = 215). Biochemical examination of saliva, histological verification of the diagnosis were carried out for all participants. The parameters of the antioxidant defense was determined spectrophotometrically. Intergroup differences were estimated by a nonparametric criterion. Saliva of lung cancer patients was characterized by imbalance in the antioxidant defense. It is shown that the activity of the enzymes of the first link of antioxidant protection (catalase, SOD) was significantly reduced (p ˂ 0.0001), whereas activity of salivary peroxidases increase (p = 0.0037). The parameters of non-enzymatic protection varied in opposite directions: the level of uric acid in lung pathologies decreases (p = 0.0399), whereas albumin concentration increased, under these conditions, it begins to exhibit prooxidant properties. Differences between adenocarcinoma and squamous cell lung cancer have been found in terms of the mode of the dynamics of antioxidant protection parameters. Probably, against the background of squamous cell lung cancer, an enzymatic link (catalase, SOD) contributes to the antioxidant protection system, whereas against adenocarcinoma - nonenzymatic (uric acid, albumin).","PeriodicalId":286037,"journal":{"name":"Biomedical Chemistry: Research and Methods","volume":"14 1","pages":"0"},"PeriodicalIF":0.0,"publicationDate":"2019-01-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"133048167","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Tissue destruction and angiogenesis play an important role in malignant tumor progression. They are responsible for the tumor growth and progress and its ability to invade and metastasize. The key role in the destructive processes belongs to matrix metalloproteinases (MMPs), which are able to cleave almost all components of the extracellular matrix (ECM). Gelatinases MMP-2 and MMP-9 hydrolyze type IV collagen, the main component of basement membranes, thereby releasing various biologically active molecules from ECM, including vascular endothelial growth factor (VEGF). VEGF is a key regulator of angiogenesis. The main mediator of the biological action of VEGF is its receptor VEGFR2. This study was aimed at assessing the relationship between the expression of the main factors of tissue destruction and angiogenesis - MMP-2, MMP-9, VEGF and VEGFR2 in the early and later stages of cervical squamous cell carcinoma (CSCC). The work was performed using samples of tumor and surrounding morphologically normal tissue, obtained from patients with or without metastases to regional lymph nodes. We have shown that MMP- 9 is significantly expressed in tumors in CSCC already at the early stages of tumor progression. At later stages of the disease (when metastases to regional lymph nodes are detected in patients), the expression of MMP-2, VEGF and VEGFR2 increases markedly both in the tumor and in the morphologically normal tissue surrounding the tumor and makes an additional contribution to the processes of destruction, angiogenesis and metastasis. We assume that MMP-2, VEGF and VEGFR2 can be considered as negative markers of the course of CSCC.
{"title":"MMP-9, MMP-2, VEGF and VEGFR-2 as Factors of Invasion and Angiogenesis in Squamous Cell Carcinoma of the Cervix","authors":"O. Timoshenko, E. V. Kugaevskaya, T. A. Gureeva","doi":"10.18097/bmcrm00187","DOIUrl":"https://doi.org/10.18097/bmcrm00187","url":null,"abstract":"Tissue destruction and angiogenesis play an important role in malignant tumor progression. They are responsible for the tumor growth and progress and its ability to invade and metastasize. The key role in the destructive processes belongs to matrix metalloproteinases (MMPs), which are able to cleave almost all components of the extracellular matrix (ECM). Gelatinases MMP-2 and MMP-9 hydrolyze type IV collagen, the main component of basement membranes, thereby releasing various biologically active molecules from ECM, including vascular endothelial growth factor (VEGF). VEGF is a key regulator of angiogenesis. The main mediator of the biological action of VEGF is its receptor VEGFR2. This study was aimed at assessing the relationship between the expression of the main factors of tissue destruction and angiogenesis - MMP-2, MMP-9, VEGF and VEGFR2 in the early and later stages of cervical squamous cell carcinoma (CSCC). The work was performed using samples of tumor and surrounding morphologically normal tissue, obtained from patients with or without metastases to regional lymph nodes. We have shown that MMP- 9 is significantly expressed in tumors in CSCC already at the early stages of tumor progression. At later stages of the disease (when metastases to regional lymph nodes are detected in patients), the expression of MMP-2, VEGF and VEGFR2 increases markedly both in the tumor and in the morphologically normal tissue surrounding the tumor and makes an additional contribution to the processes of destruction, angiogenesis and metastasis. We assume that MMP-2, VEGF and VEGFR2 can be considered as negative markers of the course of CSCC.","PeriodicalId":286037,"journal":{"name":"Biomedical Chemistry: Research and Methods","volume":"739 1","pages":"0"},"PeriodicalIF":0.0,"publicationDate":"1900-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"132451697","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
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