Removal of Double-Stranded RNA Contaminants During Template-Directed Synthesis of mRNA.

The removal of double-stranded RNA (dsRNA) contaminants during in vitro mRNA synthesis is one of the technological problems to be solved. Apparently, these contaminants are the result of the T7 RNA polymerase side activity. In this study, we used a modified method of mRNA purification based on the selective binding of dsRNA to cellulose in ethanol-containing buffer. It was shown both in vivo and in vitro that the cellulose-purified mRNA preparation leads neither to activation of the lymphocyte inflammatory marker CD69 nor to increased release of IFNα in mice, and does not contain impurities detectable by antibodies to dsRNA.

Biochemical and technological properties of moose (Alces alces) recombinant chymosin.

Recombinant chymosins (rСhns) of the cow and the camel are currently considered as standard milk coagulants for cheese-making. The search for a new type of milk-clotting enzymes that may exist in nature and can surpass the existing "cheese-making" standards is an urgent biotechnological task. Within this study, we for the first time constructed an expression vector allowing production of a recombinant analog of moose chymosin in the expression system of Escherichia coli (strain SHuffle express). We built a model of the spatial structure of moose chymosin and compared the topography of positive and negative surface charges with the correspondent structures of cow and camel chymosins. We found that the distribution of charges on the surface of moose chymosin has common features with that of cow and camel chymosins. However, the moose enzyme carries a unique positively charged patch, which is likely to affect its interaction with the substrate. Biochemical and technological properties of the moose rChn were studied. Commercial rСhns of cow and camel were used as comparison enzymes. In some technological parameters, the moose rChn proved to be superior to the reference enzymes. Сompared with the cow and camel rСhns, the moose chymosin specific activity is less dependent on the changes in CaCl2 concentration in the range of 1-5 mM and pH in the range of 6-7, which is an attractive technological property. The total proteolytic activity of the moose rСhn occupies an intermediate position between the rСhns of cow and camel. The combination of biochemical and technological properties of the moose rСhn argues for further study of this enzyme.

[Research of the structure of the mineral component of tooth enamel in connective tissue dysplasia by densitometry and atomic force microscopy in the early postpartum ontogenesis period]. / Issledovanie struktury mineral'nogo komponenta emali zubov pri displazii soedinitel'noi tkani metodami densitometrii i atomno-silovoi mikroskopii v rannem postnatal'nom periode ontogeneza.

OBJECTIVE: To give a quantitative and qualitative characteristic of the structure of the enamel's mineral component structure of impacted teeth with or without connective tissue dysplasia in different periods of early postnatal human ontogenesis using densitometry and atomic force microscopy. MATERIALS AND METHODS: The study involved 120 males with and without connective tissue dysplasia (CTD), which were divided into 3 equal subgroups (60 people with CTD and 60 people without CTD), 20 people in each, according to age: 15-20, 21-30, 31-40 years old. Each of the examined was removed either 3.8 or 4.8 tooth. To study the inorganic component of tooth enamel, a densitometric assessment of enamel's optical density was carried out using computed tomography in the Kodak Dental Systems software (Trophy 2000) and preparation of thin sections of tooth samples 3.8 or 4.8 for atomic force microscopy (AFM) according to the methods of Omsk State Medical University. RESULTS: The structure of tooth enamel in connective tissue dysplasia in the early postpartum period of ontogenesis is characterized by pronounced polymorphisms and an insufficient level of maturity. The ordering and orientation of the enamel prisms are disturbed due to insufficient packing density and a large distance between the enamel prisms at the age of 15-20, 21-30. The established changes indicate the incomplete nature of amelogenesis with connective tissue dysplasia at the indicated ages. CONCLUSIONS: In case of connective tissue dysplasia in the early postnatal period of ontogenesis, an incomplete amelogenesis is observed. This process is manifested by lower values of the mineral component's optical density, low packing of enamel prisms, a large distance between enamel prisms and their irregular shape.

Characterization of the Altai Maral Chymosin Gene, Production of a Chymosin Recombinant Analog in the Prokaryotic Expression System, and Analysis of Its Several Biochemical Properties.

For the first time, the chymosin gene (CYM) of a maral was characterized. Its exon/intron organization was established using comparative analysis of the nucleotide sequence. The CYM mRNA sequence encoding a maral preprochymosin was reconstructed. Nucleotide sequence of the CYM maral mRNA allowed developing an expression vector to ensure production of a recombinant enzyme. Recombinant maral prochymosin was obtained in the expression system of Escherichia coli [strain BL21 (DE3)]. Total milk-coagulation activity (MCA) of the recombinant maral chymosin was 2330 AU/ml. The recombinant maral prochymosin relative activity was 52955 AU/mg. The recombinant maral chymosin showed 100-81% MCA in the temperature range 30-50°C, thermal stability (TS) threshold was 50°C, and the enzyme was completely inactivated at 70°C. Preparations of the recombinant chymosin of a single-humped camel and recombinant bovine chymosin were used as reference samples. Michaelis-Menten constant (Km), turnover number (kcat), and catalytic efficiency (kcat/Km) of the recombinant maral chymosin, were 1.18 ± 0.1 µM, 2.68 ± 0.08 s-1 and 2.27± 0.10 µm M-1·s-1, respectively.

[Isolation and phylogenetic analysis of feline calicivirus in Siberia.]

The results of the study of the distribution of calicivirus infection in a population of domestic cats of different breeds, contained individually or the group method, the virus isolation in the cell culture and a comparative phylogenetic analysis of their nucleotide sequences with published sequences of reference feld and vaccine strains of Feline calicivirus (FCV) from other countries: USA, Germany, Japan, China and Korea are presented. Clinical signs of infection were found in 14.3% of the animals examined. After several passages in the primary kidney cells of the kitten embryo, seven cytopathogenic isolates FCV were isolated: 1 - from a cat with an acute infection, 5 - subclinical infection, 1 - systemic infection. They were adapted to continuous FK-81 cells in which they reached a maximum infectious activity of 10.0 ± 1.15 lg TCD 50 / cm3. Based on the sequence analysis of the open reading frame 2 region of the viral genome Eshli strain showed a close relationship with strain KM016908 from China with the identity of the nucleotide sequences between them of 81.0%. The results of the investigations showed that FCV isolates obtained from animals on the territory of Siberia are genetically different from strains included to imported vaccines used to prevent disease in Russian Federation and also among themselves. This causes a decrease in the effectiveness of preventive measures. In nurseries that do not have contacts and connections between themselves but located in the same geographic region FCV populations may have some genetic differences. A close relationship of some feld isolates with strains from other countries geographically located so far from the Siberian region has been revealed. Studies on the molecular epizootology of caliciviruses are important in the development of test systems and the monitoring of the spread of strains in Russia.

[Dynamics of LINE-1 Retrotransposon Methylation Levels in Circulating DNA from Lung Cancer Patients Undergoing Antitumor Therapy].

Malignant cell transformation is accompanied with abnormal DNA methylation, such as the hypermethylation of certain gene promoters and hypomethylation of retrotransposons. In particular, the hypomethylation of the human-specific family of LINE-1 retrotransposons was observed in lung cancer tissues. It is also known that the circulating DNA (cirDNA) of blood plasma and cell-surface-bound circulating DNA (csb-cirDNA) of cancer patients accumulate tumor-specific aberrantly methylated DNA fragments, which are currently considered to be valuable cancer markers. This work compares LINE-1 retrotransposon methylation patterns in cirDNA of 16 lung cancer patients before and after treatment. CirDNA was isolated from blood plasma, and csb-cirDNA fractions were obtained by successive elution with EDTA-containing phosphate buffered saline and trypsin. Concentrations of methylated LINE-1 region 1 copies (LINE-1-met) were assayed by real-time methylation-specific PCR. LINE-1 methylation levels were normalized to the concentration of LINE-1 region 2, which was independent of the methylation status (LINE-1-Ind). The concentrations of LINE-1-met and LINE-1-Ind in csb-cirDNA of lung cancer patients exhibited correlations before treatment (r = 0.54), after chemotherapy (r = 0.72), and after surgery (r = 0.83) (P < 0.05, Spearman rank test). In the total group of patients, the level of LINE-1 methylation (determined as the LINE-1-met/LINE-1-Ind ratio) was shown to increase significantly during the follow-up after chemotherapy (P < 0.05, paired t test) and after surgery compared to the level of methylation before treatment (P < 0.05, paired t test). The revealed association between the level of LINE-1 methylation and the effect of antitumor therapy was more pronounced in squamous cell lung cancer than in adenocarcinoma (P < 0.05 and P > 0.05, respectively). These results suggest a need for the further investigation of dynamic changes in levels of LINE-1 methylation depending on the antitumor therapy.

On mechanism of antioxidant effect of fullerenols.

Fullerenols are nanosized water-soluble polyhydroxylated derivatives of fullerenes, specific allotropic form of carbon, bioactive compounds and perspective pharmaceutical agents. Antioxidant activity of fullerenols was studied in model solutions of organic and inorganic toxicants of oxidative type - 1,4-benzoquinone and potassium ferricyanide. Two fullerenol preparations were tested: С60О2-4(ОН)20-24 and mixture of two types of fullerenols С60О2-4(ОН)20-24+С70О2-4(ОН)20-24. Bacteria-based and enzyme-based bioluminescent assays were used to evaluate a decrease in cellular and biochemical toxicities, respectively. Additionally, the enzyme-based assay was used for the direct monitoring of efficiency of the oxidative enzymatic processes. The bacteria-based and enzyme-based assays showed similar peculiarities of the detoxification processes: (1) ultralow concentrations of fullerenols were active (ca 10-17-10-4 and 10-17-10-5 g/L, respectively), (2) no monotonic dependence of detoxification efficiency on fullerenol concentrations was observed, and (3) detoxification of organic oxidizer solutions was more effective than that of the inorganic oxidizer. The antioxidant effect of highly diluted fullerenol solutions on bacterial cells was attributed to hormesis phenomenon; the detoxification was concerned with stimulation of adaptive cellular response under low-dose exposures. Sequence analysis of 16S ribosomal RNA was carried out; it did not reveal mutations in bacterial DNA. The suggestion was made that hydrophobic membrane-dependent processes are involved to the detoxifying mechanism. Catalytic activity of fullerenol (10-8 g/L) in NADH-dependent enzymatic reactions was demonstrated and supposed to contribute to adaptive bacterial response.

Is bacterial luminescence response to low-dose radiation associated with mutagenicity?

Luminous marine bacteria are widely used in bioassays with luminescence intensity being a physiological parameter tested. The purpose of the study was to determine whether bacterial genetic alteration is responsible for bioluminescence kinetics change under low-dose radiation exposure. The alpha-emitting radionuclide 241Am and beta-emitting radionuclide 3H were used as the sources of low-dose ionizing radiation. Changes of bioluminescence kinetics of Photobacterium phosphoreum in solutions of 241Am(NO3)3, 7 kBq/L, and tritiated water, 100 MBq/L, were studied; bioluminescence kinetics stages (absence of effect, activation, and inhibition) were determined. Bacterial suspension was sampled at different stages of the bioluminescent kinetics; the doses accumulated by the samples were close or a little higher than a tentative limit of a low-dose interval: 0.10 and 0.85 Gy for 241Am, or 0.11 and 0.18 Gy for 3H. Sequence analysis of the 16S ribosomal RNA gene did not reveal a mutagenic effect of low-dose alpha and beta radiation in the bacterial samples. Previous results on bacterial DNA exposed to low-dose gamma radiation (0.25 Gy) were analyzed and compared to those for alpha and beta irradiation. It is concluded that bioluminescence activation and/or inhibition under the applied conditions of low-dose alpha, beta and gamma radioactive exposure is not associated with DNA mutations in the gene sequences tested.

Exposure of luminous marine bacteria to low-dose gamma-radiation.

The study addresses biological effects of low-dose gamma-radiation. Radioactive 137Cs-containing particles were used as model sources of gamma-radiation. Luminous marine bacterium Photobacterium phosphoreum was used as a bioassay with the bioluminescent intensity as the physiological parameter tested. To investigate the sensitivity of the bacteria to the low-dose gamma-radiation exposure (≤250 mGy), the irradiation conditions were varied as follows: bioluminescence intensity was measured at 5, 10, and 20°Ð¡ for 175, 100, and 47 h, respectively, at different dose rates (up to 4100 µGy/h). There was no noticeable effect of gamma-radiation at 5 and 10°Ð¡, while the 20°Ð¡ exposure revealed authentic bioluminescence inhibition. The 20°Ð¡ results of gamma-radiation exposure were compared to those for low-dose alpha- and beta-radiation exposures studied previously under comparable experimental conditions. In contrast to ionizing radiation of alpha and beta types, gamma-emission did not initiate bacterial bioluminescence activation (adaptive response). As with alpha- and beta-radiation, gamma-emission did not demonstrate monotonic dose-effect dependencies; the bioluminescence inhibition efficiency was found to be related to the exposure time, while no dose rate dependence was found. The sequence analysis of 16S ribosomal RNA gene did not reveal a mutagenic effect of low-dose gamma radiation. The exposure time that caused 50% bioluminescence inhibition was suggested as a test parameter for radiotoxicity evaluation under conditions of chronic low-dose gamma irradiation.

Unique configuration of genes of silicon transporter in the freshwater pennate diatom Synedra acus subsp. radians.

The existence of the cluster of duplicated sit silicon transporter genes in the chromosome of the diatom Synedra acus subsp. radians was shown for the first time. Earlier, the localization of sit genes in the same chromosome and cluster formation caused by gene duplication was shown only for the marine raphid pennate diatom P. tricornutum. Only non-clustered sit genes were found in the genomes of other diatoms. It is reasonable to assume that sit tandem (sit-td) and sit triplet (sit-tri) genes of S. acus subsp. radians occurred as a result of gene duplication followed by divergence of gene copies.

[ Bioluminescent assay to detect melanocortin-1 receptor (MC1R) polymorphisms (R160W, R151C, and D294H)].

Several polymorphisms in melanocortin-1 receptor (MC1R) gene are shown to have associations with melanoma risk. In particular, rs1805007, rs1805008, and rs1805009 mutations causing the corresponding R151C, R160W, and D294H changes and associated with the phenotype ("red-hair mutations") are connected with melanoma and non-melanoma skin cancer risks. The work describes the approach to detect these polymorphisms based on primer extension reaction with the following dual bioluminescent assay. Model plasmids with polymorphic MC1R fragments as well as several clinical DNA samples were tested using the developed technique. The results were in good correlation with those obtained by Sanger sequencing.

[Phenotypic variation of spermatogenesis and a search for associations with genetic polymorphism in 13 inbred mouse strains].

Adult mice of the BALB/cLac, PT, CBA/Lac, DD/He, A/He, SWR, NZB, GR, DBA/2J, CC57Br, C57 B1/6J, A/Sn, and YT inbred strains were tested for the count, motility, and morphology of sperms from the caudal region of the epididymis. The protein-coding regions of the cytochrome P450 aromatase (CYP19a1), estrogen receptor 2 (ESR2), steroidogenic factor 1 (Nr5a1), and sex-determining (Sry) gene were sequenced. A substantial genetic heterogeneity for the genes was observed, as well as a phenotypic variation in spermatogenetic parameters, but the variation was rather discordant. The specifics of the interstrain variation in spermatogenetic parameters indicated that a physiological compensatory mechanism increases certain spermatogenetic parameters when other ones are low to maintain male fertility at a level sufficient for successful reproduction. For instance, a high sperm production compensated for a low sperm motility in DD/He males. In the issue of the protein-coding regions sequencing of the analyzed genes, 16 various mutations were observed. The decreases in proportion of motile sperms and in their velocity were attributed to mutations (I63T and W133L) of the Sry gene in the DD/He strain.

[Mutation analysis of the phenylalanine hydroxylase gene of phenylketonuria patients of Kemerovskaya Oblast' and Saha Republic].

Phenylketonuria (PKU) associated mutations in phenylalanine hydroxylase (PAH) gene were identified by direct DNA sequencing in 46 PKU patients and members of their families from Kemerovskaya Region and Saha Republic. Mutations found included both widespread known mutations (R158Q, R252W, R261Q, P281L, IVS10-11G>A, R408W, IVS12+1G>A) and several rare mutations (IVS2+5G>A, R155H, Y168H, W187R, E221_D222>Efs, A342T, Y386C, IVS11+1G>C). We observed the increase in diversity of PKU-associated alleles in the populations studied, probably due to their complex mixed ethnic structure.

Identification of new M23A mRNA of mouse aquaporin-4 expressed in brain, liver, and kidney.

Aquaporins (AQPs) belong to a transmembrane protein family of water channels that are permeable to water by the osmotic gradient. There are two isoforms of mouse AQP4 - M1 and M23. Their balance in the cell determines water permeability of the plasma membrane. These two isoforms are encoded by three mRNAs: M1 isoform is encoded by M1 mRNA and M23 isoform is encoded by M23 and M23X mRNAs. Here we found a new fourth mRNA of mouse AQP4 - M23A mRNA. The start of transcription is different for M23A mRNA from all the known AQP4 mRNAs. The 5'-untranslated region (5'-UTR) of M23A mRNA is encoded by four new exons (A, B, C, and D), which are located in the 5' region from exon-0 of the AQP4 gene. Alternative splicing between the exons-A, -B, -C, and -D leads to formation of multiple variants of M23A mRNA. We cloned six of these variants, all of which code full length M23 isoform of AQP4. Using RT-PCR we detected tissue-specific expression of the new M23A and already known M23, M23X, and M1 mRNAs. The M23A mRNA is expressed mostly in kidney, liver, and brain. Analysis of mRNA 5'-UTR structure showed low translation efficacy for M1 mRNA in comparison with high translation efficacy for M23A, M23X, and M23 mRNAs. We propose that AQP4 expression is controlled tissue-specifically by independent promoters. Thus multiple AQP4 mRNAs may allow long-term regulation of the balance between M1 and M23 AQP4 isoforms in the cell and thus water permeability of the plasma membrane.

[Effect of dDAVP, agonist of V2 vasopressin receptor on the hyaluronidase-1 and hyaluronidase-2 gene expression in the kidneys of Wistar and Brattleboro rats].

Effect of vasopressin on the expression of Hyal-1 and Hyal-2 genes in different functional zones of Wistar and homozygous vasopressin-defficient Brattlboro rat kidneys was analysed using RT-PCR mehod. It was found that, in Wistar rats the content of Hyal-1 mRNA was higher in the medulla than in other kidney zones at the normal water and food regimen. The level of Hyal-1 mRNA in the cortex and the medulla of Brattlboro rat kidney exceeded that of papilla. There were no significant differences in the Hyal-2 mRNA content detected between functional zones of Wistar and Brattlboro rat kidneys. The treatment by dDAVP, the agonist of V2 vasopressin receptor (Desmopressin, 10 microg/100 g b.w.i.p. twice a day for two days) induced an increase in urine osmolality and significant increase in the Hyal-1 and Hyal-2 mRNA content in the medulla without changes in the cortex and papilla. The effect was more pronounced in Brattlboro rat kidney. These results demonstrate that, in control conditions, genes encoding Hyal-1 and Hyal-2 were expressed independently in all functional kidney zones in the both in normal Wistar and in vasopressin-defficient Brattlboro rats. Desmopressin (dDAVP) exerts a stimulating effect on Hyal-1 and Hyal-2 gene expression in the medulla.

Functional expression and properties of Sec14p-like protein with molecular mass 45 kD from rat olfactory epithelium.

cDNA of Sec14p-like water-soluble protein with molecular mass 45 kD from rat olfactory epithelium was expressed in Escherichia coli Rosetta cells. The expression product was purified by a two-step chromatographic procedure on DEAE-Sepharose and Sephacryl S-200. The identity of structural and functional characteristics of the recombinant and native proteins was demonstrated by CD, mass spectrometry, and Western blotting. Using several lipids immobilized on nitrocellulose membranes, it was shown that phosphatidylinositol-3,4,5-triphosphate is the specific ligand for the studied protein.

Recombinant human peroxiredoxin VI: preparation and protective properties in vitro.

cDNA of human peroxiredoxin VI, one of the recently discovered novel antioxidant proteins, was expressed in Escherichia coli cells. The expression product was obtained in water-soluble form and purified by a two-step chromatographic procedure using DEAE-Sepharose and Sephacryl S-200. According to CD data, the polypeptide chain of the recombinant human peroxiredoxin VI contains approximately 40% alpha-helical region and 30% beta-structure, which is the same as for native rat peroxiredoxin VI. The protective properties of the recombinant protein determined as its ability to prevent the inactivation of glutamine synthetase from E. coli in a model oxidation system were comparable with the protective properties of native rat peroxiredoxin VI.

[The isolation and expression in E. coli of a recombinant plasmid containing the 3'-terminal fragment of the cDNA of the influenza A virus nucleoprotein gene]. / Poluchenie i ékspressiia v E. coli rekombinantnoi plazmidy, soderzhashchei 3'-kontsevoi fragment kDNK gena nukleoproteina virusa grippa A.

A recombinant plasmid pN62 determining the synthesis of a hybrid protein consisting of a full-size beta-galactosidase and C-terminus fragment of influenza A virus nucleoprotein was constructed. The complete identity of pN62 insert with the 3'-terminus cDNA fragment of influenza A virus NP-gene and conservation of beta-galactosidase reading frame was confirmed by Maxam-Gilbert sequencing of pN62. An expression of pN62 plasmid in E. coli JM103 in the presence of IPTG resulted in accumulation of fused protein as poorly soluble inclusion bodies in the bacterial cells. Analysis of E. coli JM103/pN62 bacteria lysates by 7% PAGE revealed that molecular weight of hybrid polypeptide was 18 kDa heavier than normal beta-galactosidase and corresponded to the previously deduced weight of 135 kDa.