[Combination of ARE and HRE cis- Regulatory Elements Elevates the Activity of Tumor-Specific hTERT Promoter].

Tumor-specific promoters and cis-regulatory genetic elements are used for transcriptional control of therapeutic transgene expression in cancer gene therapy. HRE (hypoxia response element) and ARE (anti-oxidant response elements) cis-regulatory elements are targets for HIF1 and Nrf2 transcriptional factors, respectively, and mediate activation of gene transcription in a response to hypoxia and oxidative stress, characteristic of most solid tumors. Due to these features HREs and AREs are used in genetic constructs for cancer gene therapy to provide tumor-specific therapeutic transgene expression or replication of oncolytic adenovi-ruses. In this work on the basis of the tumor-specific promoter hTERT we have constructed hybrid promoters carrying combinations of HRE and ARE. We showed that upon imitation of hypoxia in human lung cancer cell lines the activity of the hybrid promoter HRE-ARE-hTERT is substantially higher compared to promoters carrying only ARE or HRE. Using in vitro suicide cancer gene therapy with the CD: UPRT/5-FC (cytosine deaminase; uracil phosphoribosyl transferase/5-fluorocytosine) enzyme-prodrug system as a model we showed an enhancement of the cytotoxic effect on human lung cancer cells upon imitation of hypoxia when cytosine deaminase: uracil phosphoribosyl transferase was expressed under the control of the HRE-ARE-hTERT promoter compared to HRE-hTERT and ARE-hTERT promoters. The novel hybrid promoter HRE-ARE-hTERT could be used for transcriptional targeting of therapeutic transgene expression or oncolytic adenovirus replication upon development of novel anti-cancer gene therapeutics.

Xenobiotic Response Elements (XREs) from Human CYP1A1 Gene Enhance the hTERT Promoter Activity.

A hybrid 6XRE-hTERT promoter consisting of the hTERT tumor-specific promoter and six copies of the XRE element from the CYP1A1 human gene promoter was created. Using a human lung cancer cells as a model, we showed that XRE elements in the hybrid promoter greatly increase the activity of the hTERT promoter and ensure the reporter gene transcriptional activation in response to the treatment of the cells with the AhR ligand benzo(a)pyrene. However, similar effects were also observed in normal human bronchial epithelial cells HBEpC, which indicates the loss of the tumor-specific activity by the 6XRE-hTERT hybrid promoter. XRE elements can be used for nonspecific transcription enhancement but are unsuitable for the creation of tumor-specific promoters with enhanced activity.

[Human Antithrombin III Minigene with an Optimized Splicing Pattern].

Antithrombin III (AT3) belongs to the superfamily of serine protease inhibitors (serpins) and is a major anticoagulant in physiological conditions. Based on SERPINC1 gene, a minigene coding for human AT3, which is valuable for medicine and biotechnology, was constructed by minimizing the size of lengthy introns and preserving the splicing site-flanking sequences. An analysis of the minigene splicing pattern identified one correct AT3 transcript and two alternatively spliced transcripts, which formed either due to minigene exons 2 and 3 skipping or an aberrant exon insertion via splicing at cryptic splicing sites in intron 1 of the minigene. Site-directed mutagenesis of the cryptic splicing sites successfully optimized the splicing pattern of the AT3 minigene to completely prevent the generation of the alternative transcripts. The presence of the cryptic splicing sites in intron 1 of the minigene was confirmed with Human Splicing Finder v. 3.1 software, thus demonstrating that putative alternative splicing sites are possible to identify in minimized or hybrid introns of minigenes and to eliminate via mutagenesis before experimentally testing the minigene splicing patterns. The approach to the design of minigenes together with the bioinformatical analysis of the nucleotide sequences of minigene introns can be used to construct minigenes in order to generate transgenic animals producing economically valuable proteins in the milk.

Production of Recombinant Proteins in the Milk of Transgenic Animals: Current State and Prospects.

The use of transgenic animals as bioreactors for the synthesis of the recombinant proteins secreted into milk is a current trend in the development of biotechnologies. Advances in genetic engineering, in particular the emergence of targeted genome editing technologies, have provided new opportunities and significantly improved efficiency in the generation of animals that produce recombinant proteins in milk, including economically important animals. Here, we present a retrospective review of technologies for generating transgenic animals, with emphasis on the creation of animals that produce recombinant proteins in milk. The current state and prospects for the development of this area of biotechnology are discussed in relation to the emergence of novel genome editing technologies. Experimental and practical techniques are briefly discussed.

[Insertion of Multiple Artificial Introns of Universal Design into cDNA during Minigene Construction Assures Correct Transgene Splicing].

The presence of introns is often required for efficient transgene expression. The use of full-length genes for transgenesis is associated with technical difficulties due to the large size of the genetic construct. To solve this problem, we recently suggested a universal design of small artificial introns that ensures efficient splicing. However, the insertion of more than one intron into cDNA might result in the aberrant splicing of the minigene with exon skipping. Here, we showed that the insertion of two artificial introns of universal design into cDNA resulted in a splicing pattern that corresponds to the excision of each intron with an exon between them remaining in the transcript. No transcript formation with exon skipping was detected. Therefore, the developed design of small artificial introns assures splicing solely between the donor and the acceptor splice sites of each single intron and results in the generation of a correct transcript from minigene pre-mRNA. These findings enable the construction of minigenes for transgenesis with more than one artificial intron, with no additional cis-elements required to prevent aberrant splicing.

Characterization of the lipopolysaccharide of Escherichia coli 126.

The lipopolysaccharide (LPS) of Escherichia coli 126 was isolated and studied. The lipid A fatty acid composition of the investigated LPS was similar to that of other members of the family Enterobacteriaceae. The E. coli 126 LPS was more toxic than the LPSs of previously studied E. coli strains and of other members of the Enterobacteriaceae (Budvicia aquatica and Pragia fontium), and was less pyrogenic than pyrogenal. SDS-PAG electrophoresis showed a bimodal distribution typical of S-form LPSs. The LPS of E. coli 126 decreased the adhesive index indicating a possible competition between LPS molecules of E. coli 126 and adhesins of E. coli F-50 on rabbit erythrocytes. The LPS of E. coli 126 in a homologous system showed antigenic activity in the reactions of double immunodiffusion in agar by Ouchterlony. No serological cross-reaction of the LPS of other E. coli strains, as well as of that of the B. aquatica type strain, with the antiserum to E. coli 126 was observed. The structural components of the lipopolysaccharide obtained by mild acid hydrolysis were lipid A, the core oligosaccharide, and the O-specific polysaccharide. Based on the data of monosaccharide analysis and 1H and 13C NMR spectroscopy it was found that the O-specific polysaccharide had the structure characteristic of the representatives of E. coli serogroup O15.

A Novel Hybrid Promoter ARE-hTERT for Cancer Gene Therapy.

describe a novel hybrid tumor-specific promoter, ARE-hTERT, composed of the human TERT gene promoter (hTERT) and the antioxidant response element (ARE) from the human GCLM gene promoter. The hybrid promoter retains the tumor specificity of the basal hTERT promoter but is characterized by an enhanced transcriptional activity in cancer cells with abnormal activation of the Nrf2 transcription factor and upon induction of oxidative stress. In the in vitro enzyme-prodrug cancer gene therapy scheme, ARE-hTERT promoter-driven expression of CD : UPRT (yeast cytosine deaminase : uracil phosphoribosyltransferase) chimeric protein induced a more pronounced death of cancer cells either upon treatment with 5-fluorouracil (5FC) alone or when 5FC was combined with chemotherapeutic drugs as compared to the hTERT promoter. The developed hybrid promoter can be considered a better alternative to the hTERT promoter in cancer gene therapy schemes.

[Selection of microRNA for providing tumor specificity of transgene expression in cancer gene therapy].

The use of tumor-specific microRNA loss to inhibit transgene expression in normal cells is considered as a way to increase the specificity of gene-therapeutic antitumor drugs. This method assumes the introduction of recognition sites of suppressed in tumor cells microRNAs into transgene transcipt. In the presented work, the efficiency of the strategy for providing the tumor specificity of transgene expression depending on parameters of microRNA expression in normal and tumor cells was studied. It was established that microRNA suppression in tumor cells and the determination of absolute microRNA levels in tumor and normal cells are not sufficient for the adequate estimation of the possibility of specific microRNA usage in the scheme of cancer gene therapy, and particularly do not allow to exclude a significant decrease in the efficiency of the gene-therapeutic drug upon the introduction of microRNA recognition sites. These parameters are only suitable for the preliminary selection of microRNA. The effect of introduction of microRNA recognition sites on transgene expression level in target tumor cells should be validated experimentally. It is suggested that this should be done directly in the cancer gene therapy scheme with monitoring of the therapeutic transgene activity.

Interaction between MAK-V protein kinase and synaptopodin.

MAK-V protein kinase (also known as HUNK) was discovered more than decade ago but its functions and molecular mechanisms of action still remain mostly unknown. In an attempt to associate MAK-V with particular chains of molecular events, we searched for proteins interacting with the C-terminal domain of MAK-V protein kinase. We identified synaptopodin as a protein interaction partner for MAK-V and confirmed this interaction in various ways. Because synaptopodin is important for dendritic spine formation and plays a role in synaptic plasticity, our results might have significant impact on future studies for understanding the role of MAK-V in cells of the nervous system.

Membrane localization of the MAK-V protein kinase.

Activities of many proteins including protein kinases are often regulated by their dynamic association with specific intracellular compartments. MAK-V is an AMPK-like protein kinase with poorly characterized functions and mechanisms of action. Similarly to many other protein kinases, association of MAK-V with specific intracellular compartments could be essential for its proper functions. In this work, we studied subcellular distribution of exogenously produced and endogenous MAK-V proteins in mammalian cells using biochemical cell fractioning aiming to supplement data on MAK-V intracellular localization studied by immunocytochemical methods. We found that a significant portion of MAK-V protein in mammalian cells is associated with membranes. Moreover, MAK-V expressed in yeast was also targeted to membrane, thus suggesting an evolutionarily conservative mechanism of MAK-V membrane association. Based on the ability of various MAK-V deletion mutants to localize to membrane and comparison of MAK-V amino acid sequences from different species, we suggest a possible mechanism governing MAK-V association with intracellular membranes.

[Suppression of WIFI transcript and protein in non-small cell lung carcinomas].

Changes in WIFI expression, an extracellular inhibitor of Wnt pathway, in non-small cell lung carcinomas were analyzed. Frequent (67% cases) suppression of WIFI transcript in non-small cell lung carcinomas were found. Our results, together with previously published data, suggest that inhibition of WIFI expression often occurs in squamous cell carcinomas and is less typical of adenocarcinomas. It was also found that a decrease in the WIFI transcript in tumors is parallel to concomitant suppression of the WIFI protein level. Our results provide further evidence that the WIFI suppression is a frequent event in the lung carcinogenesis, which might lead to disregulation of Wnt signaling pathway and contribute to tumor progression.

[Solid phase ligation of synthetic DNA fragments]. / Tverdofaznoe ligirovanie sinteticheskikh fragmentov DNK.

A synthetic oligodeoxyribonucleotide (oligo) covalently bound by an internucleotide linkage to the succinylated Sephacryl S-500 support through 1.9-diaminononane spacer was used as starting compound to assemble the E. coli rec A promoter DNA fragment from synthetic oligos by means of T4 DNA ligase. The solid-phase assembly of the designed DNA was performed by two ways: stepwise ligation of two pairs of oligos (2 dyads) or simultaneous ligation of four oligos (tetrad). Both ways gave equal results with some preference in the tetrad case. The reliability of E. coli promoter DNA fragment assembly was demonstrated by cloning it in a plasmid vector and sequencing the cloned DNA by the solid-phase Maxam--Gilbert technique.

[Regional hemodynamics of the penis in erectile impotence]. / Regionarnaia gemodinamika polovogo chlena pri érektil'noi impotentsii.

Regional hemodynamics of the penis is an essential part in the complex investigation of patients with erectile impotence. It is an actual problem to find diagnostically valuable noninvasive techniques allowing one to make a reliable assessment of penile erection mechanisms in various groups of the patients. In this line the authors faced the problem to find out the diagnostic value of dopplerography in measuring the linear rate of the blood flow (LRBF) in the penile arteries during the complex study of penile hemodynamics with regard to various groups of patients. The authors employed the following techniques: measurement of penile systolic pressure; determination of the penile-brachial index; rheophallography and determination of rheographic index; dopplerography and LRBF measurements. A total of 60 males were under study: group I--controls (20 males); group II--sexologically compromised patients without any disease of major vessels; group III--patients with Leriche's syndrome. The findings were analysed in variance. The coefficient of paired correlation was derived. Comparing each parameter between the groups showed a significant difference in each method between groups 2 and 3, as for parameters, only LRBF varied greatly in these groups.

[Chemical-enzymatic synthesis and cloning of DNA, coding the signal for secretion of proteins in gram-negative bacteria]. / Khimiko-fermantativnyi sintez i klonirovanie DNK, kodiruiushchei signal dlia sekretsii belkov v gramootritsatel'nykh bakteriaiakh.

Chemical-enzymatic synthesis of an artificial gene encoding leader peptide and 22 N-terminal amino acids of mature carboxypeptidase G2 from Pseudomonas sp. followed by enterokinase signal sequence (Asp4Lys) has been accomplished. The resulted DNA was fused with semi-synthetic gene coding for polypeptide 4-157 of mature human tumour necrosis factor (TNF) and then placed under control of early promoters of T7 bacteriophage. The expression products of the construct obtained was analysed using anti-TNF anti-serum. In E.coli leader peptide was cleaved off during translocation through inner membrane and the resultant product was found in membrane fraction.

[Synthesis of 5'-phosphorylated oligodeoxyribonucleotides by the H-phosphonate method]. / Sintez 5'-fosforilirovannykh oligodezoksiribonukleotidov H-fosfonatnym metodom.

N,O-protected 2'-deoxyribonucleotide-3'-H-phosphonates and 2-(p-nitrophenylethyl)-H-phosphonate were prepared and used in solid-phase synthesis of 5'-phosphorylated DNA. H-Phosphonate condensation is performed with 1:2 ratio of P-component to activator (pivaloyl chloride). Protection groups were removed either by sequential treatment with conc. NH3 and DBU or by combination of these reagents.