Assessment of Autophagy in Tumor Cells of Human Skin Melanoma of Different Stages.

High levels of autophagy can increase the viability of tumor cells as well as their resistance to chemotherapy. Evaluation of the dynamics of autophagy processes at different stages of carcinogenesis can extend our understanding of melanoma pathogenesis to develop new therapeutic approaches. We performed a comparative study of tumor cell autophagy in stages II and III human skin melanoma. Tumor cells were characterized by high content of structures associated with autophagy (autophagosomes and autolysosomes). In stage III melanoma characterized by the presence of regional metastases in the lymph nodes, tumor cells showed higher expression of the autophagy marker protein LC3beta in comparison with stage II melanoma cells, which can indicate the involvement of autophagy processes in tumor progression and the formation of metastases in the lymph nodes.

Expression of Molecular Markers of Angiogenesis, Lymphangiogenesis, and Proliferation Depending on the Stage of Skin Melanoma.

The expression of molecular markers characterizing activity of the tumor process and metastases (proliferation marker Ki-67, angiogenesis marker CD34, and lymphangiogenesis markers podoplanin and LYVE-1) was assessed by immunohictochemical method in the primary tumor specimens collected during surgery for cutaneous melanoma (40 patients). Proliferative activity of the tumor tissue and volume density of peritumoral blood and lymph vessels increased with increasing tumor malignancy, which could indicate the risk of metastases.

[The realization of the program of medical care to patients with vascular pathology in the Central Russia: a three year experience of the Saratov Regional Vascular Center]. / Realizatsiia programmy po okazaniiu meditsinskoĭ pomoshchi patsientam s sosudistoĭ patologieĭ v Tsentral'noĭ Rossii: trekhletniĭ opyt regional'nogo sosudistogo tsentra Saratova.

UNLABELLED: Creation of vascular centers in Russian regions is one of the ways of prevention of spreading of vascular diseases. AIM: To analyze three year (2012-2014) activity of the Saratov Regional Vascular Center (RVS). MATERIAL AND METHODS: Several stages of creation and development of RVS as well as the progress achieved in the treatment of acute coronary and cerebrovascular pathology have been analyzed. RESULTS AND CONCLUSION: The realization of protocols, standards, procedures of medical care to patients with cerebrovascular diseases by RVS personnel, wide use of high-technology methods of diagnosis and treatment during 3 years allowed not only to achieve positive results but to find unrealized possibilities of the activity in this direction.

Application of universal primers for identification of Foot-and-mouth disease virus and Swine vesicular disease virus by PCR and PCR-ELISA.

Two approaches for simultaneous identification of both Foot-and-mouth disease virus (FMDV) and Swine vesicular disease virus (SVDV) are described: (1) a single-step reverse transcription-PCR with three primers and (2) a PCR-ELISA assay with two universal primers for genome amplification and two virus-specific probes for identification. These methods are based on the use of 3D gene universal PCR primers, the structure of which was optimized and refined due to the close relationship between the two viruses belonging to different genera of the Picornaviridae family. In procedure (1), a three-primer PCR containing one universal antisense primer and two virus-specific primers was shown to differentiate between FMDV and SVDV in one reaction, due to the different length of the amplified DNA fragments (600 and 340 base pairs, respectively). In procedure (2), the two viruses were identified by PCR-ELISA, i.e. PCR for the 3D gene followed by two parallel hybridizations with FMDV and SVDV-specific probes in microplate wells and ELISA detection. The application of universal primers could halve the number of PCR experiments in both cases, as compared to the usual virus-specific PCR procedures. Also, we investigated the 3D gene structure of several SVDV strains isolated at different times. No essential changes were detected in the regions coding for conserved motifs of the RNA-dependent RNA polymerase recognized by our universal primers. The multi-primer PCR was successfully tested on 38 FMDV and 15 SVDV strains, and the PCR-ELISA on 32 FMDV and 16 SVDV strains including clinical material from disease cases.

[Phylogenetic analysis of swine fever virus strains]. / Filogeneticheskii analiz shtammov virusa chumy plotoiadnykh.

Amplification of H-gene fragment in combination with cDNA nucleotide sequencing can be used for indication and strain differentiation of classical swine fever virus.

[The nucleotide sequences of the HN gene and F gene fragment of Newcastle disease virus strains BOR74 and BOR82]. / Nukleotidnye posledovatel'nosti gena HN i fragmenta gena F shtammov BOR74 i BOR82 virusa N'iukaslskoi bolezni.

The complete nucleotide sequence of HN gene, the region of F gene, and intergene regions (M-F, F-HN, and HN-L) of the BOR74 and BOR82 strains of Newcastle disease virus have been determined. Based on the nucleotide and amino acid sequences, the speeds of the nucleic and amino acid changes were calculated (approximately 10(-3) nucleotides or amino acids/year). The BOR strains were grouped phylogenetically with the asymptomatic strains. These strains and the BOR strains have the same motif of the cleavage site (112GKQGR116-L117), but the HN protein of BOR strains has the 572 amino acids which differ the BOR strains from all other strains (571, 577, and 616 amino acids).

[Molecular basis of changes in biological properties of foot and mouth disease virus of subtype A22]. / Izuchenie molekuliarnykh osnov izmeneniia nekotorykh biologicheskikh svoistv virusa iashchura podtipa A22.

Primary structure of capsid proteins and RNA polymerase of three closely related strains of foot and mouth disease virus (FMDV), subtype A22, differing by biological properties (the initial epitheliotropic strain A22 550 and its derivatives: thermoresistant myotropic A22 550/4 and thermosensitive attenuated A22 645) are compared by nucleic acid sequencing and analysis of the amino acid sequencing. The study revealed 1 substitute in VPI and 8 in RNA polymerase in the myotropic variant and 1 substitute in VP2, 2 in VP3, 13 in VP1, and 3 in RNA polymerase. Alteration of A22 550/4 tropism is probably due to a single substitution Gly 145-->Thr in the RGD site of capsid protein VP1. Analysis of the origin and biological properties of the attenuated strain A22 645 and the results of studies of the primary structure of proteins permit us to hypothesize that attenuation is polygenic, caused by adaptation to a heterologous host (continuous porcine cell culture), and can be expressed by changes in the structure of virus antireceptor providing its binding to cell receptors. Sites responsible for the reproduction of A22 FMDV at certain temperatures are presumably located in RNA polymerase.

[Comparative analysis of the VP2 variable region of the gene from infectious bursal disease virus isolates]. / Sravnitel'nyi analiz variabel'noi oblasti gena VP2 izoliatov virusa infektsionnoi bursal'noi bolezni.

Variable cDNA regions in the VP2 gene of 24 isolates of infectious bursal disease virus (IBDV) isolated in Russia in 1993-1996 were amplified by the "nested" PCR and sequenced. The primary structure analysis of the VP2 gene variable region revealed 2 major groups of IBDV isolates. The first group consisted of the isolates with the structure identical or closely related to the highly virulent European strains CS89, 74/89A, 661, JY86, and DV86, the second group included the isolates with a high level of homology to the vaccine strains PBG98 and Cu-1. In addition, two isolates with original structure were identified, which differed from previously studied strains.

[Automated synthesis of oligodeoxyribonucleotides. VI. Properties of segmental supports based on cellulose]. / Avtomaticheskii sintez oligodezoksiribonukleotidov. VI. Issledovanie svoistv segmentnykh nositelei na osnove tselliulozy.

The course of development of the rapid automated synthesis of oligodeoxyribonucleotides, various cellulose supports have been studied. Mild acid or aqueous ammonia treatment of Whatman or Filtrak paper disks is shown to improve characteristics of the supports, increasing the capacity in terms of the first immobilized nucleoside. New segmental supports based on cotton or flax were prepared and used successfully in automated synthesis of oligodeoxyribonucleotides on "Victoriya-2" synthesizer.

[Rapid automated synthesis on paper disks of oligodeoxyribonucleotides constituting the promoter fragment of the vaccinia virus genome]. / Bystryi avtomatizirovannyi sintez na bumazhnykh diskakh oligodeoksiribonukleotidov, sostavliaiushchikh promotornyi fragment genoma virusa ospovaktsiny.

For automation of segmental solid-phase synthesis a simple approach leading to the optimal scheme of synthesis of a large numbers of oligonucleotides in one reaction vessel has been proposed. An advantage of the scheme as compared with synthesis in four reaction vessels is a lower number of condensation steps and increased economy of the process. Sixteen oligodeoxyribonucleotides constituting promoter fragment of the viral genome have been synthesised by the modified segmental method on "Victoriya-2" synthesizer according to the optimal scheme.

[Chemico-enzymatic synthesis of genetic elements for expression of synthetic genes in Bacillus subtilis cells]. / Khimiko-fermentativnyi sintez geneticheskikh élementov dlia ékspressii iskusstvennykh genov v kletkakh Bacillus subtilis.

In order to obtain the recombinant Bacillus subtilis strain, a transcriptional-translational control unit of the alpha-amylase gene of B. amyloliquefaciens was synthesized. The oligodeoxyribonucleotides were prepared by the modified triester method in solution and by the solid-phase approach. Then these oligonucleotides were joined by DNA ligase into two fragments which were cloned in the phage M13mp9 DNA and the plasmid pBR327. A plasmid harboring the site regulating the transcription of the alpha-amylase gene may be employed as vector for cloning the promoter-containing fragments in E. coli cells.