Parkinson's Disease-Associated Changes in the Expression of Neurotrophic Factors and their Receptors upon Neuronal Differentiation of Human Induced Pluripotent Stem Cells.

Parkinson's disease (PD) is a neurodegenerative pathology resulting from the degeneration of dopaminergic (DA) neurons in the substantia nigra (SN). Neurotrophic factors (NTFs) and their receptors are key regulators of the survival, differentiation, and development of neurons. However, the role of these factors in the pathogenesis of PD is still unclear. Here, we analyzed the expression of NTFs and their receptors in human induced pluripotent stem cells (iPSCs) derived from the fibroblasts of patients with PD and healthy donors (HDs). Four PD-derived iPSC lines with different mutations and three cell lines from HDs at different stages of neuronal differentiation were used for RT-qPCR analysis and ELISA. We found that the mRNA levels of most analyzed genes were altered in PD-derived cells compared with those in HD-derived cells at all stages. Importantly, irrespective of PD-associated mutations, the mRNA levels of the BDNF and GDNF genes were mostly increased or unchanged in predominantly DA terminally differentiated neurons (TDNs) compared with those in HD-derived cells. Strikingly, in contrast to BDNF and GDNF mRNA levels, BDNF and GDNF protein levels were lower in almost all PD-derived TDNs than in HD-derived cells, thus indicating the dysregulation of NTF expression at the post-transcriptional level. We suggest that this dysregulation is one of the important signs of PD development.

[Radical cystectomy: therapeutic aspects of preoperative and early postoperative management].

AIM: to improve the results of radical cystectomy by optimization of patient preparation for surgery and early postoperative care. MATERIALS AND METHODS: A total of 136 patients who underwent radical cystectomy and either orthotopic ileal neobladder or ileal conduit formation were included in the study. Brikkers operation was performed in 92 patients (76% men and 24% women) aged from 39 to 83 years, while in 44 patients (97.7% men, 2.3% women) aged from 32 to 75 years the Studer ileal neobladder was created. All patients underwent preoperative comprehensive examinations in order to determine type and extent of surgical treatment. RESULTS: A complication rate after radical cystectomy with urine derivation using bowel segment was 49.2%. Mortality rate in early postoperative period was 3.9%. CONCLUSION: An algorithm of postoperative care after radical cystectomy with formation of either orthotopic neobladder or ileal conduit and consideration of comorbid status and preparation which we have used in clinical practice was developed. According to the results, after implementation of algorithm of management in preoperative and early postoperative period a decrease in complications and mortality rate has been found.

The Transcriptional Changes of trim Genes Associated with Parkinson's Disease on a Model of Human Induced Pluripotent Stem Cells.

Over the last few years, in vitro models, based on patient-derived induced pluripotent stem cells (iPSCs), have received considerable attention for modeling different neurodegenerative disorders. Using this model, we analyzed transcription of 15 tripartite motif (trim) genes in iPSCs, derived from the different groups: Parkinson's disease (PD) patients bearing mutations in different genes, patient with the sporadic form of PD, and the healthy individuals. The transcription was observed during neuronal differentiation of the cells in vitro into neuronal stem cells and terminally differentiated neurons. The transcription of over 50 % of these genes, belonging to different sub-groups of the TRIM family, varied between PD patients and healthy individuals during the reprogramming of fibroblasts into iPSCs and the following neuronal differentiation. Moreover, the transcription of the trim6 and trim24 genes was different between cells, derived from PD patients, and control cells at all stages. The transcription of the four trim genes (trim5α, 26, 27, 31) remained unchanged during almost all investigated stages, compared with the controls. We suppose that the revealed changes in the transcription of several trim genes reflect their possible role in neurodegenerative processes at the early stages of PD. These genes may act as a gear unit between the PD progression and the deregulation of the immune system.

[Effect of Ultrasmall Gold Nanoparticles on the Murine Native Sperm Chromatin].

The effects of ultrasmall (2-3 nm) gold nanoparticles on native epididymal sperm chromatin of CBAxC57BL/6 hybrid mice and 129/IMG mice with a mutation in the DNA-polymerase iota gene were studied. It is shown that for both mouse strains after sperm incubation in a solution containing Au nanoparticles, at 23, 37 and 60 degrees C for 30 min followed by 1 hour treatment in dithiothreitol solution, a decrease in the number of nuclei with fully decondensed chromatin was observed compared with the control. Though, the manifestation of this effect in the population of 129/IMG mice mature sperm, was weaker. Also we have demonstrated that sperm of both strains that were incubated in a sol of Au nanoparticles at 60 degrees C behave differently under the action of dithiothreitol. A considerable part (-80%) of sperm of CBAxC57BL/6 hybrid mice treated with Au nanoparticles showed high resistance to the action of dithiothreitol, whereas in the case of 129/IMG mice only -30% did, and a partial or complete chromatin decondensation takes place in the remaining sperm. In general, using the method of nuclear chromatin decondensation in vitro for the native sperm, the patterns that we have identified in earlier studies on previously demembranized sperm are confirmed.

[Effect of Mn(II) on the error-prone DNA polymerase iota activity in extracts from human normal and tumor cells].

The DNA polymerase iota (Pol iota), which has some peculiar features and is characterized by an extremely error-prone DNA synthesis, belongs to the group of enzymes preferentially activated by Mn2+ instead of Mg2+. In this work, the effect of Mn2+ on DNA synthesis in cell extracts from a) normal human and murine tissues, b) human tumor (uveal melanoma), and c) cultured human tumor cell lines SKOV-3 and HL-60 was tested. Each group displayed characteristic features of Mn-dependent DNA synthesis. The changes in the Mn-dependent DNA synthesis caused by malignant transformation of normal tissues are described. It was also shown that the error-prone DNA synthesis catalyzed by Pol iota in extracts of all cell types was efficiently suppressed by an RNA aptamer (IKL5) against Pol iota obtained in our work earlier. The obtained results suggest that IKL5 might be used to suppress the enhanced activity of Pol iota in tumor cells.

[Activity of error-prone DNA polymerase iota in different periods of house mouse Mus musculus ontogeny].

Analysis of incorrect activity of error-prone DNA polymerase iota in M. musculus ontogeny demonstrated considerable changes in its activity, which peaks in most organs during prenatal development and decreases in the adult body. We propose that the capacity of error-prone DNA polymerases to synthesize on damaged DNA regions is critical for the realization of rapidly changing genetic program in mammalian embryogenesis, which relieves the replication block and prevents cell death.

[Targeting the gene for platelet-derived growth factor A-chain (PDGF-A) and preparation of chimeric mice with a "knockout" genome]. / Targeting gena A-tsepi trombotsitarnogo faktora rosta (PDGF-A) i poluchenie khimernoi myshi s "nokautirovannym" genom.

A vector was selected for homologous recombination to generate mouse embryonic stem cell clones with disrupted platelet-derived growth factor A (PDGF-A) chain gene. A chimeric mouse with targeted PDGF-A gene has been created.

[Localization of fragments binding nuclear proteins in the promotor region of the human alpha-1-antitrypsin gene]. / Lokalizatsiia v promotornoi oblasti gena alpha-1-antitripsina cheloveka uchastkov sviazyvaniia s iadernymi belkami.

Human and mice nuclear extracts from livers and mice spleen extract were analysed in an attempt to find any proteins capable of binding to the human alpha 1-antitrypsin gene promoter. The nuclei of all studied tissues contain such proteins. The proteins were partially purified on DEAE-trisacryl, heparin sepharose and phosphocellulose columns. The multiple sites for liver nuclear proteins binding to the human alpha 1-antitrypsin gene promoter were found by the DNAse I footprinting technique.

[Induction of unstable mutations in Drosophila melanogaster by microinjection of oncogenic virus DNA into the embryo polar plasma. Insertional nature of mutations]. / Induktsiia nestabil'nyukh mutatsii u Drosophila melanogaster mikroin"ektsiei DNK onkogennykh virusov v poliarnuiu plazmu émbrionov. Insertsionnaia priroda mutatsii.

We have demonstrated that mutations induced in Drosophila melanogaster by the microinjections of adenovirus Sa7 DNA in early embryos are of insertional nature. The role of insertional elements is played by the Drosophila transposons, but not by the virus DNA. The ability of oncoviral DNA to induce transpositions of mobile elements in recipient genome is the molecular basis of this system of genetic instability.

INDUCTION OF UNSTABLE MUTATIONS IN DROSOPHILA MELANOGASTER BY THE MICROINJECTION OF ONCOGENIC VIRAL DNA INTO THE POLAR PLASM OF EMBRYOS: THE INSERTIONAL NATURE OF THE MUTATIONS.

MUTATIONS INDUCED BY THE MICROINJECTION OF ADENOVIRUS SA7 DNA INTO THE POLAR PLASM OF DROSOPHILA MELANOGASTER EMBRYOS WERE SHOWN TO BE OF THE INSERTIONAL TYPE. THE INSERTED ELEMENTS RESPONSIBLE FOR THESE MUTATIONS WERE ENDOGENOUS TRANSPOSONS RATHER THAN VIRAL SEQUENCES. THUS, GENETIC INSTABILITY ACTIVATED BY ONCOVIRAL DNA RESULTED FROM THE ABILITY OF THESE DNA SEQUENCES TO INDUCE TRANSPOSITION OF MOBILE ELEMENTS IN THE RECIPIENT GENOMES.

[Several copies of angiogenin gene are present in the mammalian genome]. / Gen angiogenina predstavlen neskol'kimi kopiiami v genomakh mlekopitaiushchikh.

Human cDNAs coding for angiogenin were isolated from Li7 hepatoma. Analysis of the nucleotide sequences of isolated cDNA clones and its comparison with recently published sequences of cDNA and human angiogenin gene permitted to suggest that an intron is present in the 5' region of the gene, dividing the coding and 5' untranslating regions. The size of the intron exceeds 1700 b.p. Up to now it has been known that the angiogenin gene is a single copy gene. However our results of blot-hybridization with genomic DNA of some mammals showed that there are 2-3 copies of the gene in their genomes. According to our results the angiogenin gene is conserved in mammalian genomes.

[Molecular and cytologic analyses of the transcription of evolutionarily conservative moderately repetitive sequences of vertebrate genomes]. / Molekuliarnyi i tsitologicheskii analiz transkriptsii évoliutsionno konservativnykh umerenno povtoriaiushchikhsia posledovatel'nostei genomov pozvonochnykh.

Transcription of several families of moderately repeated sequences, conserved through the evolution of vertebrates, has been studied in different types of pigeon, chicken and mouse cells. It is shown both by hybridization with isolated RNA and by in situ hybridization that the families of repeats, dispersed in bird genomes and organized in clusters, are differentially expressed in pigeon erythroid cells with different degrees of specialization; in addition, they are transcribed in different types of chick embryo cells and on lampbrush chromosomes in chicken oocytes. Sequences homologous to these repeats were transcribed in different types of newborn mouse cells. Another family of conservative moderate repeats (family T1) dispersed in the mouse genome was also transcribed in a large variety of tissues in both new-born mice and chick embryos. A comparison of structural and transcription features of conservative moderate repeats represented in genomes of the number of vertebrates made it possible to regard them as "housekeeping" elements. The conservation in the evolution as well as the character of transcription of similar genome elements testify to their important role in the organism functioning at different stages of development.

[Structural features of the integration site of foreign DNA in the transgenic mouse genome]. / Strukturnye osobennosti uchastka integratsii chuzherodnoi DNK v genome transgennoi myshi.

The structure of the transgenic mouse DNA region containing an integrated transgene (fragment of pBR322 sequence) was analysed. In one of the sequences flanking the transgene, short direct and inverted overlapping repeats were revealed at a distance of 60 bp from the integration site. In the same flanking sequence, there is an extended sequence (3.5 kbp) 0.3-1 kbp away from the transgene. It repeats 100-300 times in the mouse genome and is highly conservative (the homologs of the repeat have been revealed in other mammalian, bird, fish and insect genomes). This up-to-date unknown family of highly-conserved dispersed repeats has been denoted by T1. We believe that both the revealed short inverted repeats capable of forming hairpins with loops and the T1 repeat are structures involved in the process of non-homologous insertion of foreign DNA into the region of the transgenic mouse genome.

[Molecular cloning of the human insulin gene]. / Molekuliarnoe klonirovanie gena insulina cheloveka.

Isolation of genomic clones containing the human insulin gene by screening the human genomic library for this gene using the cDNA rat insulin probe is reported. The analysis of promoter-enhancer region and exons sequences has revealed their identity to analogous sequences determined earlier.

[Transcription, in frog oocytes and transgenic mice, of recombinant plasmids with long terminal repeats of retrovirus proviruses]. / Transkriptsiia v ootsitakh liagushki i transgennykh myshakh rekombinantnykh plazmid s dlinnymi kontsevymi povtorami provirusov retrovirusov.

RNA-DNA hybridization was used to study the transcription efficiency of the genes controlled by the long terminal repeats (LTR) from two different retroviral proviruses (exogenous provirus of the chicken Rous sarcoma virus and endogenous xenotropic provirus of the mouse). The oocyte nuclei of Xenopus laevis and the liver of transgenic mice were used as the transcription systems. The transcription efficiency of genes with the above mentioned promoters was shown to be roughly the same for both systems. Thus, the LTR of two proviruses of different origin possess the promoters of comparable strength and do not show any marked tissue or species specificity.

Rearrangements of microinjected recombinant DNA in the genome of transgenic mice.

Previously, mouse zygotes were microinjected with the recombinant plasmid pMA3, which contains the Herpes simplex virus thymidine kinase gene attached to the promoter region of the Rous sarcoma virus (Gazaryan et al. 1984a). In the present work the pMA3 fragment with the flanking genomic sequences was isolated from the DNA of one transgenic Fo mouse by the "plasmid rescue" technique. The rescued plasmid (pMAR1) lacked all virus-specific sequences and retained only some pBR322 sequences. The flanking region at one end of the integrated pBR322-specific fragment contained a highly conserved mouse repetitive sequence. The possible mechanisms of rearrangement of foreign DNA in germ line cells are discussed.

[Cloning of the DNA fragment of a transgenic mouse containing an integrated recombinant plasmid]. / Klonirovanie fragmenta DNK transgennoi myshi, soderzhashchego integrirovannuiu rekombinantnuiu plazmidu.

The "plasmid rescue" method has been used to isolate the recombinant plasmid pMA3 fragment and flanking sequences from the transgenic mouse genome containing the fragment in integrated form. The "rescued" plasmid, pMAR1, lacks all virus sequences and retains only those regions of pBR322 that are responsible for the plasmid replication and Escherichia coli ampicillin resistance. The plasmid pMA3 deletion has occurred at its integration into the mouse genome after microinjection into the zygote. The integrated fragment of the plasmid is adjacent to the genome repeated sequence that is highly conservative in evolution.