[Long-Chain Free Fatty Acids Influence Lipid Accumulation, Lysosome Activation and Glycolytic Shift in Various Cells In Vitro].

Hydrophobic molecules may be toxic when present in excess. When dissolved in membranes, hydrophobic molecules disrupt membrane function. Studies on the effects of free fatty acids (FFA) on cultured cells contradict each other. Here we describe the effects of FFA on various human cells in culture. The addition of long-chain FFA (oleic, palmitic, linoleic, linolenic, etc.) to cultured cells led to lipid accumulation in hepatocytes and muscle cells, initiation of autophagy, and uncoupling of oxidative phosphorylation. Although treated cells increase their oxygen consumption, metabolic shifts in favor of glycolysis were observed. All these effects were expressed to varying degrees in different cells and with the addition of different FFAs. The mechanisms of these FFA effects are discussed, as well their practical implications.

[Clinical and metabolic features of nonalcoholic fatty liver disease in men and women]. / Klinicheskie i metabolicheskie osobennosti nealkogol'noi zhirovoi bolezni pecheni u muzhchin i zhenshchin.

AIM: To evaluate clinical features and metabolic disorders in men and women with non-alcoholic fatty liver disease (NAFLD).

NON-ALCOHOLIC FATTY LIVER DISEASE IN ELDERLY.

AIM: The study of the clinical course and metabolic disorders in patients nonalcoholic fatty liver disease (NAFLD) elderly. SUBJECTS AND METHODS: 153 patients with NAFLD was investigated, including 97 men and 56 women. In comparative terms we studied clinical manifestations of NAFLD. All patients NAFLD was verified for the first time. We studied the functional state of the liver function, lipid, carbohydrate and porphyrin metabolism, insulin resistance. RESULTS: Revealed comorbid pathology, which is predominantly observed in elderly patients. Disturbances in lipid metabolism and insulin resistance hyperinsulinemiya and proved to be more significant in young patients. Disorders of porphyrin metabolism observed in most patients. Disorders are variable. Do young people have dominated the initial disorder, on the other hand more often in elderly patients were observed faction (later) porphyrin metabolism disorders. CONCLUSION: Studies suggest that the main pathophysiologic and pathogenetic processes of formation of NAFLD (insulin resistance and dyslipidemia) significantly more pronounced in younger patients. This fact suggests that NAFLD is mainly formed at a young age. Elderly patients have comorbid pathology.

Modification of human fibroblast properties in microtransplant.

We studied the properties of human skin fibroblast in filamentous polyglycolic microtransplant. Fibroblast adhesion to the microtransplant filaments is followed by the formation of a network cross-linked with fibroblasts. The cells rapidly proliferate during the first few days; after transfer of the microtransplant to the standard culture flask, the cells migrate to the plastic and continue proliferation. The cells are uniform and exhibit high colony-formation capacity. The bundles of microtransplant filaments persist in the culture for several days and through the cells completely leave them, the area around these filaments remains the most populated for 40 days. Mitotic cells are seen in the immediate proximity to the degrading filaments of the transplant. The effect of cell "rejuvenation" in the microtransplant can be explained by selection of cells by their adhesion to relatively thin (about 15 µ) filaments, which excludes large old cells.

[Preparation stimulating hair growth possibly acts by inhibiting hair follicle ageing experiments on mice and transcriptome analysis].

Preparation stimulating hair growth (PSHG) was studied on mice of various strains (Balb/c, CBA, C57BI/6, and outbred). It was shown that a long-term (44 months) application of PSHG does not reliably affect the appearance of young healthy mice but does induce increase in the hair follicle size. No adverse consequences of the PSHG application were observed. Naturally occurring propagating regenerative hair waves peculiar to mice were preserved. In older mice (more than 2 years) with signs of alopecia, application of PSHG caused an overgrowing of bald patches within two months. Transcriptome analysis of the PSHG effect performed in fibroblast cell culture showed that PSHG stimulates processes of tissue development and remodeling. These observations together with previous findings showing that PSHG stimulates autophagy and induces death of cells subjected to oxidative stress may suggest that the mechanism of the PSHG effect involves stimulation of regeneration of skin and its derivatives owing to more efficient elimination of senescent and damaged follicle cells.

[Possible role of autophagy activation in the stimulation of regeneration].

Human cell senescence occurs unevenly and senescent cells in tissues frequently can disturb the function of neighbouring nonsenescent ones. Setting of tissues regeneration can have profound practical significance in medicine, especially in geriatrics. One of the approaches to solve the problem is selective elimination of senescent and damaged cells from the tissues that can be the first phase of the process. During the investigation of the mechanisms of action of the preparation for hair growth stimulation it was discovered that this preparation does not stimulate proliferation of various human cells and does not increase the resistance of cells to stress. On the contrary the preparation becomes cytotoxic at the conditions of oxidative stress although on its own account it did not induce elevation of production of reactive oxygen species. Further investigations showed that the preparation increases transcriptional activity of p53 gene, increase autophagy level and induce weak adipogenic differentiation. The hypothesis of autophagic regeneration is discussed. As a result, the selective autophagic cell death of any senescent and damaged cells that undergoes oxidative stress triggers the regeneration process which can be increased by both the rejuvenation effect of increased autophagy and at the expense of nutrients released during the autophagy.

[Increasing the resolution of chromosome analysis using pyrido[1,2alpha]benzimidazoles].

We studied the influence of three derivatives of pyrido[1,2alpha]benzimidazoles (PBIs), which have DNA-intercalating properties, on plant mitotic chromosome condensation, in order to increase the resolution of chromosome analysis. The efficiency of the influence of these agents was assessed using the median chromosome length on chromosome slides, as well as by the number and size of chromosome DAPI bands. We used the third chromosome of Linum grandiflorum Desf. in these experiments. The chromosome was identified on the slides using its DAPI band pattern and a molecular marker, viz., the 5S rDNA site, which is located in the proximal region of the long arm of the chromosome. The influence of the well-known 9-aminoacridine (9-AMA) DNA intercalator, which is widely used in karyotype studies of short-chromosome organisms, was used as a control in all of the experiments. It was found that the influence of each of the three PBIs in the study on the root meristem of L. grandiflorum resulted in an increase in the median length of the third chromosome, the linear centromeric DAPI band size, and the number ofintercalary DAPI bands. All three PBIs acted more efficiently than 9-AMA. The median chromosome length was increased by 15-40% and the number of intercalary bands increased by 1.5-3 times after PBI treatment, as compared to 9-AMA treatment. At the same time, 7-CF3-PBI, in a similar manner to 9-AMA, did not change the relative size of the centromeric DAPI band, while 7-NH2-PBI and 7-CF3-9-NH2-PBI gradually increased this parameter. It is concluded that these substances can be used as intercalating agents in cytogenetic studies in order to increase the resolution of chromosome analysis.

[Comparative cytogenetic study of the forms of Macleaya cordata (Willd.) R. Br. from different localities].

A comparative cytogenetic study of two introduced forms of Makleaya cordata (Willd.) R. Br. = syn. Bocconia cordata Willd. grown in different ecological and geographical regions (Moscow and Donetsk areas) was carried out. In the study, a complex of methods utilizing various chromosomal markers, i.e., C- and DAPI-banding technique, fluorescence in situ hybridization (FISH) with probes of26S and 5S rDNA, as well as estimation of the total area of C-positive regions (C-HCH) in prophase nucleoli and meiosis analysis, was used. In the karyotypes (2n = 20), each chromosome was identified on the basis of C-banding and FISH patterns and the chromosome ideograms were built. Pericentrometric and telomeric C-positive bands in chromosomes of the Moscow form karyotype were found to be smaller and intercalary bands, larger than the corresponding bands in the M. cordata form grown in Donetsk. It was found that the content of C-HCH in prophase nucleoli in the form of M. cordata grown in Donetsk was higher than in the form grown in Moscow. In both forms sites of 26S rDNA and 5s rDNA were localized on satellite chromosome 1 and on chromosome 4 respectively but the signals were more intensive in the plant form grown in Donetsk. The results of this study enable selecting M. cordata forms for use in pharmacology and recommending them for cultivation in various ecological and geographical regions.

[Genetic polymorphism of flax Linum usitatissimum based on use of molecular cytogenetic markers].

Using a set of approaches based on the use of molecular cytogenetic markers (DAPI/C-banding, estimation of the total area of DAPI-positive regions in prophase nuclei, FISH with 26S and 5S rDNA probes) and the microsatellite (SSR-PCR) assay, we studied genomic polymorphism in 15 flax (Linum usitatissimum L.) varieties from different geographic regions belonging to three directions of selection (oil, fiber, and intermediate flaxes) and in the k-37 x Viking hybrid. All individual chromosomes have been identified in the karyotypes of these varieties on the basis of the patterns of differential DAPI/C-banding and the distribution of 26S and 5S rDNA, and idiograms of the chromosomes have been generated. Unlike the oil flax varieties, the chromosomes in the karyotypes of the fiber flax varieties have, as a rule, pericentromeric and telomeric DAPI-positive bands of smaller size, but contain larger intercalary regions. Two chromosomal rearrangements (chromosome 3 inversions) were discovered in the variety Luna and in the k-37 x Viking hybrid. In both these forms, no colocalization of 26S rDNA and 5S rDNA on the satellite chromosome was detected. The SSR assay with the use of 20 polymorphic pairs of primers revealed 22 polymorphic loci. Based on the SSR data, we analyzed genetic similarity of the flax forms studied and constructed a genetic similarity dendrogram. The genotypes studied here form three clusters. The oil varieties comprise an independent cluster. The genetically related fiber flax varieties Vita and Luna, as well as the landrace Lipinska XIII belonging to the intermediate type, proved to be closer to the oil varieties than the remaining fiber flax varieties. The results of the molecular chromosomal analysis in the fiber and oil flaxes confirm their very close genetic similarity. In spite of this, the combined use of the chromosomal and molecular markers has opened up unique possibilities for describing the genotypes of flax varieties and creating their genetic passports.

[Comparative genome analysis in pea Pisum sativum L. varieties and lines with chromosomal and molecular markers].

C banding, Ag-NOR staining, FISH with pTa71 (45S rDNA) and pTa794 (5S rDNA), and RAPD-PCR analysis were used to study the genome and chromosome polymorphism in four varieties (Frisson, Sparkle, Rondo, and Finale) and two genetic lines (Sprint-2 and SGE) of pea Pisum sativum L. A comparison of the C-banding patterns did not reveal any polymorphism within the varieties. The most significant between-variety differences were observed for the size of C bands on satellite chromosomes 4 and 7. All grain pea varieties (Frisson, Sparkle, and Rondo) had a large C band in the satellite of chromosome 4 and a medium C band in the region adjacent to the satellite thread on chromosome 7. C bands were almost of the same size in the genetic lines and vegetable variety Finale. In all accessions, 45S rDNA mapped to the secondary constriction regions of chromosomes 1, 3, and 5. The signal from chromosome 5 in the lines was more intense than in the varieties. Ag-NOR staining showed that the transcriptional activity of the 45S rRNA genes on chromosome 7 was higher than on chromosome 4 in all accessions. No more than four Ag-NOR-positive nucleoli were observed in interphase nuclei. Statistical analysis of the total area of Ag-NOR-stained nucleoli did not detect any significant difference between the accessions examined. RAPD-PCR analysis revealed high between-variety and low within-variety genomic polymorphism. Chromosomal and molecular markers proved to be promising for genome identification in pea varieties and lines.

[Ligands with affinity to specific sequence of DNA base pairs. XII. The synthesis and cytological studies of dimeric Hoechst 33258 molecules].

We synthesized dimeric Hoechst dye molecules composed of two moieties of the Hoechst 33258 fluorescent dye phenolic hydroxy groups of which were tethered via pentamethylene, heptamethylene, or triethylene oxide linkers. A characteristic pattern of differential staining of chromosome preparations from human premonocytic leukemia HL60 cells was observed for all the three fluorescent dyes. The most contrast pattern was obtained for the bis-Hoechst analogue with the heptamethylene linker; its quality was comparable with the picture obtained in the case of chromosome staining with 4',6-diamidino-2-phenylindole. The ability to penetrate into the live human fibroblasts was studied for the three bis-Hoechst compounds. The fluorescence intensity of nuclei of live and fixed cells stained with the penta- and heptamethylene-linked bis-Hoechst analogues was found to differ only slightly, whereas the fluorescence of the nuclei of live cells stained with triethylene oxide-linked bis-Hoechst was considerably weaker than that of the fixed cells. The bis-Hoechst molecules are new promising fluorescent dyes that can both differentially stain chromosome preparations and penetrate through cell and nuclear membranes and effectively stain cell nuclei.

[Chromosomal localization of 5S and 45S ribosomal DNA in species of Linum L. section Linum (syn=Protolinum and Adenolinum)]. / Khromosomnaia lokalizatsii 5S i 45S ribosomnoi DNK u vidov roda Linum L. sektsii Linum (syn.=Protolinum i Adenolinum).

Fluorescence in situ hybridization (FISH) was for the first time used to study the chromosomal location of the 45S (18-2.5S-26S) and 5S ribosomal genes in the genomes of five flax species of the section Linum (syn. Protolinum and Adenolinum). In L. usitatissimum L. (2n = 30), L. angustifolium Huds. (2n = 30), and L. bienne Mill. (2n = 30), a major hybridization site of 45S rDNA was observed in the pericentric region of a large metacentric chromosome. A polymorphic minor locus of 45S rDNA was found on one of the small chromosomes. Sites of 5S rDNA colocalized with those of 45S rDNA, but direct correlation between signal intensities from the 45S and 5S rDNA sites was observed only in some cases. Other 5S rDNA sites mapped to two chromosomes in these flax species. In L. grandiflorum Desf. (2n = 16) and L. austriacum L. (2n = 18), large regions of 45S and 5S rDNA were similarly located on a pair of homologous satellite-bearing chromosomes. An additional large polymorphic site of 45S and 5S rDNA was found in the proximal region of one arm of a small chromosome in the L. usitatissimum. L. angustifolium, and L. bienne karyotypes. The other arm of this chromosome contained a large 5S rDNA cluster. A similar location of the ribosomal genes in the pericentric region of the pair of satellite-bearing metacentrics confirmed the close relationships of the species examined. The difference in chromosomal location of the ribosomal genes between flax species with 2n = 30 and those with 2n = 16 or 18 testified to their assignment to different sections. The use of ribosomal genes as chromosome markers was assumed to be of importance for comparative genomic studies in cultivated flax, a valuable crop species of Russia, and in its wild relatives.

[Genome comparisons of three closely related flax species and their hybrids with chromosomal and molecular markers]. / Sravnenie genomov trekh blizkoorodstvennykh vidov l'na i ikh gibridov s ispol'zovaniem khromosomnykh i molekuliarnykh markerov.

Chromosome C-banding patterns were analyzed in three closely related flax species (Linum usitatissimum L., 2n = 30; L. angustifolium Huds., 2n = 30; and L. bienne Mill., 2n = 30) and their hybrids. In each case, the karyotype included metacentrics, submetacentrics, and one or two satellite chromosomes. Chromosomes of the three flax species were similar in morphology, size (1-3 microns), and C-banding pattern and slightly differed in size of heterochromatic regions. In all accessions, a large major site of ribosomal genes was revealed by hybridization in the pericentric region of a large metacentric. A minor 45S rDNA site was observed on a small chromosome in L. usitatissimum and L. bienne and on a medium-sized chromosome in L. angustifolium. Upon silver staining, a nucleolus-organizing region (NOR) was detected on a large chromosome in all species. In L. angustifolium, an Ag-NOR band was sometimes seen on a medium-sized chromosome. In the karyotypes of interspecific hybrids, silver-stained rDNA loci were observed on satellite chromosomes of both parental species. RAPD analysis with 22 primers revealed a high similarity of the three species. The greatest difference was observed between L. angustifolium and the other two species. The RAPD patterns of L. bienne and L. usitatissimum differed in fewer fragments. A dendrogram of genetic similarity was constructed for the three flax species on the basis of their RAPD patterns. Genome analysis with chromosome and molecular markers showed that L. bienne must be considered as a subspecies of L. usitatissimum rather than a separate species. The three species were assumed to originate from a common ancestor, L. angustifolium being closest to it.

[Comparative study of genomes of two species of flax by C-banding of chromosomes]. / Sravnitelnoe issledovanie genom dvukh vidov l'na po risunkam C-okraski khromosom.

C-banding patterns of the karyotypes of two closely related wild flax species, Linum austriacum L. (2n = 18) and Linum grandiflorum Desf. (2n = 16), were studied. The karyotypes of both species were similar in the chromosome morphology and size. In each species, metacentric and acrocentric chromosomes (1.7-4.3 microns) and one satellite chromosome were observed. In the karyotypes of the species studied, all homologous chromosome pairs were identified, and quantitative ideograms were constructed. Eight chromosome pairs in the two species had similar C-banding patterns. A low level of intraspecific polymorphism in the intercalary and telomeric C-bands was shown in both species. The results indicate that the genomes of two flax species originated from one ancestral genome with the main chromosome number of 8 or 9. Apparently, the doubling of chromosome number or loss of one chromosome with subsequent redistribution of the chromosome material in the ancestral form resulted in the divergence into two species, L. austriacum L. and L. grandiflorum Desf. A considerable similarity of chromosomes in these species provides evidence for their close phylogenetic relatedness, which makes it possible to place them in one section within the Linum genus.

Polymorphism of heterochromatic regions of flax chromosomes.

The C-banding technique was used to study flax chromosomes (Linum usitatissimum L., 2n = 30). Heterochromatin was located mainly in pericentromeric regions of chromosomes. In spite of small size (1.5-3.5 microm), all 15 pairs of homologous chromosomes were identified on the basis of the C-banding pattern and morphology. An idiogram of C-banded chromosomes of L usitatissimum L. is presented. Polymorphism of chromosomal heterochromatic regions was studied in karyotypes of three flax samples: L usitatissimum L., accession K-603 (L usitatissimum var. usitatissimum), and accession K-594 (L. usitatissimum var. humile (Mill.)). A common C-banding pattern was observed in all forms studied, although there were some distinctions in the individual band size. The fibre flax (accession K-603) karyotype had the C-banding pattern similar to that of L usitatissimum L., but some intercalary and telomeric C-bands were somewhat larger, and a satellite (NOR) was observed in the short arm of chromosome I. In crown flax, (K-594) chromosomal C-banding pattern exhibited smaller pericentromeric and larger intercalary bands; telomeric bands were present on almost all chromosomes. Thus, the intraspecies polymorphism revealed in the chromosomal C-banding pattern makes possible the use of C-bands as chromosome markers in the studies of genetic and genomic polymorphism of this species.

[Localization of DNA probes for human ribosomal genes on barley chromosomes]. / Lokalizatsiia na khromosomakh iachmenia DNK-prob ribosomnykh genov cheloveka.

To estimate the possibility of plant genome mapping using human genome probes, the probes fluorescent in situ hybridization (FISH) of human 18S-28S rDNA (clon 22F9 from the LA-13NCO1 library) was carried out on chromosomes of the spring barley Hordeum vulgare L. As a control, wheat rDNA probe (clon pTa71) was taken. Hybridization of the wheat DNA probe revealed two major labelling sites on mitotic barley chromosomes 5I (7H) and 6I (6H), as well as several minor sites. With the human DNA probe, signals were detected in the major sites of the ribosomal genes on chromosomes 5I (7H) and 6I (6H) only when the chromosome preparations were obtained using an optimized technique with obligatory pepsin treatment followed by hybridization. Thus, this study demonstrates that physical mapping of plant chromosomes with human DNA probes that are 60 to 75% homologous to the plant genes is possible. It suggests principal opportunity for the FISH mapping of plant genomes using probes from human genome libraries, obtained in the course of the total sequencing of the human genomes and corresponding to the coding regions of genes with known functions.

Telomerase activity of cells during alterations of their proliferative states.

We showed that the telomerase activity (TAC) of human promyelocytic leukemic cells U-937 and HL-60 sharply decreased after induction of macrophagal differentiation. Dedifferentiation which occurred several days after removing the inductor was accompanied by the resumption of proliferation and increase of TAC. Telomerase activity significantly decreased also when U-937 cells ceased to proliferate in response to long-term inhibition of the telomerase function by azidothymidine. TAC was observed to decrease slowly for 6-8 days in the course of transition of mouse fibroblasts 3T3 Swiss to the quiescent state. TAC decreased both in serum-deprived cells and in slowly proliferating high-density inhibited cells. During the exit from quiescence and in dedifferentiation, the increase in TAC preceded the resumption of proliferation. In all the cases described the alterations of TAC correlated with alterations in the nonspecific polymerase activity which we had found earlier (D. N. Chernov, Y. E. Yegorov, and S. S. Akimov, DokL Biochemistry 349:55-58 (1996)). The problems of TAC regulation are discussed.