Showing papers in "Tsitologiia in 2003"

A role for Cajal bodies in assembly of the nuclear transcription machinery

Abstract: Cajal bodies (CBs) are small nuclear organelles that contain the three eukaryotic RNA polymerases and variety of factors involved in transcription and processing of all types of RNA. A number of these factors, as well as subunits of polymerase (pol) II itself, are rapidly and specifically targeted to CBs when injected into the cell. It is suggested that pol I, pol II and pol III transcription and processing complexes are preassembled in the CBs before transport to the sites of transcription on the chromosomes and in the nucleoli.

HIV-1 gag expression is quantitatively dependent on the ratio of native and optimized codons.

Abstract: There is a significant variation of codon usage bias among different species and even among genes within the same organisms. Codon optimization, this is, gene redesigning with the use of codons preferred for the specific expression system, results in improved expression of heterologous genes in bacteria, plants, yeast, mammalian cells, and transgenic animals. The mechanisms preventing expression of genes with rare or low-usage codons at adequate levels are not completely elucidated. Human immunodeficiency virus (HIV) represents an interesting model for studying how differences in codon usage affect gene expression in heterologous systems. Construction of synthetic genes with optimized codons demonstrated that the codon-usage effects might be a major impediment to the efficient expression of HIV gag/pol and env gene products in mammalian cells. According to another hypothesis, the poor expression of HIV structural proteins even without HIV context is attributed to the so-called cis-acting inhibitory elements (INS), which are located within the protein-coding region. They consist of AU-rich sequences and may be inactivated through the introduction of multiple mutations over the large regions of gag gene. In our work, we evaluated expression of hybrid HIV-1 gag mRNAs where wild-type (A-rich) gag sequences were combined with artificial sequences. In such "humanized" gag fragments with adapted codon usage, AT-content was significantly reduced in favor of G and C nucleotides without any changes in protein sequence. We show that wild-type gag sequences negatively influence expression of gag-reporter, and the addition of fragments with optimized codons to gag mRNA partially rescues its expression. The results demonstrate that the expression of HIV-1 gag is determined by the ratio of optimized and rare codons within mRNA. Our data also indicates that some wtgag fragments counteract the influence of the other wtgag sequences, which cause the inhibition of gag expression. The presented data do not contradict the concept of INS; yet, it makes the definition of INS more complex. This supports the idea of a broader role of the selected codon usage in influencing the expression of HIV proteins in mammalian cells.

Codon usage-mediated inhibition of HIV-1 gag expression in mammalian cells occurs independently of translation.

Abstract: Codon usage is considered one of the critical factors that limit the expression rate of heterologous genes. Impaired translation efficiency, specifically insufficient amount of corresponding tRNAs and changed startcodon context, are believed to account for the low translation initiation and elongation rates during the protein biosynthesis in unicellular organisms. Translational efficiency is probably not the primary factor influencing codon usage diversity in mammalian cells. However, the other possible mechanisms preventing expression of genes with low-usage such as mRNA stability, processing and nucleocytoplasmic transport, are not adequately explored. In our work, we addressed the question of whether codon usage differences affect exclusively translational efficiency of mammalian gene products. We demonstrated that the CMV-induced expression of gag-reporter in human H1299 cell line was influenced by the nucleotide composition of the mRNA, and the limitation of gag expression appeared to be inversely related to the level of codon optimization. However, cytoplasmic expression of the gag-reporter driven by vaccinia virus/T7 RNA polymerase hybrid system rescued its expression independently of HIV-1 gag mRNA nucleotide content. We concluded that impaired HIV-1 gag expression may be caused by translation-independent mechanisms, which probably play a major role in codon usage-mediated defects in heterologous gene expression in mammalian cells.

Inhibition of liver mitochondrial monoamine oxidase activity by alkaloids isolated from Chelidonium and Macleaya and by their derivative drugs

Abstract: It has been shown that the major alkaloids from plants Chelidonium majus L. and Macleaya (Bocconia) cordata and microcarpa, namely, berberine, sanguinarine, chelidonine, and drugs "Ukrain" (thiophosphoric acid derivative of a sum of the alkaloids isolated from Ch. majus L.) and "Sanguirythrine" (a mixture of the alkaloids sanguinarine and chelerythrine, w/w 3:7, isolated from Macleaya), are irreversible inhibitors of oxidative deamination reaction of serotonin and tyramine as substrates, catalyzed by rat liver mitochondrial monoamine oxidase (MAO). At the same time these substances do not influence the oxidative deamination reaction of benzylamine as substrate (in concentration 1 mM or less). The substrate specificity of this inhibition manifests that mainly the oxidative deamination reactions catalyzed by MAO form A are inhibited by the agents studied. Among the examined agents, alkaloid chelidonine and drug "Ukrain" are the strongest inhibitors of the reaction. Alkaloids berberine and sanguinarine and drug "Sanguirythrine" exhibit a weaker action. Judging from the data obtained, sanguinarine and chelerythrine appear to exert similar inhibitory effects in this reaction, since sanguinarine and "Sanguirythrine" have similar values of bimolecular rate constants of their interaction with mitochondrial MAO. As it is well known, the MAO inhibitors appear to be, as a rule, pronounced antidepressants. The combination of malignotoxicity and antidepressive activity in drug "Ukrain" seems to be favourable for its clinical applications.

[Analysis of living motor nerve ending of a frog by endocytotic fluorescent marker FM 1-43].

Abstract: In our experiments on motor nerve endings of the frog cutaneous pectoris muscle, using fluorescent marker FM 1-43, the intensity and topography of endocytosis were investigated after the initiation of massive exocytosis of synaptic vesicles by increasing the extracellular potassium concentration. Using FM 1-43, fluorescent spots were shown to appear, looking as accumulations of synaptic vesicles in the active zone region. The forms and sizes of luminous spots and the distances between them were analysed. Considerable variations in brightness and total areas of fluorescent spots per a length unit in different regions of the nerve ending were revealed in addition to a proximal-distal gradient of these parameters along the nerve terminal. Peculiarities of topography and intensities of luminescence in the most terminal regions of the nerve ending are described. The obtained data are discussed in terms of the exo- and endocytosis cycle of synaptic vesicles in the active zone region, and from the point of view of the plasticity of the motor nerve ending and active zones. The factors involved in the transmitter release nonuniformity are analysed.

The spore wall and polar tube proteins of the microsporidian Nosema grylli: the major spore wall protein is released before spore extrusion.

Abstract: Incubation of Nosema grylli spores in alkaline-saline solution (10 mM KOH, 170 mM KCl) leads to solubilization of the major spore wall protein of 40 kDa (p40). Both the compounds of this solution are crucial for p40 solubilization. After spore incubation in 170 mM KCl no proteins were released in the medium. In contrast, 10 mM KOH causes a release of many spore proteins but only a small amount of p40. A long storage of spores (over a year) in water or 0.02 % sodium azide results in a sharp decrease of p40 content. Specific polyclonal antibodies were obtained by immunization of rabbits with isolated p40. The specificity of serum was confirmed by immunoblotting. IFA showed reliable reaction on the envelopes of sporonts and sporoblasts, whereas only part of spores reacted with antibodies. This distinction may be due to changing surface antigens during spore maturation. Solubilization of p40 under alkaline conditions could be associated with spore extrusion, since a subsequent transfer of spores to neutral solution leads to their discharge. Subsequent wash of discharged spores with 1-3 % SDS, 9 M urea and treatment by 100 % 2-ME result in solubilization of protein of 56 kDa (p56). The maximum concentration of 2-ME is important for isolation of pure p56. Evidence has been provided that p56 is a protein of N. grylli polar tubes. Treatment of discharged spores by 2-ME in the presence of SDS results in solubilization of four additional proteins with molecular weights about 46, 34, 21 and 15 kDa.

Initial steps of infection of the ciliate paramecium with bacteria Holospora sp.

Abstract: New light and electron microscope data on the initial steps of endocytobiosis establishment between the ciliate Paramecium and specific intranuclear bacteria Holospora are provided. At the cytoplasmic step of infection bacteria of all Holospora species are found in a vesicle originating from the membrane of the host cell phagosome. The association between host cell microfilaments and the bacterium bearing vesicle may suggest a possible involvement of the ciliate cytoskeleton in the transportation of bacteria to the host cell nucleus. The authors subdivide the process of infection into 6 steps. Some strains of P. caudatum never take up infectious Holospora bacteria in the course of phagocytosis.

Low efficiency of DNA repair system in mitochondria

Abstract: Under the action of endogenous reactive oxygen species and exogenous chemical and physical agents, significantly more lesions occur in mitochondrial DNA (mtDNA) than in nuclear DNA (nDNA). However, the mechanisms of DNA repair in mitochondria are less efficient that in the nuclei. The mechanisms of nucleotide excision repair capable of removing UV-induced lesions or other complex adducts induced by chemical compounds are not operative in mitochondria at all. At the same time, mitochondria of some kinds contain a photoreactivation enzyme providing monomerization of cyclobutane pyrimidine dimers. Also, the enzyme system for DNA base excision repair (BER) and O6-alkylguanine-DNA alkyl transferase are functional in mitochondria. However, the rate of BER-controlled repair of lesions in mtDNA is lower than that in nDNA. The literature data suggest that the controlling system for the delay of DNA replication till the repair complexion (cell cycle checkpoint) cannot be provided in mitochontria. Besides, it remains unclear whether the mismatch repair mechanisms are operable in mammalian mitochondria. On the other hand, double-strand breaks in mammalian mtDNA are possibly repaired by involving the DNA-dependent protein kinase complex, and the process of BER is affected by poly(ADP-ribosyl)ation of proteins. Possible consequences of induction of the increased level of damage in mtDNA and the low efficiency of repair systems in mitochondria are discussed in this review.

[STAT1 and STAT3 activation by oxidative stress in A431 cells involves Src-dependent EGF receptor transactivation].

Abstract: Different cellular signal transduction cascades are affected by environmental stressors (UV-radiation, gamma-irradiation, hyperosmotic conditions, oxidants). In this study, we examined oxidative stress-evoked signal transduction pathways leading to activation of STATs in A431 carcinoma cells. Oxidative stress, initiated by addition of H2O2 (1-2 mM) to A431 cells, activates STAT3 and, to a lesser extent, STAT1 in dose- and time-dependent manner. Maximum phosphorylation levels were observed after a 2 minutes stimulation at 1-2 mM H2O2. Phosphorylation was blocked by AG1478, a pharmacological inhibitor of the epidermal growth factor receptor tyrosine kinase, implicating intrinsic EGF receptor tyrosine kinase in this process. Consistent with this observation, H2O2-stimulated EGFR tyrosine phosphorylation was abolished by specific Src kinase family inhibitor CGP77675, implicating Src in H2O2-induced EGFR activation. An essential role for Src and JAK2 in STATs activation was suggested by three findings. 1. Src kinase family inhibitor CGP77675 blocked STAT3 and STAT1 activation by H2O2 in a concentration-dependent manner. 2. In Src-/-fibroblasts, activation of both STAT3 and STAT1 by H2O2 was significantly attenuated. 3. Inhibiting JAK2 activity with the specific inhibitor AG490 reduced the level of H2O2-induced STAT3 phosphorylation, but not STAT1 in A431 cells. These data show essential roles for Src and JAK2 inactivation of STAT3. In contrast, H2O2-mediated activation of STAT1 requires only Src kinase activity. Herein, we postulate also that H2O2-induced STAT activation in carcinoma cells involves Src-dependent EGFR transactivation.

Effect of agents changing the intracellular level of reactive oxygen species on the cell cycle phase distribution in 3T3 and 3T3SV40 cell lines

Abstract: A comparison of cell cycle phase distribution of 3T3 cells and their transformants 3T3SV40 treated with different substances changing the intracellular level of reactive oxygen species (ROS) has been made. In this study the following glutathione synthesis modulating agents were tested: two precursors of intracellular glutathione, antioxidant N-acetyl-L-cysteine (NAC) (-)-2-oxo-4-thiazolidine-carboxylic acid (OTZ), and inhibitor of glutathione synthesis, DL-buthionine-S, R-sulfoximine (BSO). It has been shown that both NAC (10-20 mM) and OTZ (20 mM) decreased the intracellular level of ROS in both cell lines. OTZ was more potent than NAC. However, only NAC caused changes in cell cycle progression of both cell types in dose-dependent manner. These changes differed in 3T3 and 3T3SV40 cells. Flow cytometric analysis of cell cycle phase distribution indicated that NAC (20 mM) blocked cell cycle in the G1 phase. The G1--arrest was completely reversible after removal of NAC from the medium. NAC (10-20 mM) caused a decrease in S and G2/M phases of transformants 3T3SV40. Moreover, a part of the population died apoptoticaly. Different mechanisms of NAC effect on normal and transformed cells are discussed. It is suggested that there is no strong correlation between cell cycle progression and intracellular level of ROS.

The karyotypic structure of cell populations in vitro as an integral system.

Abstract: This review describes regularities of karyotypic variability maintaining karyotypic stabilization of continuous cell lines. Statistical analysis of individual karyotypes of "marker" and "markerless" cell lines show that survival of cell population in vitro is maintained by a certain ratio of cells with different structural variants of karyotype (SVK). Characteristic feature of karyotypic variability in the "markerless" cell lines during long-term cultivation under various conditions is dicentric formation due to telomeric associations. These dicentrics seem to form genetical structures providing adaptation to conditions in vitro of the cell population as an autonomous system. Correlations between the numerical variability reflecting in SVK, and structural variability (dicentric formation) are manifestations of an integral cell-populational function. Experimental data allow to suggest that integrity of the karyotypic structure of cell populations is maintained not only by selection of random variations, but also by programmed (adaptive) changes of karyotype. As a whole, in the cell population the state is realized that can be called karyotypic homeostasis; the observed phenomena characterize processes maintaining such homeostasis.

[Genetic variation in two forms of the common spadefoot toad Pelobates fuscus (Pelobatidae, Anura, Amphibia) distinguished by genome size].

Abstract: Genetic differences (presumed 23 loci) between two cryptic forms of Pelobates fuscus, differing in genome size, were examined by means of polyacrylamide-gel electrophoresis. This method allowed to discriminate between these forms. Average genetic distance (DN) between both the forms was equal to 0.311, ranging from 0.055 to 0.563. As a rule, differences within these groups were smaller (0.021-0.388). The data show obvious genetic differentiation between these two cryptic forms of P. fuscus. Differences between these forms and P. syriacus were significantly higher (in average 0.943). Genetic distances in relation to speciation and species concepts are discussed.

Hormonal signal system of the lower eukaryotes

Abstract: The literary and the authors' own data on the structural and functional organization of hormonal signaling systems in the lower eukaryotes (yeasts, trypanosomes, ciliates, slide mold Dictyostelium discoideum) have been summarized and analysed. On the basis of a comparative analysis of the primary structures of signal proteins in the lower and higher eukaryotes (G-protein alpha-subunits, enzymes-cyclases-adenylyl and guanylyl cyclases) some possible pathways of the evolution of proteins are suggested. At the level of unicellular organisms, the main blocks of hormone-sensitive signaling systems of the higher eukaryotes were created. Moreover, signaling systems of the lower eukaryotes ar more invariant than these of the higher eukaryotes. It may be associated with the fact that of functional blocks, typical for signaling systems of multicellular animals, fungi and plants, were selected from the numerous variants of signaling system blocks of unicellular organisms.

[Water and ion balance in rat thymocytes under apoptosis induced with dexamethasone or etoposide. Ion-osmotic model of cell volume decrease].

Abstract: Cell ion and water balance was studied with respect to analysis of the osmotic model of apoptotic volume decrease (AVD) in rat thymocytes under dexamethasone (1 microM, 4-6 h) or etoposide (50 microM, 5 h) treatment. Intracellular water content was determined by measurement of cell buoyant density in continuous Percoll gradient, while intracellular potassium and sodium contents were determined by flame emission analysis. Apoptosis was verified by an increase in cell buoyant density, fluorescence of cells stained with Acridine orange and Ethidium bromide (flow cytometry), by changes in the cell cycle and the appearance of sub-diploid peak in the DNA histogram (flow cytometry), and by a decrease in cell size examined with light microscope. A separate fraction of dense cells with reduced size was found to appear after dexamethasone or etoposide treatment. This fraction was considered as apoptotic. An increase in buoyant density of apoptotic cells corresponded to a decrease in cell water content. In apoptotic cells vs. cells with normal buoyant density, the intracellular potassium content was lower, but sodium content was higher. The sum of potassium and sodium contents was lower in apoptotic cells. Taken into account the loss of anions, associated with the loss of cations, the bulk decrease in ions content has been sufficient to be accounted for cell volume decrease on the basis of the ion-osmotic model.

[Morphological changes in the kidney after 5/6 nephrectomy and low-dose radiation].

Abstract: In the present study, we investigated the effect of low-dose irradiation of experimental nephrectomized rats. We hypothized that the low-dose irradiation may slow down the development of focal-segmented glomerulosclerosis (FSGS) after 5/6 nephrectomy. Experiments were performed with 32 male Wistar rats, divided into four groups. The first group contained only operated animals. Animals in the second and third groups were irradiated on the next day after operation with 1 and 3 Gy, respectively. The healthy animals made the forth, control group. Attention was focused on physiological and morphological changes after low-dose (1 and 3 Gy) irradiation. We measured blood pressure, proteinuria, serum creatinin and cysC. Morphological changes of glomerulus and tubules were studied. Animals of the first group had significantly thicker glomerular basement membrane, compared to animals of other groups. The morphological study demonstrated degeneration of the tubular epithelium, tubular atrophy and FSGS. Besides, it was shown that changes in the third group (3 Gy) were less than in nephrectomized (first group) and 1 Gy (second group). The animals of the third group (3 Gy) had significantly lower proteinuria and FSGS. We conclude that our hypothesis, suggesting that low-dose irradiation slows down the development of FSGS, was confirmed.

Dynamics of cytoskeleton microtubules in higher plant meiosis. I. The perinuclear band of microtubules and the meiotic spindle formation

Abstract: A planar meridional perinuclear band of microtubules was observed at the late meiotic prophase I in a range of higher plant species A distinct high-organized structure and a long time of existence allow to consider it as a new class of MTs dependent on the cell cycle in plant meiosis MTs of the perinuclear band convert into meiotic spindle through a complex process of spatial rearrangements

Cajal bodies in insect oocytes. I. Identification and immunocytochemical characteristics of Cajal bodies in vitellogenic oocytes of the yellow mealworm beetle

Abstract: In vitellogenic oocytes of Tenebrio molitor (inactive stage), numerous fibrogranular nuclear bodies (NBs) are present. Using immunofluorescent microscopy, these NBs were shown to contain pre-mRNA splicing factors (small nuclear [sn] RNPs and SR-protein, SC35) as well as RNA polymerase II. A limited set of NBs also contained coilin, a marker protein for Cajal bodies (CBs). We suggest that in T. molitor oocytes, coilin-containing NBs, which also contain splicing factors and RNA polymerase II, seem to represent CBs. In the species studied, no morphological features of CBs were established as compared with other NBs, which do not contain coilin. Microinjectons in oocytes of myc-tagged coilin mRNA, followed by revealing newly translated protein with antibody specific for this tag, have shown targeting of myc-coilin with CBs. The own and literary data on the morphology and molecular composition of CBs are discussed in terms of searching for criteria for CB identification in cells of different origin, and at active and inactive stages.

Cytopathological effects of protein synthesis inhibitor emetine on HeLa cells and their nucleoli

Abstract: Eukaryotic cell nucleolus is a highly dynamic structure, which is sensitive to all changes within or outside cell borders. Numerous data are available on changes of the nucleolar structure and functions under different treatments. However, almost nothing is known about the action of translation inhibitors on the nucleolus, although these substances, together with TNF-alpha, are commonly used for apoptosis induction, both for scientific and therapeutic purposes. Emetine is one of such inhibitors. We have shown that emetine suppresses cell viability, decreases mitotic index, and induces apoptosis in HeLa cells. Emetine action is irreversible, and it sensitizes cells to unfavourable external conditions. The emetine action causes redistribution of UBF, one of RNA-polymerase I factor, from the nucleolus to nucleoplasm even after a short exposure, i.e. when the morphology of the nucleus and chromatin still keeps its native pattern. It is important that other nucleolar proteins, such as fibrillarin and B23, are not recognized in the nucleoplasm until the very late stages of apoptotic process. A suggestion is made that changes in UBF localization may be associated with the onset of ribosomal repeat cleavage and migration of rDNA-"free" fragments from the nucleolus to nucleoplasm. It looks likely that these changes can serve as an initial morphological indication of apoptosis.

Regulation of Na(+)-K(+)-2Cl- cotransporter activity in rat skeletal muscle and intestinal epithelial cells.

Abstract: In mammalian cells, Na + -K + -2Cl - cotransporter activity participates in regulation of ion and volume homeostasis. This makes NKCC indispensable for normal cell growth and proliferation. We recently reported the existence of two mechanisms that can regulate NKCC activity in mature skeletal muscle. In isosmotic conditions, signaling through ERK MAPK pathway is necessary, while inhibition of the cAMP-dependent protein kinase A (PKA) pathway stimulates NKCC activity during hyperosmotic challenge. Both pathways are involved in regulating cell proliferation in wide variety of cells of epithelial and non-epithelial origin, so we tested which pathway regulated NKCC activity in cultured cells. In cultured rat skeletal muscle (L6) and intestinal epithelial (IEC-6) cells, NKCC activity in the basal state comprised 30 to 50 % of total potassium influx, assessed as bumetanide-sensitive 8 6 Rb-uptake. This NKCC activity could not be abolished by inhibitors of ERK MAPK (PD98059 and U0126), PKC (GF109203X), or PI 3-K (wortmannin, LY294002). In L6 myoblasts and in IEC-6 cells, elevation of cAMP levels with isoproterenol or forskolin led to a 60 % inhibition on NKCC activity. In contrast, incubation of IEC-6 cells with the PKA-inhibitor H-89 resulted in 50 % increase of NKCC activity compared with the basal level. In conclusion, it appears that in cultured cells the cAMP-PKA pathway regulates NKCC activity. This resembles hyperosmotic regulation of NKCC activity.

Epigenetic heredity in evolution

Abstract: We discuss the role of cell memory in heredity and evolution. We describe the properties of the epigenetic inheritance systems (EISs) that underlie cell memory and enable environmentally and developmentally induced cell phenotypes to be transmitted in cell lineages, and argue that transgenerational epigenetic inheritance is an important and neglected part of heredity. By looking at the part EISs have played in the evolution of multicellularity, ontogeny, chromosome organization, and the origin of some post-mating isolating mechanisms, we show how considering the role of epigenetic inheritance can sometimes shed light on major evolutionary processes.

[Histone deacetylase inhibitor blocks proliferation of cells transformed with oncogenes E1A and cHa-ras].

Abstract: Rat embryonic fibroblasts, transformed with E1A and cHa-ras oncogenes, are unable to stop in the cell cycle checkpoints under growth factor withdrawal and genotoxic stresses (Bulavin et al., 1999). In the present paper, we showed that sodium butyrate, an inhibitor of histone deacetyase activity, decreased the share of cells being in S-phase, and caused G1/S and G2/M blocks of the cell cycle in the transformants. By means of RT-PCR and immunoblotting, we found that NaB significantly changed the expression of genes involved in proliferation: cyclins D1, A, E and cyclin-dependent kinases Cdk2 and Cdk4, whereas the amount of p21Waf1 and p27Kip1 inhibitors greatly increased. Along with accumulation of p21Waf1 protein content, that of Cdk2-bound p21 increases. Taken together, these data allow to suggest that NaB treatment does evidently restore the capability of p21Waf1 to inhibit cyclin-kinase activity. One may suppose that inhibition of HDAC activity by sodium butyrate leads to activation of yet unknown HDAC-dependent genes, which is followed by restoration of p21Waf1 function in spite of the E1A oncogene expression.

Hemocytes of the blowfly Calliphora vicina and their dynamics during larval development and metamorphosis

Abstract: On the basis of in vitro observation of live cells and examination of stained slides of larval and prepupal Calliphora vicina hemolymph, seven types of hemocytes have been detected: prohemocytes, stable and unstable hyaline cells, thrombocytoids, spindle cells, larval plasmatocytes, and plasmatocytes I-IV, a. The last representing sequential stages of one cell line differentiation. Prohemocytes are basic cells, from which other forms of hemocytes derive outside the hemopoietic tissue, i.e. in free hemolymph. At the last larval instar, three waves of hemopoiesis occur. Either wave tends to increase the general number of cells and to change the quality of hemocyte population. The first wave occurs at the close of larva feeding and is accompanied by increase in the number of hyaline hemocytes, thrombocytoids and larval plasmatocytes. The second wave of hemopoiesis occurs after the larva's crop emptying. In this period the main increase of hemocyte population occurs at the expense of prohemocytes and plasmatocytes I. The most significant (five-fold) explosion of the population of free hemocytes takes place at the onset of pupariation and correlates with the rise of ecdysone titer. At the first stage of this peak, the amount of plasmatocytes I sharply increases. Further on these are rapidly differentiated into plasmatocytes II and III. After the puparium formation, hemocytes are reduced in number. Plasmatocytes III phagocytose fragments of destroyed larval tissues, pass to the stage of plasmatocytes IV (macrophages), and partially settle on tissues.

Recognition of internal (TTAGGG)n repeats by telomeric protein TRF1 and its role in maintenance of chromosomal stability in Chinese hamster cells

Abstract: Mammalian telomeres contain long tandem (TTAGGG)n repeats, which are protected by a complex of different proteins Telomeric repeat-binding factors TRF1 and TRF2 play the key role in protection of telomeres through the formation of terminal loops (called T-loop) A T-loop isolates the 3' strand telomeric end and with this mechanism protects telomeres from the influence of enzymes of DNA reparation and telomere fusions and also interferes with the interaction of telomerase with telomeres Many vertebrate species also contain large blocks of (TTAGGG)n sequences in pericentric and interstitial chromosome bands The Chinese hamster genome contains a total of 18 arrays of these non-telomeric internal (TTAGGG)n sequences (ITs) Chromosome bands containing these arrays are unstable and should be protected with the help of another mechanism, rather than that using telomeres In this study we analysed association of Green Fluorescent Protein (GFP)-tagged TRF1 in Chinese hamster V79 cells with ITs We found that in these cells GFP-TRF1 associates with ITs in the interphase nucleus We detected a little overlap between IT-associated GFP-TRF1 and random DSB sites visualized after the treatment of V79 cells with ionizing radiation We found that the treatment of V79 cells with WM significantly increases the frequency of spontaneous chromosome aberrations These WM effects are possible due to inhibiting phosphorylation of TRF1 by ATM TRF1 is known to be eliminated from telomeres by overexpression of TANK1, which induces TRF1 poly(ADP-ribosyl)ation We transfected V79 cells by plasmid encoding TANK1 and found that the frequency of chromosome rearrangements increased in these cells independently of their treatment by IR Taken together, our results suggest that TRF1 may be involved in the sequence-specific protection of internal non-telomeric (TTAGGG)n repeats

Mating types in the ciliate Dileptus anser. Genetical instability in the mating type system

Abstract: In F1 and F2 from a cross between two clones of Dileptus anser isolated from natural sources (MT 1 x x MT III; MT = mating type), along with "normal" clones, many clones were observed demonstrating abnormal phenotype with respect to the MT-character. Irregular features of the latter were as follows: a) a delay in maturation; b) temporary reversion to immature or adolescent state, which means instability of maturity state; c) expression of MT I and MT III, rather than MT II as in properly matured clones; d) changes in MT (i.e., MT instability); e) appearance of totally unexpected MTs in terms of the scheme of genetic control of MTs in D. anser previously suggested by Afon'kin and Yudin (1987)--e.g., of all three MTs in F1 from the initial (analysing!) cross. Amazingly, these abnormal D. anser clones closely resembled some selfer-clones of Tetrahymena pigmentosa, previously reported as an example of genetic instability in the ciliate MT system (Simon, 1980; Simon, Orias, 1987).

[Matrix metalloproteinases MMP-2 and MMP-9 of wound and burn exudates and their effect on extracellular matrix proteins].

Abstract: The dynamics of matrix metalloproteinases (MMP), as well as of fibronectin concentration in wound and burn fluids was traced. The wound fluid proteolytic activity was studied by gelatin zymography method. The data on degradation of fibronectin and various laminin isoforms by wound fluid proteases show that laminin-1, laminin-2/4 and fibronectin were degraded by wound fluid into small fragments. Remodelling of extracellular matrix proteins occurs. Dynamics of MMP-2 and MMP-9 content in wound or burn fluids as well as that of adhesive protein fibronectin content could be used as a base for development of method of controlling the extracellular matrix remodelling process.

Activation of transcription of ribosome genes following human embryo fibroblast infection with cytomegalovirus in vitro

Abstract: Cytomegalovirus (CMV) infection of human diploid embryo fibroblasts in vitro causes a massive cell death on the 3-4th day of infection with a high primary infection coefficient (1-5 U/cell) Cytopathological effects of viral infection on the 3-4th day includes diminishes of the cell size, changes in their form, compaction of the nuclear chromatin, and disorganization and inactivation of the nucleolus However, the early stages of the viral infection progression (the 1st-2nd day) are accompanied by unequivocal activation of rDNA (ribosomal gene) and the bulk of chromatin transcription There are several features to support this conclusion: In the early CMV-infected cell 1) the nucleolar size is increased; 2) the number of intranucleolar foci binding the specific RNA-polymerase I transcription factor (UBF) is augmented; 3) the Ag-NOR staining is enhanced; 4) 3H-uridine incorporation to the nucleoli is activated; 5) the ultrastructure of the nucleolus is changed Altogether, these data argue in favor of activation of rDNA transcription in human fibroblasts in vitro at the initial stages of infection

[RNA polymerase II and pre-mRNA splicing factors in diplotene oocyte nuclei of the giant African gastropod Achatina fulica].

Abstract: The nuclear distribution of pre-mRNA splicing factors (snRNPs and SR-protein SC35) and unphosphorylated from of RNA polymerase II (Pol II) was studied using fluorescent and immunoelectron cytochemistry in diplotene oocytes of the gastropod Achatina fulica. Association of Pol II and splicing factors with oocyte nuclear structures was analysed. The antibodies against splicing factors and Pol II were shown to label perichromatin fibrils at the periphery of condensed chromatin blocks as well as those in interchromatin regions of nucleoplasm. The revealed character of distribution of snRNPs, SC35 protein, and Pol II, together with the decondensed chromatin and absence of karyosphere, enable us to suggest that oocyte chromosomes maintain their transcriptional activity at the diplotene stage of oogenesis. In A. fulica oocytes, sparse nuclear bodies (NBs) of a complex morphological structure were revealed. These NBs contain snRNPs rather than SC35 protein. NBs are associated with a fibrogranular material (FGM), which contains SC35 protein. No snRNPs were revealed in this material. Homology of A. fulica oocyte nuclear structures to Cajal bodies and interchromatin granule clusters is discussed.

Isolation and characterisation of continuous human embryonic stem cell lines

Abstract: A long-term cultivation (5-8 months) of human blastocyst-derived embryonic cells (hES) was performed. Several properties of hESs were examined to prove the state of continuous cell lines. These cells have passed through 100-175 population doublings with the average population doubling time equal to 37.0 +/- 1.5 h. Isolated hESs, referred to as HESC-1, HESC-2, HESC-3, HESC-4, cultivated on mitotically inactivated mouse embryonic fibroblasts (STO continuous cell line), formed multilayer colonies of various shape. The cells maintained stable proliferative activity, high activity of alkaline phosphatase and expression of transcription factor Oct4, and all this characterizes embryonic stem cells of different origin. Expression of hES specific cell surface antigens (SSEA-3, SSEA-4, TRA-1-81 and TRA-11-81 and TRA-1-60) was confirmed by immunofluorescence analysis with the corresponding monoclonal antibodies. An additional prove for species specificity of HESC lines is the lack of expression of mouse specific surface antigen SSEA1. The cell cycle of HESC-1 undifferentiated cells and embryoid bodies was analysed cytofluorimetrically.

Chromosome mutations in marshland and dry valley populations of Abies sibirica Ledeb

Abstract: Results of chromosome mutation investigation in Abies sibirica populations growing in fen and dry valley in Western Siberia are presented. The frequency of chromosome aberrations and mitotic irregularities is higher in dry valley population of the fir, than in its fen population. However, the spectrum of abnormalities registered in metaphase and ana-telophase cells of the fir from fen is broader, compared to that from dry valley. These features may be due to the fir genome reorganization in different environmental conditions, and indicate the plant reaction to stress factors.

Enhancement of growth promoting activity of human blood on keratinocytes after its irradiation in vivo (transcutaneously) and in vitro with visible and infrared polarized light

Abstract: To stimulate wound healing, current medicine uses various methods of phototherapy. The induced activation of proliferative processes in the wound occurs due to development of not only local, but also systemic processes, whose nature remains largely uninvestigated. The present work provides evidences that as early as 30 min after irradiation of a small area of the volunteer's body surface with polychromatic visible light + infrared polarized light (400-3400 nm, 95% of polarization) at a therapeutic dose (12 J/cm2), soluble factors appear in the circulating blood, which are able to stimulate proliferation of human keratinocytes in primary culture. A similar effect was also revealed after a direct blood irradiation. A proof is provided in favor of a hypothesis that a rapid rise of growth promoting activity of the entire circulating blood may be a consequence of transcutaneous photomodification of the small amount of light-modified blood in superficial skin vessels, and of the effect of such blood on its entire circulating volume. A possibility of a release into plasma of growth factors from blood cells and complexes with alpha 2-macroglobulin is discussed.