[Recognition of lymphatic tumors of the mediastinum].
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Biomedical subjects
Publications and source records attributed to I A Vorob'ev.
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The inner cytosine methylation was analyzed in the CCWGG sequences of the 5'-terminal region of the human calcitonin gene from peripheral blood and bone marrow cells in various forms of leukemia. Since these sequences remain nonmethylated both in norm and in various leukemia forms, the CpG dinucleotide hypermethylation of the 5'-terminus of the human calcitonin gene, characteristic for the development of leukemias, does not spread over adjacent CpNpG sequences.
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One of the spindle poles of mitotic PK cells was irradiated with UV microbeam at anaphase. After irradiation, cell division completed with a minor delay and two daughter cells were spreading synchronously. Later on, cells with irradiated centrosomes slightly shrunk, while their sister cells enlarged normally. Sister cells entered S-phase, some of them undergoing mitosis. In the cells with irradiated centrosomes the formation of nucleoli was disturbed and numerous primary nucleoli remained for 50 h (the maximum time of observation). RNA synthesis in the cells with irradiated centrosomes was twice less than in the sister cells, with ribosomal RNA synthesis being suppressed predominantly. Cells with irradiated centrosomes did not enter S-phase for as long as 24 h. The same irradiation of a portion of cytoplasm outside the spindle performed during anaphase did not change the pattern behaviour in daughter cell. In is concluded that the centrosome regulates progression throughout the cell cycle, and that centrosome irradiation induces specific and irreversible damage of interphase cells.
After addition of 20 mM calcium ionophore A23187 to cultured PK (pig kidney embryo) cells, [Ca++] in cytosol increased by more than 10 times. The maximum [Ca++] concentration was observed 1-2 min after drug introduction. Later on [Ca++] gradually decreased, and after 30 min of incubation with A23187 [Ca++] its concentration was 3-5 times higher than in the norm. 1 min after introduction of the calcium ionophore, mitotic spindles shortened for 1/3 and the angle of divergence of spindle microtubules from the centrosome extended. These changes remained for 5 min of treatment. After nocodazole treatment the length of the mitotic spindle reduced (2 min), then mitotic spindle and the metaphase plate were disrupted. The rate of mitotic spindle shortening after addition of the ionophore is about the same as after addition of nocodazole, but after ionophore treatment the metaphase plate remained for more than 5 min. Based on the results obtained we suggest that the maximum distance between spindle poles at metaphase is in the intact cell, and after any perturbation of normal microtubule dynamics the spindle may rapidly collapse. The collapsed mitotic spindle becomes more stable and its size is determined predominantly by kinetochore fibers. The metaphase spindle is completely and rapidly destroyed when the microtubule growth is prohibited, but it is preserved when this growth is restricted.
Addition of 20 microM calcium ionophore A23187 to cultured PK (pig kidney embryo) cells gave an increase of Ca2+ in cytosol by more than 10 times. The maximum of [Ca2+] was achieved in 1-2 min after introduction of the drug. Later on [Ca2+] gradually decreased, and after 30 min of incubation with A23187 [Ca2+] was 3-5 times above normal level. Immunofluorescent and electron microscope studies showed no alterations in the microtubule system of interphase cells after 1-30 min treatment. The electro microscope study showed that-following 1.5 min introduction of the drug the random orientation of material and daughter centrioles changed: most of them settled down at an angle more, than 74 degrees to the substrate surface. After 3 min of A23187 treatment more than half of maternal centrioles were oriented perpendicular to the substrate surface. After 5 min of A23187 treatment, the percentage of maternal centrioles with perpendicular orientation was the same and this orientation remained for 30 min. The percentage of perpendicular daughter centrioles decreased after 3 min of treatment, and after 30 min their orientation was random. We suggest that the perpendicular orientation of centrioles to the substrate surface is mediated through centrosome-associated calcium-binding proteins.
Saltatory movements of large (0.3-0.8 micron) granules in the cytoplasm of PK cells were described in norm and after nocodazole and sodium azide treatments. In untreated cells the length of single movements was up to 2 micron and even more (sometimes up to 5 micron). Nocodazole at a concentration of 0.2 micron and sodium azide at a concentration of 20 micron inhibited all rapid movements longer than 1.2 micron, but did not affect the frequency of movements shorter than 1 micron. The effect of nocodazole was reversible: the long rapid movements were seen resumed after its removal. A comparison of histograms (length versus frequency) of the rapid movements of granules in norm and after nocodazole and sodium azide treatments made it possible to conclude that real saltations (ATP and microtubule-dependent motions) are rapid translocations which exceed 1 micron.
Cytoplasts of cultured L fibroblasts, obtained by enucleation at the end of G1 of synchronized cells collected in mitosis, have usually two centrioles. Starting from 8 h after enucleation, the centrioles in all cytoplasts were seen replicating. The formed procentrioles had an average length of 0.2 micron and did not grow within the next 16 h. When the same synchronized L cells were treated from the end of G1 with actinomycin D, inhibiting more than 96% of RNA synthesis, procentrioles were not observed for about 16 h of incubation. It is concluded that centrioles in cultured cells could replicate in the absence of RNA synthesis, and that the nucleus may not only regulate the centriole replication, but also suppress this process.
Centrioles in the enterocytes of murine small intestine are located far away from the nuclei and close to the apical surface of cells (1-3 microns from the brush border). Centrioles never form a primary cilium and are not attached to the plasma membrane. Centrosomes (cell center) in enterocytes undergo subsequent involution in respect to the position of cell in the crypt-villus system. Five zones were studied separately: the bottom of the crypt (approx. 1/3 of the appendix), the upper part of the crypt (before its transition into the villus), the basal part, the middle and top of the villus. A centrosome in the crypt contains two centrioles (maternal and daughter) located close to each other. In 10 of 27 cells studied centrioles were replicating. A maternal centriole has 2-3 pericentriolar satellites and appendages. Several cytoplasmic microtubules run towards mother centrioles. Centrioles at the upper part of the crypt split at a distance up to 1.5 microns from each other. The average number of cytoplasmic microtubules and pericentriolar satellites decreases. At the basal part of the villus, centrioles split a distance up to 5 microns from each other; seldom microtubules may be found in a radius of 1 micron around centrioles and none of them is attached to centrioles or satellites. Starting from the middle of the villus, centrioles sometimes may have incomplete triplets of microtubules at the distal and (or) the proximal end. At the upper part of the villus there is only one centriole per cell, that was found in 10 of 50 cells studied on a complete series of 0.2 micron thick sections.
It has been shown that after enucleation of the PE cells with cytochalasin D the centrioles remain in approximately 80% of cytoplasts. Some cytoplasts contain only single centriole, either a mother (active) of a daughter (inactive) one. 20% cytoplasts have no centrioles. 2h after enucleation the centrosome structure in the cytoplasts did not differ from that in normal cells. 14-16 h after enucleation in many cytoplasts large secondary lysosomes and lipid droplets appeared around the centrosome. At this time in some cytoplasts in the centrosome we observed free microtubule convergence foci. 14-16 h after the enucleation, some cytoplasts have doubling centrioles. Under the influence of ouabain (30 min), the number of active centrioles oriented perpendicularly to the substrate plane in the cytoplasts increased. We suggest that the preferentially perpendicular orientation of centrioles to the substrate plane does not depend on the nuclear activity.
A 30-min action of energy transfer inhibitors (2,4-dinitrophenol, deoxyglucose, azide and calcium ionophore A23187) on tissue culture cells results in a significant increase in the quantity of microtubules around the centrosome. After the action of all the inhibitors, mostly increases the number of long microtubules with free proximal end oriented towards the centrosome. It is suggested that energy transfer inhibitors may stimulate foundation of microtubules on the centrosome and stabilize free microtubules, while they exert no effect on the frequency of detachment of microtubules from the centrosome.
One of the spindle poles of mitotic PK cells was irradiated with UV microbeam in metaphase or in anaphase. Electron microscopy showed that immediately after irradiation the microtubules around the centrosome were maintained, and that the ultrastructure of both irradiated and nonirradiated poles was similar. After microirradiation of the centrosome in metaphase, the mitotic halo around this centrosome was retained, but in due time the number of microtubules was getting less compared to that around the nonirradiated centrosome. When daughter cells with irradiated centrosomes are passing into the interphase, their centrioles are not separated from each other, no primary cilia are formed, and no replication of centrioles occurs. In the interphase cells with irradiated centrosomes, satellites are formed on the active centriole, but centrosome-attached microtubules are practically absent.
Ultraviolet (280 nm) microbeam irradiation of the centrosome (spindle pole) in the early anaphase slows down and then stops chromosome movement towards the irradiated pole. This happens as a result of rapid (in 1-2 min) disorganization of the half-spindle. Chromosome movement towards the opposite pole continues normally. Irradiation of the centrosome also affects cystotomy--the residual body is formed later than in the normal cell. In some cases additional constrictions are formed or the cytoplasm starts blebbing. Immediately after division the microtubule network in two daughter cells (one of them with irradiated centrosome) is similar. Two hours later in the irradiated cell the amount of microtubules is often less than in the sister cell. Incubation with nocodazole (0.5-1.5 h, 0.15 microgram/ml) shows that in the irradiated cells microtubules radiating from the centrosome are practically absent. Irradiation of other regions of the cytoplasm does not cause any of the effects described above.
Blue light (wavelength 350-480 nm) irradiation of the early mitotic (prophase and prometaphase) tissue culture cells at the dose of 50-3000 J/cm2 delay mitosis or completely block it at the metaphase. Cell sensitivity to the near UV light (wavelength 360 nm) was few times more as compared with the sensitivity to the visible light (wavelength 400-480 nm). Mitotic cells irradiated with the green light (wavelength more than 500 nm; dose up to 7500 J/cm2) completed division normally. The effect of the blue light did not depend on the presence of phenol red in tissue culture medium. Rhodamin 123 staining did not show any changes in the mitochondrial system in the irradiated mitotic cells. Blue light irradiation with the dose enough for the induction of mitotic delay appears to be insufficient to affect the proliferation of interphase cells.
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A 5 second UV microirradiation of the centrosome during the early metaphase leads to a rapid (within 5 minutes) chromosome shift towards the normal pole and disorganizes the spindle. The later metaphase plate is also disorganized, chromosomes being situated chaotically in the central part of the cell. Numerous (up to 10 or more) microtubule convergence centers are observed instead of the spindle. 2-4 hours after the microirradiation some cells may enter cytotomy. The microirradiation of chromosomes and cytoplasm in similar and greater doses (exposure up to 15 seconds) did not lead to disorganization of the spindle and did not effect the normal completion of mitosis. Sometimes the 5 second microirradiation in the middle metaphase also blocked anaphase, but the microirradiation within the last 5 minutes of the metaphase always failed to block anaphase and normal completion of division.
Carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP), ouabain and calcium ionophore A23187 caused a centrosome restructuring expressed in mean number increase of satellites on the active (mother) centriole, in an increase of the mean slope of centrioles to the substrate surface and in the more frequent occurrence of primary cilia. In the presence of FCCP the effect appeared only in 10 min and retained for 2 h. The ouabain caused the separation of active and inactive centrioles in more than in a half of the cells. The data obtained permit to conclude that depolarization of the plasmatic membrane only is needed for the initiation of centrosome restructuring. The authors propose that this reaction of centrosome is a component of the cell overall response to nonspecific lesions.
The disruption of microtubules with nocodazole or microfilaments with cytochalasin B did not prevent mother centrioles from nonrandom, preferentially perpendicular orientation with respect to the substrate plane after FCCP treatment. The microtubules affect negatively the reorientation of centrioles, because after their disruption by nocodazole the percentage of centrioles with the perpendicular orientation (the angle is tipped to the substrate plane by over 74 degrees) is seen to increase. The microfilaments have the positive effect, because after their disruption by cytochalasin B the share of centrioles with the perpendicular orientation decreases. Thus, our observations do not support the hypothesis that the long microtubules can provide the perpendicular orientation of centrioles anchoring them in the cytoplasm.