Asunto(s)
Diferenciación Celular , Histonas/metabolismo , Rayos X , Animales , Diferenciación Celular/efectos de la radiación , Núcleo Celular/metabolismo , Corazón/efectos de la radiación , Humanos , Riñón/citología , Riñón/metabolismo , Riñón/efectos de la radiación , Ratones , Miocardio/citología , Miocardio/metabolismo , Fosforilación/efectos de la radiaciónRESUMEN
Newly replicated DNA segments (RDS) have been shown to form discrete foci in the mammalian nucleus. Comparison of the number of such foci in formaldehyde-fixed cell nucleus with estimated number of simultaneously active replication forks (RF) suggests that each replication focus contains a cluster of about 10 to 20 closely associated RF. That implied the cluster of synchronously activated replicons as the primary unit of mammalian DNA replication. It still remains unclear whether such clustering of RF does mean adjacency of the replicons in a genomic location (structural clustering, model 1), or it arises from transient clustering of the replicons from different DNA domains at the functioning replication machinery (functional clustering, model 2). In this study we used conventional fluorescence microscopy of the hypotonically treated nuclei preparations to investigate replication foci at the optical resolution limit. Human K562 cells were labeled with 5'-iododeoxyuridine for different time periods. We synchronized the cell culture with hydroxyurea to be able to measure an average increase in DNA content during labeling period using DNA cytometry. Under these conditions, RDS appear as multiple small foci (mini-foci, MF). Further studies revealed that most of such mini-foci of replication represent optical diffraction spots, which are standard in size and different in brightness. The number of the "spots" and variation of their brightness mostly depend on the extent of hypotonic treatment. Flow cytometry control of the synchronized cells peak movement allowed us to measure mean DNA content of the MF. In case of most effective hypotonic treatment, a MF contains about 40 Kbp of labeled DNA, and the general number of the MF approaches the number of replicons that are simultaneously active in a given moment of S-phase. Influence of the effect of hypotonic treatment on overall number of observed MF suggests that replication foci in early and mid S-phase cells do not represent stable structures, but rather arise from functional clustering of comparatively distant replicating regions, thus supporting model 2.
Asunto(s)
Núcleo Celular/genética , Replicación del ADN , Línea Celular Tumoral , ADN/análisis , ADN/fisiología , Citometría de Flujo , Humanos , Soluciones Hipotónicas , Microscopía Fluorescente , ReplicónRESUMEN
Many types of DNA lesions in template strands block DNA replication and lead to a stalling of replication forks. This block can be overcome (bypassed) by special DNA polymerases (for example, DNA polymerase eta, Pol eta) that perform translesion synthesis on damaged template DNA. The phenomenon of completing DNA replication, while DNA lesions remain in the template strands, has been named post-replication repair (PRR). In yeast Saccharomyces cerevisiae, PRR includes mutagenic and error-free pathways under the regulation of the RAD6/RAD18 complex, which induces ubiquitylation of PCNA. In mammalian cells, Pol eta accumulates in replication foci but the mechanism of this accumulation is not known. Pol eta possesses a conserved PCNA binding motif at the C terminal and phosphorylation of this motif might be essential for its interaction with PCNA. We have shown previously that staurosporine, an inhibitor of protein kinases, inhibits PRR in human cells. In this study we examined whether the accumulation of Pol eta in replication foci after DNA damage is dependent on phosphorylation of the PCNA binding motif. We also studied DNA damage-induced phosphorylation of GFP-tagged human Rad18 (hRad18) and its accumulation in replication foci. Our data indicate that (1) Pol eta is not phosphorylated in response to UV irradiation or MMS treatment, but its diffusional mobility is slightly decreased, and (2) hRad18 accumulates in MMS-treated cells, and considerable amount of the protein co-localizes with detergent insoluble PCNA in replication foci; these responses are sensitive to staurosporine. Our data suggest that hRad18 phosphorylation is the staurosporine-sensitive PRR step.
Asunto(s)
Reparación del ADN , Replicación del ADN , Proteínas de Unión al ADN/metabolismo , ADN Polimerasa Dirigida por ADN/metabolismo , Antígeno Nuclear de Célula en Proliferación/metabolismo , Secuencias de Aminoácidos , Animales , Transporte Biológico , Línea Celular , Cricetinae , Daño del ADN , Replicación del ADN/efectos de los fármacos , Proteínas de Unión al ADN/química , ADN Polimerasa Dirigida por ADN/química , Humanos , Mamíferos , Metilmetanosulfonato/farmacología , Fosforilación/efectos de los fármacos , Antígeno Nuclear de Célula en Proliferación/química , Estaurosporina/farmacología , Ubiquitina-Proteína LigasasRESUMEN
For the first time the karyotypes of diploid (2n = 2x = 18) and tetraploid (2n = 4x = 36) species of Lonicera from the Caeruleae subsection: L. altaica Pall., L. boczkarnikowii Plekh. (L. regeliana Boczkarn.), L. edulis Turcz. ex Freyn (2x, 4x), L. emphyllocalyx Maxim., L. iliensis Pojark., L. kamtschatica Pojark., L. pallasii Ledeb., L. stenantha Pojark., L. turczaninowii Pojark., L. villosa (2x, 4x) (Michx.) Muhl. are described. The species karyotypes from 23 natural populations have shown the considerable generic resemblance that expressed in the similar chromosome morphology and variation range of their length from 1 to 3 microns. The species with the same level of ploidy had the same karyotype formula: 2m + 6sm + 1st in diploids and 4m + 11sm + 3st in tetraploids, respectively. The amphiploid origin of the tetraploid Lonicera species has been shown. Diploid and tetraploid forms of L. edulis and L. villosa were the particular karyotypes but not the 2x and 4x races of the same species, respectively. Specific differences were revealed in the total chromosome length in the haploid set and in the number of satellites and secondary constrictions. Generic resemblance and specific peculiarities of Lonicera karyotypes indicate a common center of the blue honeysuckle origin and a common initial population of karyotypes which evolved into several phylogenetic branches of the Caeruleae subsection: the Central Asiatic--L. iliensis and L. stenantha; the Siberian--L. altaica, L. edulis, and L. pallasii; the Beringian--L. emphyllocalyx, L. kamtschatica, and L. villosa; the Manchurian--L. boczkarnikowii (L. regeliana), and L. turczaninowii.
Asunto(s)
Cromosomas de las Plantas/genética , Lonicera/genética , Mitosis , Ploidias , Cariotipificación , Lonicera/clasificación , Filogenia , Brotes de la Planta/clasificación , Brotes de la Planta/genética , Especificidad de la EspecieRESUMEN
XPA repair protein is absolutely needed for nucleotide excision repair (NER). It preferentially binds UV-irradiated DNA in vitro and possibly takes place in the recognition of pyrimidine dimers, the main type of UV-lesions in DNA. Using immunofluorescent microscopy and immunoblotting technique we have found that XPA protein is fully extractable by Triton X-100 solution from non-irradiated normal human fibroblasts, but after UV-irradiation its extractability decreases in UV-dose dependent manner. UV-induced XPA-immobilization was observed in human cell lines with different types of repair defects, but XPA-extractability from unirradiated cells of these lines was significantly lower in comparison with normal fibroblasts. These data do not permit to make conclusion concerning the distinct connection of this phenomenon with different pathways of NER. Histone deacetylase inhibitor, sodium butyrate, did not change the level of extractability in unirradiated and UV-irradiated normal human cells and CHO cells, defective in global genome repair, that indicated the independence of XPA-immobilization from the level of histone acetylation. It was established with the help of confocal microscopy that XPA-foci in detergent-treated UV-irradiated cell were partially colocalized with the focal sites of PCNA, an auxiliary protein of DNA polymerases delta and epsilon. It may mean that a part of detergent-resistant XPA foci correspond to DNA repair synthesis sites, but the major part of immobilized XPA reflects the early step of repair proteins assembly formation needed for the repair of the lesions.
Asunto(s)
Reparación del ADN , Proteínas de Unión al ADN/metabolismo , Línea Celular , Reparación del ADN/efectos de la radiación , Proteínas de Unión al ADN/efectos de la radiación , Humanos , Especificidad de Órganos , Rayos Ultravioleta , Proteína de la Xerodermia Pigmentosa del Grupo ARESUMEN
An auxiliary protein of DNA polymerases delta and epsilon, the proliferating cell nuclear antigen (PCNA), is necessary for efficient DNA replication in vivo and in vitro, and also for the repair synthesis in vitro, but its role in the excision repair of genome in vivo is not exactly established. In S-phase of unirradiated cells, PCNA is tightly bound to focal centers of DNA replication and is not removed by treatment with detergent Triton X-100, but is completely extracted from non-S-phase cells by the indicated detergent. It was shown earlier that after UV-irradiation PCNA could not be removed by the detergent even from non-S-phase cells. It was interpreted as the evidence of PCNA integration into the repair complex and of the participation of this protein in repair synthesis in vivo. In the present work the data were obtained indicating that the role of PCNA in cell response to UV-damage was not confined only to its possible involvement in repair synthesis. With the help of confocal microscopy it was established that in Triton X-100-extracted normal cells PCNA did not colocalize with the well known excision repair protein XPB/ERCC3, defective in cells from Xeroderma pigmentosum (complementation group B) patients. XPB-protein is induced by UV-irradiation in normal cells, and this induction is not observed in repair deficient cells. However, in such cells UV-light induces a detergent-resistant form of PCNA, and this form is obviously not connected with repair. It cannot be excluded that a rapid PCNA immobilization immediately after UV-irradiation of cells is needed for the facilitation of photochemical damage bypass during the subsequent replication of genome.
Asunto(s)
Antígeno Nuclear de Célula en Proliferación/efectos de la radiación , Rayos Ultravioleta/efectos adversos , Adenocarcinoma , Neoplasias de la Mama , Células Cultivadas , Daño del ADN , ADN Helicasas , Reparación del ADN/efectos de la radiación , Proteínas de Unión al ADN/metabolismo , Proteínas de Unión al ADN/efectos de la radiación , Humanos , Microscopía Fluorescente , Antígeno Nuclear de Célula en Proliferación/metabolismo , Coloración y Etiquetado/métodos , Células Tumorales Cultivadas , Xerodermia PigmentosaRESUMEN
Effects of various antituberculosis remedies (ATR)--isoniazid (INA), saluzid (SA), streptosaluzid (SS), ethambutol (EB), sodium para-aminosalicylate (SPAS) on phagosome-lysosome (PL) fusion, on F-actin content in mouse macrophages and on G-actin polymerization in vitro were studied. The ATR of choice have been shown to stimulate the PL fusion. INA (0.2 mM), SA (0.02 mM), SS (0.05 mM), EB (0.08 mM) and SPAS (0.5 mM) increased the F-actin content and changed its localization within macrophages. ATR changed the character of G-actin polymerization in vitro. Possible mechanisms of interrelation between cytoskeleton (actin part) changes and PL fusion are discussed. The results obtained suggest to use the ATR-induced changes in PL fusion and F-actin content in the cells for estimating therapeutic effects of respective antituberculosis remedies.
Asunto(s)
Actinas/efectos de los fármacos , Antituberculosos/farmacología , Lisosomas/efectos de los fármacos , Macrófagos Peritoneales/efectos de los fármacos , Fagosomas/efectos de los fármacos , Actinas/metabolismo , Animales , Células Cultivadas , Relación Dosis-Respuesta a Droga , Fluorescencia , Lisosomas/metabolismo , Lisosomas/ultraestructura , Macrófagos Peritoneales/metabolismo , Macrófagos Peritoneales/ultraestructura , Ratones , Ratones Endogámicos C57BL , Fagosomas/metabolismo , Fagosomas/ultraestructura , Polímeros , ConejosRESUMEN
The influence of natural and synthetic polyamines, phalloidin, cytochalasin D, vinblastine, colchicine, puromycin, chlorpromazine, urea, glutaraldehyde, and ethanol on the phagosome-lysosome fusion and the content of F-actin in murine peritoneal macrophages has been studied. Fluorescent phallotoxin FITC-phalloidin was used to stain F-actin. Natural polyamines (spermine, spermidine, putrescine), phalloidin, ethanol (0.1 M) stimulated the phagosome-lysosome fusion and increased the mid-content of F-actin in macrophages. Cytochalasin D, vinblastine, colchicine, puromycin, chlorpromazine, urea, glutaraldehyde, ethanol (0.15 and 0.2 M) inhibited this process and decreased the mid-content of F-actin. Possible mechanisms of the interconnection of cytoskeleton and the phagosome-lysosome fusion are discussed.
Asunto(s)
Actinas/efectos de los fármacos , Lisosomas/efectos de los fármacos , Macrófagos/efectos de los fármacos , Fagosomas/efectos de los fármacos , Actinas/análisis , Animales , Fusión Celular , Células Cultivadas/efectos de los fármacos , Células Cultivadas/ultraestructura , Lisosomas/ultraestructura , Macrófagos/química , Macrófagos/ultraestructura , Ratones , Ratones Endogámicos C57BL , Cavidad Peritoneal/citología , Fagosomas/ultraestructura , Coloración y Etiquetado/métodos , Factores de TiempoRESUMEN
Automated fluorescent microscope developed by the authors permits photometry of microquantities of cellular suspensions in scanning standard 60-well plates with a flat bottom. Automated and semiautomated modes of operation are possible. Fluorescent stains and schemes of staining the examined cells that yield stable results in the lymphocytotoxic test have been selected. Comparative analysis of the efficacies of histocompatibility antigens detection by the routine and the fluorescent technique has shown a number of advantages of the fluorescent method. Specific features of fluorescent stains used for staining live cells are described.
Asunto(s)
Prueba de Histocompatibilidad/instrumentación , Prueba de Histocompatibilidad/métodos , Humanos , Microscopía Fluorescente/instrumentación , Microscopía Fluorescente/métodos , Fotometría/instrumentación , Fotometría/métodosRESUMEN
Morphometric parameters were measured and specific light optic features of epitheliocyte nuclei examined in the cytologic preparations from 67 patients with various abnormalities of the cervix uteri multi-layer squamous epithelium by a present-day long-distance microimage analyzer. The most informative biometric parameters were detected, role of DNA cytophotometry defined, and algorithm of differential diagnosis between benign conditions, dysplasia, and carcinoma developed.
Asunto(s)
Displasia del Cuello del Útero/patología , Neoplasias del Cuello Uterino/patología , Femenino , Humanos , Frotis VaginalRESUMEN
Morphometrical and optical density (including DNA content) parametres of nuclei of the endometrial epithelial cells in smears of endometrial aspirates obtained from 57 women without endometrial disorders with hyperplastic, precancer conditions and endometrial cancer have been measured by using television image analyzer IBAS-2. Biometrical data have demonstrated a good correlation with cytomorphological diagnostic criteria and in 85.7% they have facilitated discrimination of the normal and hyperplastic epithelium from precancer and high differentiated endometrial cancer.
Asunto(s)
Endometrio/patología , Enfermedades Uterinas/diagnóstico , Adenocarcinoma/diagnóstico , Adenocarcinoma/patología , Biometría , Núcleo Celular/patología , Citodiagnóstico , ADN/análisis , ADN de Neoplasias/análisis , Diagnóstico Diferencial , Hiperplasia Endometrial/diagnóstico , Hiperplasia Endometrial/patología , Epitelio/patología , Femenino , Humanos , Ciclo Menstrual , Lesiones Precancerosas/diagnóstico , Lesiones Precancerosas/patología , Enfermedades Uterinas/patología , Neoplasias Uterinas/diagnóstico , Neoplasias Uterinas/patologíaRESUMEN
Smears of the endocervical channel taken from 58 patients with various endocervical conditions (reserve cell hyperplasia and dysplasia, reserve cell carcinoma in situ. Adenocarcinoma in situ, invasive glandular and poorly differentiated carcinoma) were stained by Feulgen method. Morphometrical (surface, perimeter, maximum and minimum diameter) and light microscopic (mean optical density of staining, its dispersion, DNA content) parameters of the epithelial cell nuclei were measured by means of telemetric image analyzer. The most informative biometrical indexes are found, the role of the DNA cytophotometry in the differential diagnosis is determined, the algorithm of the automated cytomorphological diagnosis of the dysplasia and endocervical carcinoma is developed.
Asunto(s)
Lesiones Precancerosas/diagnóstico , Neoplasias del Cuello Uterino/diagnóstico , Adenocarcinoma/análisis , Adenocarcinoma/diagnóstico , Adenocarcinoma/ultraestructura , Algoritmos , Biometría/métodos , Carcinoma in Situ/análisis , Carcinoma in Situ/diagnóstico , Carcinoma in Situ/ultraestructura , Núcleo Celular/análisis , Núcleo Celular/ultraestructura , Cuello del Útero/análisis , Cuello del Útero/ultraestructura , Citodiagnóstico , ADN de Neoplasias/análisis , Diagnóstico Diferencial , Epitelio/análisis , Epitelio/ultraestructura , Femenino , Humanos , Lesiones Precancerosas/análisis , Lesiones Precancerosas/ultraestructura , Displasia del Cuello del Útero/análisis , Displasia del Cuello del Útero/diagnóstico , Displasia del Cuello del Útero/ultraestructura , Neoplasias del Cuello Uterino/análisis , Neoplasias del Cuello Uterino/ultraestructura , Frotis VaginalRESUMEN
Cytohistological and biometric analysis identified certain cytomorphological features of reserve cell hyperplasia, dysplasia (atypical hyperplasia of reserve cells), reserve cell carcinoma in situ and adenocarcinoma in situ in the cervical canal. The role of TV analyser of microimages was established and their application to aid the practising cytologists in making diagnosis, particularly, in cases of conflicting cytologic and histologic evidence is discussed.
Asunto(s)
Transformación Celular Neoplásica/patología , Cuello del Útero/patología , Adenocarcinoma/metabolismo , Adenocarcinoma/patología , Adulto , Anciano , Biopsia , Carcinoma in Situ/metabolismo , Carcinoma in Situ/patología , Transformación Celular Neoplásica/metabolismo , Cuello del Útero/metabolismo , ADN de Neoplasias/análisis , Epitelio/metabolismo , Epitelio/patología , Femenino , Histocitoquímica , Humanos , Persona de Mediana Edad , Lesiones Precancerosas/metabolismo , Lesiones Precancerosas/patología , Displasia del Cuello del Útero/metabolismo , Displasia del Cuello del Útero/patología , Neoplasias del Cuello Uterino/metabolismo , Neoplasias del Cuello Uterino/patología , Frotis VaginalRESUMEN
Nucleic acids and total protein contents in hepatocytes and erythrocytes of diploid (Bufo viridis, 2n = 22) and tetraploid (B. danatensis, 2n = 44) green toads were measured cytophotometrically. The RNA (table 1) and protein (table 2) contents in the tetraploid cells are twice as much as that in diploid ones. According to these results tetraploidy in green toads may have a recent origin. The relation between structural and functional polyploidization in tetraploid amphibian species is discussed.