Your browser doesn't support javascript.
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 2.832
Filtrar
1.
Einstein (Sao Paulo) ; 17(4): eAO4742, 2019 Sep 09.
Artigo em Inglês, Português | MEDLINE | ID: mdl-31508660

RESUMO

OBJECTIVE: To evaluate the induction of DNA damage in peripheral blood mononuclear cells of patients with sickle cell disease, SS and SC genotypes, treated with hydroxyurea. METHODS: The study subjects were divided into two groups: one group of 22 patients with sickle cell disease, SS and SC genotypes, treated with hydroxyurea, and a Control Group composed of 24 patients with sickle cell disease who were not treated with hydroxyurea. Peripheral blood samples were submitted to peripheral blood mononuclear cell isolation to assess genotoxicity by the cytokinesis-block micronucleus cytome assay, in which DNA damage biomarkers - micronuclei, nucleoplasmic bridges and nuclear buds - were counted. RESULTS: Patients with sickle cell disease treated with hydroxyurea had a mean age of 25.4 years, whereas patients with sickle cell disease not treated with hydroxyurea had a mean age of 17.6 years. The mean dose of hydroxyurea used by the patients was 12.8mg/kg/day, for a mean period of 44 months. The mean micronucleus frequency per 1,000 cells of 8.591±1.568 was observed in the Hydroxyurea Group and 10.040±1.003 in the Control Group. The mean frequency of nucleoplasmic bridges per 1,000 cells and nuclear buds per 1,000 cells for the hydroxyurea and Control Groups were 0.4545±0.1707 versus 0.5833±0.2078, and 0.8182±0.2430 versus 0.9583±0.1853, respectively. There was no statistically significant difference between groups. CONCLUSION: In the study population, patients with sickle cell disease treated with the standard dose of hydroxyurea treatment did not show evidence of DNA damage induction.


Assuntos
Anemia Falciforme/genética , Dano ao DNA/efeitos dos fármacos , Hidroxiureia/farmacologia , Inibidores da Síntese de Ácido Nucleico/farmacologia , Adolescente , Adulto , Anemia Falciforme/tratamento farmacológico , Criança , Pré-Escolar , Citocinese , Dano ao DNA/genética , Feminino , Humanos , Hidroxiureia/efeitos adversos , Hidroxiureia/uso terapêutico , Masculino , Testes para Micronúcleos , Pessoa de Meia-Idade , Testes de Mutagenicidade , Mutação/efeitos dos fármacos , Inibidores da Síntese de Ácido Nucleico/efeitos adversos , Inibidores da Síntese de Ácido Nucleico/uso terapêutico , Adulto Jovem
2.
Exp Parasitol ; 204: 107727, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31344389

RESUMO

BACKGROUND: Trypanosoma rangeli is a protozoan parasite that is non-virulent to the mammalian host and is morphologically and genomically related to Trypanosoma cruzi, whose proliferation within the mammalian host is controversially discussed. OBJECTIVES: We aimed to investigate the T. rangeli cell cycle in vitro and in vivo by characterizing the timespan of the parasite life cycle and by proposing a molecular marker to assess cytokinesis. METHODOLOGY: The morphological events and their timing during the cell cycle of T. rangeli epimastigotes were assessed using DNA staining, flagellum labelling and bromodeoxyuridine incorporation. Messenger RNA levels of four genes previously associated with the cell cycle of trypanosomatids (AUK1, PLK, MOB1 and TRACK) were evaluated in the different T. rangeli forms. FINDINGS: T. rangeli epimastigotes completed the cell cycle in vitro in 20.8 h. PLK emerged as a potential molecular marker for cell division, as its mRNA levels were significantly increased in exponentially growing epimastigotes compared with growth-arrested parasites or in vitro-differentiated trypomastigotes. PLK expression in T. rangeli can be detected near the flagellum protrusion site, reinforcing its role in the cell cycle. Interestingly, T. rangeli bloodstream trypomastigotes exhibited very low mRNA levels of PLK and were almost entirely composed of parasites in G1 phase. MAIN CONCLUSIONS: Our work is the first to describe the T. rangeli cell cycle in vitro and proposes that PLK mRNA levels could be a useful tool to investigate the T. rangeli ability to proliferate within the mammalian host bloodstream.


Assuntos
Proteínas de Ciclo Celular/genética , Ciclo Celular/genética , Citocinese/fisiologia , Proteínas Serina-Treonina Quinases/genética , Proteínas Proto-Oncogênicas/genética , RNA Mensageiro/análise , Trypanosoma rangeli/citologia , Animais , Bromodesoxiuridina/metabolismo , Ciclo Celular/efeitos dos fármacos , Citocinese/genética , DNA de Protozoário/química , DNA de Protozoário/isolamento & purificação , Citometria de Fluxo , Imunofluorescência , Hidroxiureia/farmacologia , Camundongos , Camundongos Endogâmicos BALB C , Inibidores da Síntese de Ácido Nucleico/farmacologia , RNA de Protozoário/genética , RNA de Protozoário/isolamento & purificação , Fatores de Tempo , Trypanosoma rangeli/efeitos dos fármacos , Trypanosoma rangeli/enzimologia , Trypanosoma rangeli/genética , Tripanossomíase/parasitologia
3.
Mol Biol (Mosk) ; 53(3): 446-455, 2019.
Artigo em Russo | MEDLINE | ID: mdl-31184610

RESUMO

Ran is an evolutionarily conserved GTPase crucial in regulating various cell divisions, including mitosis and meiosis. A previous study showed that the knockdown of RAN1 inhibited macronuclear amitosis with the abnormal organization of intramacronuclear microtubules in Tetrahymena thermophila. This study aimed to further investigate the effects of the inducible expression of wild-type Ran1 (Ran1WT), GTP-bound Ran1-mimetic (Ran1Q70L), and GDP-bound Ran1-mimetic (Ran1T25N) on cytoplasmic microtubule assembly during amitosis of T. thermophila, based on previous studies about their effects on intramacronuclear microtubule. The mutant strains of T. thermophila for inducible expression of Ran1WT/T25N/Q70L by Cd^(2+) were constructed. The inducibly expressed HA-Ran1Q70L/T25N distributed asymmetrically across the macronuclear envelope during amitosis. At the lower level of inducible expression, only Ran1T25N showed a significant decreasing effect on T. thermophila reproduction, macronuclear amitosis and cytokinesis. At the higher level of inducible expression, Ran1WT/Q70L/T25N inhibited T. thermophila reproduction, macronuclear amitosis and cytokinesis, and the inhibitive effect of Ran1T25N was the most significant. The inducible expression of Ran1WT/Q70L/T25N led to defects in amitosis and cytokinesis with abnormal cytoplasmic microtubule assembly. These results further confirmed the regulatory function of Ran1 on amitosis and suggested a novel role of Ran1 in cytokinesis and the alignment of cytoplasmic microtubules in T. thermophila.


Assuntos
Citocinese , Guanosina Difosfato/metabolismo , Guanosina Trifosfato/metabolismo , Microtúbulos/metabolismo , Mutação , Proteínas de Protozoários/metabolismo , Tetrahymena thermophila , Proteína ran de Ligação ao GTP/metabolismo , Microtúbulos/patologia , Mitose , Proteínas de Protozoários/genética , Tetrahymena thermophila/citologia , Tetrahymena thermophila/genética , Tetrahymena thermophila/metabolismo , Proteína ran de Ligação ao GTP/genética
4.
mSphere ; 4(3)2019 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-31043517

RESUMO

The early divergent protozoan parasite Trypanosoma brucei alternates between the insect vector and the mammalian hosts during its life cycle and proliferates through binary cell fission. The cell cycle control system in T. brucei differs substantially from that in its mammalian hosts and possesses distinct mitosis-cytokinesis checkpoint controls between two life cycle stages, the procyclic form and the bloodstream form. T. brucei undergoes an unusual mode of cytokinesis, which is controlled by a novel signaling cascade consisting of evolutionarily conserved protein kinases and trypanosome-specific regulatory proteins in the procyclic form. However, given the distinct mitosis-cytokinesis checkpoints between the two forms, it is unclear whether the cytokinesis regulatory pathway discovered in the procyclic form also operates in a similar manner in the bloodstream form. Here, we showed that the three regulators of cytokinesis initiation, cytokinesis initiation factor 1 (CIF1), CIF2, and CIF3, are interdependent for subcellular localization but not for protein stability as in the procyclic form. Further, we demonstrated that KLIF, a regulator of cytokinesis completion in the procyclic form, plays limited roles in cytokinesis in the bloodstream form. Finally, we showed that the cleavage furrow-localizing protein FRW1 is required for cytokinesis initiation in the bloodstream form but is nonessential for cytokinesis in the procyclic form. Together, these results identify conserved and life cycle-specific functions of cytokinesis regulators, highlighting the distinction in the regulation of cytokinesis between different life cycle stages of T. brucei IMPORTANCE The early divergent protozoan parasite Trypanosoma brucei is the causative agent of sleeping sickness in humans and nagana in cattle in sub-Saharan Africa. This parasite has a complex life cycle by alternating between the insect vector and the mammalian hosts and proliferates by binary cell fission. The control of cell division in trypanosomes appears to be distinct from that in its human host and differs substantially between two life cycle stages, the procyclic (insect) form and the bloodstream form. Cytokinesis, the final step of binary cell fission, is regulated by a novel signaling cascade consisting of two evolutionarily conserved protein kinases and a cohort of trypanosome-specific regulators in the procyclic form, but whether this signaling pathway operates in a similar manner in the bloodstream form is unclear. In this report, we performed a functional analysis of multiple cytokinesis regulators and discovered their distinct functions and regulations in the bloodstream form.


Assuntos
Citocinese , Proteínas de Protozoários/genética , Trypanosoma brucei brucei/genética , Trypanosoma brucei brucei/fisiologia , Tripanossomíase Bovina/sangue , Animais , Bovinos , Regulação da Expressão Gênica , Estágios do Ciclo de Vida , Proteínas Quinases/metabolismo , Proteínas de Protozoários/metabolismo
5.
DNA Cell Biol ; 38(6): 532-540, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30985224

RESUMO

Anillin is an actin binding protein and plays crucial roles during mitotic cell cycle progression in metazoan. However, the sequence and functions of the Anillin gene have not been yet characterized in the silkworm, Bombyx mori. In this study, we cloned the full-length cDNA sequence of the silkworm Anillin (BmAnillin) gene. The deduced amino acid sequence for BmAnillin protein comprises an Anillin homology region (AHR) covering an Anillin homology domain and a pleckstrin homology domain. Phylogenetic analysis and multiple alignments of the Anillin genes from silkworm and other organisms indicated evolutionary conservation in the AHR containing conserved phosphorylation sites. Reverse transcription-PCR experiments confirmed that the BmAnillin gene was highly expressed during larval development of gonads in which cells undergo mitotic cycles and exhibited an unexpected high expression in silk gland with endocycle during larval molting. RNA interference-mediated knockdown of the BmAnillin gene in silkworm BmN4-SID1 cells derived from ovary disrupted chromosome separation and resulted in a loss of the F-actin filament at cleavage furrow during anaphase, suggesting that the BmAnillin gene is essential for cytokinesis in silkworm.


Assuntos
Bombyx/genética , Proteínas Contráteis/genética , Animais , Bombyx/metabolismo , Células Cultivadas , Segregação de Cromossomos , Clonagem Molecular , Proteínas Contráteis/metabolismo , Proteínas Contráteis/fisiologia , Citocinese , Expressão Gênica , Genes de Insetos , Filogenia , Alinhamento de Sequência , Análise de Sequência
6.
Asian Pac J Cancer Prev ; 20(4): 1045-1050, 2019 Apr 29.
Artigo em Inglês | MEDLINE | ID: mdl-31030472

RESUMO

Objectives: Vast number of studies show the relationship between aneuploidy and cancer. Ionizing radiation in addition to induce all kinds of damages to the cells and structure of chromosomes, is also able to induce aneuploidy through direct damages to chromosome division apparatus. Also irradiation of the cells induces mutations in several genes which might be involved in cell division fidelity and play a role in reversing the effect of aneugens. Therefore, irradiation of cells and tissues might produce sensitivity to agents with aneugenic capability in irradiated cells. Methods: To investigate the persistent genomic effect of ionizing irradiation on chromosomal instability, L929 cells were gamma irradiated with the dose of 2 Gy. Cells were left to recover from the harmful effect of irradiation. They were treated with low dose of vinblastine (0.5 ng.ml-1) 72h post-gamma irradiation. Finally, the induced chromosomal abnormalities were scored using micronucleus assay in cytokinesis-blocked binucleated cells (MnBi). Results: Irradiation-recovered L929 cells treated with vinblastine showed a statistically higher frequency of MnBi compared to non-irradiated and vinblastine treated cells. Conclusion: The results indicate that gamma irradiation, in addition to direct induction of chromosomal damages, is also able to create persisting genomic sensitivity in the cells to chromosomal instability, which is detectable when exposed to the second stimulus.


Assuntos
Antineoplásicos Fitogênicos/farmacologia , Núcleo Celular/genética , Aberrações Cromossômicas , Citocinese , Fibroblastos/patologia , Raios gama , Vimblastina/farmacologia , Divisão Celular , Células Cultivadas , Fibroblastos/efeitos dos fármacos , Fibroblastos/efeitos da radiação , Humanos , Testes para Micronúcleos
7.
Eur J Pharm Sci ; 135: 103-112, 2019 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-31034983

RESUMO

Tuberculosis, caused by Mycobacterium tuberculosis has been one of the primal afflictions to human, and owing to the current scenario of drug resistance, newer drugs, and alternate targets are required to mitigate the disease. FtsZ is a GTP hydrolyzing protein, conserved in prokaryotes that plays a central role in Z-ring formation during cell division cytokinesis stage. This study employs the use of pharmacophore models derived from two different datasets based on Mtb-FtsZ GTPase inhibition and whole cell antibacterial activity, to virtually screen and shortlist novel compounds from In-house small molecule library as Mtb-FtsZ inhibitors and evaluate their in-vitro and ex-vivo activity. The results revealed Piperine (IC50 = 21.2 ±â€¯0.7 µM), 4-Bromo di-methoxy coumarin (IC50 = 13.0 ±â€¯1.6 µM) and Di-ethyl amino methyl coumarin (IC50 = 19.4 ±â€¯1.1) as potent Mtb-FtsZ GTPase inhibitors which showed considerable antibacterial activity (84.0 ±â€¯2.6 µM, 56.0 ±â€¯4.3 µM and 108 ±â€¯7.1 µM respectively) against M. smegmatis. They appear to be bacteriostatic, as well as treatment with these compounds led to a 3× increase in cell length of M. smegmatis. Further these molecules also altered the FtsZ gene expression by 3-fold when compared to untreated. In addition compound Aloin, an Aloe exudate showed potent Mtb-FtsZ inhibition (IC50 = 16.7 ±â€¯0.4 µM) but exhibited poor anti-mycobacterial activity (>500 µM).


Assuntos
Antibacterianos/farmacologia , Proteínas de Bactérias/metabolismo , Proteínas do Citoesqueleto/metabolismo , Mycobacterium smegmatis/efeitos dos fármacos , Alcaloides/farmacologia , Proteínas de Bactérias/genética , Benzodioxóis/farmacologia , Divisão Celular/efeitos dos fármacos , Cumarínicos/farmacologia , Citocinese , Proteínas do Citoesqueleto/genética , Bases de Dados de Compostos Químicos , Avaliação Pré-Clínica de Medicamentos/métodos , GTP Fosfo-Hidrolases/antagonistas & inibidores , Humanos , Simulação de Acoplamento Molecular , Mycobacterium smegmatis/citologia , Mycobacterium tuberculosis/metabolismo , Piperidinas/farmacologia , Alcamidas Poli-Insaturadas/farmacologia , Relação Estrutura-Atividade
8.
J Exp Clin Cancer Res ; 38(1): 158, 2019 Apr 12.
Artigo em Inglês | MEDLINE | ID: mdl-30979377

RESUMO

BACKGROUND: RASSF1A, a tumor suppressor gene, is frequently inactivated in lung cancer leading to a YAP-dependent epithelial-mesenchymal transition (EMT). Such effects are partly due to the inactivation of the anti-migratory RhoB GTPase via the inhibitory phosphorylation of GEF-H1, the GDP/GTP exchange factor for RhoB. However, the kinase responsible for RhoB/GEF-H1 inactivation in RASSF1A-depleted cells remained unknown. METHODS: NDR1/2 inactivation by siRNA or shRNA effects on epithelial-mesenchymal transition, invasion, xenograft formation and growth in SCID-/- Beige mice, apoptosis, proliferation, cytokinesis, YAP/TAZ activation were investigated upon RASSF1A loss in human bronchial epithelial cells (HBEC). RESULTS: We demonstrate here that depletion of the YAP-kinases NDR1/2 reverts migration and metastatic properties upon RASSF1A loss in HBEC. We show that NDR2 interacts directly with GEF-H1 (which contains the NDR phosphorylation consensus motif HXRXXS/T), leading to GEF-H1 phosphorylation. We further report that the RASSF1A/NDR2/GEF-H1/RhoB/YAP axis is involved in proper cytokinesis in human bronchial cells, since chromosome proper segregation are NDR-dependent upon RASSF1A or GEF-H1 loss in HBEC. CONCLUSION: To summarize, our data support a model in which, upon RASSF1A silencing, NDR2 gets activated, phosphorylates and inactivates GEF-H1, leading to RhoB inactivation. This cascade induced by RASSF1A loss in bronchial cells is responsible for metastasis properties, YAP activation and cytokinesis defects.


Assuntos
Movimento Celular/genética , Citocinese/genética , Inativação Gênica , Genes Supressores , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Supressoras de Tumor/genética , Animais , Biomarcadores Tumorais , Linhagem Celular Tumoral , Modelos Animais de Doenças , Transição Epitelial-Mesenquimal/genética , Xenoenxertos , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/mortalidade , Neoplasias Pulmonares/patologia , Camundongos , Metástase Neoplásica , Proteínas Nucleares/metabolismo , Fenótipo , Fosforilação , Prognóstico , Fatores de Transcrição/metabolismo , Proteína rhoB de Ligação ao GTP/metabolismo
9.
Methods Cell Biol ; 151: 29-36, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30948013

RESUMO

I happen to have been trained in cell and developmental biology in the early 1970s, which was near the beginning of the explosive growth of the field of cell biology. The American Society for Cell Biology had been founded in 1960 and so the field was in its early days. Cell biology research was dominated by the use of the electron microscope and by protein biochemistry. Molecular biology and the use of genetics were in their infancy. When we track the path of discoveries in cell biology contributed by research using echinoderm eggs, we follow the development of new technologies in genetics, molecular biology, biochemistry and biophysics, bioengineering, and imaging. The changes in approaches and methods have led to many key discoveries in cell biology through the use of sea urchin, sand dollar and sea star eggs. These include the discovery of cyclin, cytoplasmic dynein, rho activation for cytokinesis, new membrane addition as a late event in cytokinesis, multiple kinesins playing multiple roles, how flagella beat, the dynamics of microtubules in the mitotic apparatus, control over centrosomes and cell cycle checkpoints, the process of nuclear envelope breakdown for cell division, the discovery of 1-methyl adenine (hormones) as the trigger for meiotic maturation, Ca++ transients controlling cell activation and exocytosis among others. What I hope to provide in this perspective is to highlight some of those wonderful discoveries as my own career evolved to contribute to the field.


Assuntos
Biologia Celular/história , Equinodermos/crescimento & desenvolvimento , Desenvolvimento Embrionário/genética , Óvulo/crescimento & desenvolvimento , Animais , Centrossomo/metabolismo , Citocinese/genética , Equinodermos/genética , Fertilização/genética , História do Século XX , História do Século XXI , Mitose/genética
10.
PLoS Negl Trop Dis ; 13(3): e0007256, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30897087

RESUMO

Aurora kinases constitute a family of enzymes that play a key role during metazoan cells division, being involved in events like centrosome maturation and division, chromatin condensation, mitotic spindle assembly, control of kinetochore-microtubule attachments, and cytokinesis initiation. In this work, three Aurora kinase homologues were identified in Trypanosoma cruzi (TcAUK1, -2 and -3), a protozoan parasite of the Kinetoplastida Class. The genomic organization of these enzymes was fully analyzed, demonstrating that TcAUK1 is a single-copy gene, TcAUK2 coding sequence is present in two different forms (short and long) and TcAUK3 is a multi-copy gene. The three TcAUK genes are actively expressed in the different life cycle forms of T. cruzi (amastigotes, trypomastigotes and epimastigotes). TcAUK1 showed a changing localization along the cell cycle of the proliferating epimastigote form: at interphase it is located at the extremes of the kinetoplast while in mitosis it is detected at the cell nucleus, in close association with the mitotic spindle. Overexpression of TcAUK1 in epimastigotes leaded to a delay in the G2/M phases of the cell cycle due a retarded beginning of kinetoplast duplication. By immunofluorescence, we found that when it was overexpressed TcAUK1 lost its localization at the extremes of the kinetoplast during interphase, being observed inside the cell nucleus throughout the entire cell cycle. In summary, TcAUK1 appears to be a functional homologue of human Aurora B kinase, as it is related to mitotic spindle assembling and chromosome segregation. Moreover, TcAUK1 also seems to play a role during the initiation of kinetoplast duplication, a novel role described for this protein.


Assuntos
Aurora Quinases/metabolismo , Estágios do Ciclo de Vida , Mitocôndrias/fisiologia , Trypanosoma cruzi/enzimologia , Aurora Quinases/genética , Núcleo Celular/metabolismo , Centrossomo/metabolismo , Segregação de Cromossomos , Citocinese , Humanos , Mitose , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Fuso Acromático/metabolismo , Trypanosoma cruzi/genética , Trypanosoma cruzi/crescimento & desenvolvimento , Trypanosoma cruzi/fisiologia
11.
Mol Biol Cell ; 30(10): 1160-1169, 2019 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-30865554

RESUMO

The apicomplexan centrosome has a unique bipartite structure comprising an inner and outer core responsible for the nuclear cycle (mitosis) and budding cycles (cytokinesis), respectively. Although these two cores are always associated, they function independently to facilitate polyploid intermediates in the production of many progeny per replication round. Here, we describe the function of a large coiled-coil protein in Toxoplasma gondii, TgCep250, in connecting the two centrosomal cores and promoting their structural integrity. Throughout the cell cycle, TgCep250 localizes to the inner core but, associated with proteolytic processing, is also present on the outer core during the onset of cell division. In the absence of TgCep250, stray centrosome inner and outer core foci were observed. The detachment between centrosomal inner and outer cores was found in only one of the centrosomes during cell division, indicating distinct states of mother and daughter centrosomes. In mammals, Cep250 processing is required for centrosomal splitting and is mediated by Nek phopsphorylation. However, we show that neither the nonoverlapping spatiotemporal localization of TgNek1 and TgCep250 nor the distinct phenotypes upon their respective depletion support conservation of this mechanism in Toxoplasma. In conclusion, TgCep250 has a tethering function tailored to the unique bipartite centrosome in the Apicomplexa.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Centrossomo/metabolismo , Quinase 1 Relacionada a NIMA/metabolismo , Proteínas de Protozoários/metabolismo , Toxoplasma/metabolismo , Animais , Apicomplexa/metabolismo , Autoantígenos/metabolismo , Ciclo Celular/genética , Núcleo Celular/metabolismo , Citocinese/fisiologia , Replicação do DNA/fisiologia , Humanos , Mitose/fisiologia , Toxoplasma/citologia
12.
Food Chem Toxicol ; 128: 1-7, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30910685

RESUMO

Methamphetamine (METH) is a widely consumed psychostimulant drug; its acute toxic effects in brain and liver are well known, furthermore, there is some evidence in regard to its DNA damaging properties in humans. Therefore, we studied the impact of the drug on genomic stability in human derived hepatoma (HepG2) cells, which reflect the activation/detoxification of drugs better than other cell lines. Furthermore, experiments with human buccal derived cells (TR146) were conducted as the drug is consumed orally. Induction of DNA damage in both cell types with doses reflecting the exposure in abusers was found in single cell gel electrophoresis (SCGE) assays (which detect single and double strand breaks as well as apurinic sites). Furthermore, induction of micronuclei (formed as a consequence of structural and numerical chromosomal aberrations) and formation of nuclear buds resulting from gene amplifications was detected. Additional experiments with lesion-specific enzymes showed that the drug causes oxidation of purines and pyrimidines, indicating that its genotoxic effects may be due to oxidation of the DNA. Our findings support the assumption that the drug may cause adverse health effects (such as cancer and infertility) in long-term users which are causally related to DNA damage.


Assuntos
Transtornos Relacionados ao Uso de Anfetaminas/sangue , Aberrações Cromossômicas , Ensaio Cometa/métodos , Dano ao DNA , DNA/efeitos dos fármacos , Metanfetamina/toxicidade , Mutagênicos/toxicidade , Linhagem Celular , Citocinese/efeitos dos fármacos , DNA/metabolismo , DNA-Formamidopirimidina Glicosilase/metabolismo , Relação Dose-Resposta a Droga , Endodesoxirribonucleases/metabolismo , Células Hep G2 , Humanos , Metanfetamina/administração & dosagem , Testes para Micronúcleos , Mutagênicos/administração & dosagem , Oxirredução , Testes de Toxicidade Aguda
13.
Radiat Res ; 191(4): 342-351, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30779694

RESUMO

The cytokinesis-block micronucleus (CBMN) assay has become a fully-validated and standardized method for radiation biodosimetry. The assay is typically performed using microscopy, which is labor intensive, time consuming and impractical after a large-scale radiological/nuclear event. Imaging flow cytometry (IFC), which combines the statistical power of traditional flow cytometry with the sensitivity and specificity of microscopy, has been recently used to perform the CBMN assay. Since this technology is capable of automated sample acquisition and multi-file analysis, we have integrated IFC into our Rapid Automated Biodosimetry Technology (RABiT-II). Assay development and optimization studies were designed to increase the yield of binucleated cells (BNCs), and improve data acquisition and analysis templates to increase the speed and accuracy of image analysis. Human peripheral blood samples were exposed ex vivo with up to 4 Gy of c rays at a dose rate of 0.73 Gy/min. After irradiation, samples were transferred to microtubes (total volume of 1 ml including blood and media) and organized into a standard 8 × 12 plate format. Sample processing methods were modified by increasing the blood-to-media ratio, adding hypotonic solution prior to cell fixation and optimizing nuclear DRAQ5 staining, leading to an increase of 81% in BNC yield. Modification of the imaging processing algorithms within IFC software also improved BNC and MN identification, and reduced the average time of image analysis by 78%. Finally, 50 ll of irradiated whole blood was cultured with 200 ll of media in 96-well plates. All sample processing steps were performed automatically using the RABiT-II cell: :explorer robotic system adopting the optimized IFC-CBMN assay protocol. The results presented here detail a novel, high-throughput RABiT-IFC CBMN assay that possesses the potential to increase capacity for triage biodosimetry during a large-scale radiological/nuclear event.


Assuntos
Citocinese/efeitos da radiação , Citometria de Fluxo , Testes para Micronúcleos , Radiometria/métodos , Robótica , Triagem , Adulto , Automação , Calibragem , Relação Dose-Resposta à Radiação , Feminino , Humanos , Masculino , Pessoa de Meia-Idade
14.
Mol Biol Cell ; 30(8): 992-1007, 2019 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-30726162

RESUMO

Cell division is critical for development, organ growth, and tissue repair. The later stages of cell division include the formation of the microtubule (MT)-rich central spindle in anaphase, which is required to properly define the cell equator, guide the assembly of the acto-myosin contractile ring and ultimately ensure complete separation and isolation of the two daughter cells via abscission. Much is known about the molecular machinery that forms the central spindle, including proteins needed to generate the antiparallel overlapping interzonal MTs. One critical protein that has garnered great attention is the protein regulator of cytokinesis 1, or Fascetto (Feo) in Drosophila, which forms a homodimer to cross-link interzonal MTs, ensuring proper central spindle formation and cytokinesis. Here, we report on a new direct protein interactor and regulator of Feo we named Feo interacting protein (FIP). Loss of FIP results in a reduction in Feo localization, rapid disassembly of interzonal MTs, and several defects related to cytokinesis failure, including polyploidization of neural stem cells. Simultaneous reduction in Feo and FIP results in very large, tumorlike DNA-filled masses in the brain that contain hundreds of centrosomes. In aggregate, our data show that FIP acts directly on Feo to ensure fully accurate cell division.


Assuntos
Proteínas de Drosophila/antagonistas & inibidores , Proteínas de Drosophila/metabolismo , Proteínas Associadas aos Microtúbulos/antagonistas & inibidores , Proteínas Associadas aos Microtúbulos/metabolismo , Anáfase/fisiologia , Animais , Divisão Celular/fisiologia , Centrossomo/metabolismo , Citocinese , Proteínas de Drosophila/fisiologia , Drosophila melanogaster/metabolismo , Desenvolvimento Embrionário , Proteínas Associadas aos Microtúbulos/fisiologia , Microtúbulos/metabolismo , Miosinas/metabolismo , Domínios e Motivos de Interação entre Proteínas/fisiologia , Fuso Acromático/metabolismo
15.
Mol Biol Cell ; 30(8): 933-941, 2019 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-30759055

RESUMO

In many eukaryotes, cytokinesis is facilitated by the contraction of an actomyosin ring (AMR). The exact mechanisms that lead to this contractility are unknown, although some models posit that actin turnover in the AMR is essential. The effect of reduced actin dynamics during AMR formation has been well studied in Schizosaccharomyces pombe; however, the corresponding effects on AMR contraction are not well understood. By using mutants of the fission yeast actin severing protein Adf1, we observed that contracting AMRs display a "peeling" phenotype, where bundles of actin and myosin peel off from one side of the AMR, and are pulled across to the opposite side. This occurs multiple times during cytokinesis and is dependent on the activity of myosins Myo2, Myp2, and Myo51. We found that the distribution of Myo2 in the AMR anticorrelates with the location of peeling events, suggesting that peeling is caused by a nonuniform tension distribution around the AMR, and that one of the roles of actin turnover is to maintain a uniform tension distribution around the AMR.


Assuntos
Actinas/metabolismo , Actomiosina/fisiologia , Citocinese/fisiologia , Citoesqueleto de Actina/metabolismo , Fatores de Despolimerização de Actina/metabolismo , Actomiosina/metabolismo , Divisão Celular/fisiologia , Proteínas dos Microfilamentos/metabolismo , Cadeias Pesadas de Miosina/metabolismo , Miosina Tipo II/metabolismo , Miosinas/metabolismo , Fenótipo , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/metabolismo
16.
Mol Biol Cell ; 30(9): 1051-1059, 2019 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-30785846

RESUMO

Membrane blebs are specialized cellular protrusions that play diverse roles in processes such as cell division and cell migration. Blebbing can be divided into three distinct phases: bleb nucleation, bleb growth, and bleb retraction. Following nucleation and bleb growth, the actin cortex, comprising actin, cross-linking proteins, and nonmuscle myosin II (MII), begins to reassemble on the membrane. MII then drives the final phase, bleb retraction, which results in reintegration of the bleb into the cellular cortex. There are three MII paralogues with distinct biophysical properties expressed in mammalian cells: MIIA, MIIB, and MIIC. Here we show that MIIA specifically drives bleb retraction during cytokinesis. The motor domain and regulation of the nonhelical tailpiece of MIIA both contribute to its ability to drive bleb retraction. These experiments have also revealed a relationship between faster turnover of MIIA at the cortex and its ability to drive bleb retraction.


Assuntos
Vesícula/metabolismo , Miosina não Muscular Tipo IIA/fisiologia , Actinas/metabolismo , Animais , Células COS , Membrana Celular/metabolismo , Estruturas da Membrana Celular/metabolismo , Movimento Celular/fisiologia , Extensões da Superfície Celular/metabolismo , Cercopithecus aethiops , Citocinese/fisiologia , Citoplasma/metabolismo , Proteínas do Citoesqueleto/metabolismo , Células HeLa , Humanos , Miosina Tipo II/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Miosina não Muscular Tipo IIA/metabolismo , Miosina não Muscular Tipo IIB/metabolismo
17.
Nat Commun ; 10(1): 420, 2019 01 24.
Artigo em Inglês | MEDLINE | ID: mdl-30679428

RESUMO

Septins are cytoskeletal filaments that assemble at the inner face of the plasma membrane. They are localized at constriction sites and impact membrane remodeling. We report in vitro tools to examine how yeast septins behave on curved and deformable membranes. Septins reshape the membranes of Giant Unilamellar Vesicles with the formation of periodic spikes, while flattening smaller vesicles. We show that membrane deformations are associated to preferential arrangement of septin filaments on specific curvatures. When binding to bilayers supported on custom-designed periodic wavy patterns displaying positive and negative micrometric radii of curvatures, septin filaments remain straight and perpendicular to the curvature of the convex parts, while bending negatively to follow concave geometries. Based on these results, we propose a theoretical model that describes the deformations and micrometric curvature sensitivity observed in vitro. The model captures the reorganizations of septin filaments throughout cytokinesis in vivo, providing mechanistic insights into cell division.


Assuntos
Membrana Celular/química , Citoesqueleto/química , Septinas/química , Divisão Celular , Membrana Celular/ultraestrutura , Citocinese , Citoesqueleto/ultraestrutura , Imagem Tridimensional , Bicamadas Lipídicas/química , Microscopia de Fluorescência , Modelos Teóricos , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/química , Septinas/ultraestrutura , Lipossomas Unilamelares
18.
Int J Med Microbiol ; 309(2): 130-142, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30665874

RESUMO

Giardia trophozoites have developed resistance mechanisms to currently available compounds, leading to treatment failures. In this context, the development of new additional agents is mandatory. Sirtuins, which are class III NAD+-dependent histone deacetylases, have been considered important targets for the development of new anti-parasitic drugs. Here, we evaluated the activity of KH-TFMDI, a novel 3-arylideneindolin-2-one-type sirtuin inhibitor, on G. intestinalis trophozoites. This compound decreased the trophozoite growth presenting an IC50 value lower than nicotinamide, a moderately active inhibitor of yeast and human sirtuins. Light and electron microscopy analysis showed the presence of multinucleated cell clusters suggesting that the cytokinesis could be compromised in treated trophozoites. Cell rounding, concomitantly with the folding of the ventro-lateral flange and flagella internalization, was also observed. These cells eventually died by a mechanism which lead to DNA/nuclear damage, formation of multi-lamellar bodies and annexin V binding on the parasite surface. Taken together, these data show that KH-TFMDI has significant effects against G. intestinalis trophozoites proliferation and structural organization and suggest that histone deacetylation pathway should be explored on this protozoon as target for chemotherapy.


Assuntos
Antiprotozoários/farmacologia , Giardia lamblia/efeitos dos fármacos , Inibidores de Histona Desacetilases/farmacologia , Trofozoítos/efeitos dos fármacos , Células CACO-2 , Citocinese/efeitos dos fármacos , Giardia lamblia/citologia , Giardia lamblia/crescimento & desenvolvimento , Humanos , Concentração Inibidora 50 , Microscopia , Microscopia Eletrônica , Testes de Sensibilidade Parasitária , Trofozoítos/citologia , Trofozoítos/crescimento & desenvolvimento
19.
J Environ Sci Health B ; 54(2): 147-154, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30668218

RESUMO

The effect of doramectin (DOR) was tested on two experimental somatic bovine cells in vitro: peripheral lymphocytes (PL) and cumulus cells (CC). The cytotoxicity and genotoxicity of DOR were assessed using 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay, single cell gel electrophoresis assay (SCGE) and cytokinesis-block micronucleus cytome (CBMN Cyt) assay. Both cells were treated with three concentrations of DOR (20, 40, 60 ng mL-1) for 24 h. The results obtained from PL demonstrated that DOR was able to induce cytotoxic effect and DNA damage with all concentrations tested. Additionally, DOR increased micronuclei (MNi) frequency and nuclear buds (NBuds) with 20, 40, 60 ng mL-1, and nucleoplasmic bridges (NPBs) only with 40 ng mL-1. On the other hand, the three concentrations of DOR were not able to induce cytotoxic effect and DNA damage using SCGE in the bovine CC. Nevertheless, the two higher concentrations of DOR (20, 40 µg mL-1) significantly increased the frequency of micronucleus formation in bovine CC. These results represent the first experimental evidence of genotoxic and cytotoxic effects exerted by DOR on bovine PL and CC.


Assuntos
Células do Cúmulo/efeitos dos fármacos , Ivermectina/análogos & derivados , Linfócitos/efeitos dos fármacos , Animais , Bovinos , Células Cultivadas , Citocinese , Dano ao DNA/efeitos dos fármacos , Eletroforese/métodos , Feminino , Humanos , Ivermectina/administração & dosagem , Ivermectina/toxicidade , Testes para Micronúcleos , Análise de Célula Única/métodos , Testes de Toxicidade/métodos , Drogas Veterinárias/toxicidade
20.
Curr Genet ; 65(3): 663-668, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30600396

RESUMO

During cell division, the timing of mitosis and cytokinesis must be ordered to ensure that each daughter cell receives a complete, undamaged copy of the genome. In fission yeast, the septation initiation network (SIN) is responsible for this coordination, and a mitotic checkpoint dependent on the E3 ubiquitin ligase Dma1 and the protein kinase CK1 controls SIN signaling to delay cytokinesis when there are errors in mitosis. The participation of kinases and ubiquitin ligases in cell cycle checkpoints that maintain genome integrity is conserved from yeast to human, making fission yeast an excellent model system in which to study checkpoint mechanisms. In this review, we highlight recent advances and remaining questions related to checkpoint regulation, which requires the synchronized modulation of protein ubiquitination, phosphorylation, and subcellular localization.


Assuntos
Pontos de Checagem do Ciclo Celular , Proteínas de Ciclo Celular/metabolismo , Citocinese , Mitose , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/metabolismo , Análise Espaço-Temporal , Fosforilação , Ubiquitinação
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA