The Schizosaccharomyces pombe cdc14 gene is required for septum formation and can also inhibit nuclear division.

A conditional heat-sensitive mutation in the cdc14 gene of the fission yeast Schizosaccharomyces pombe results in failure to form a septum. Cells become highly elongated and multinucleate as growth and nuclear division continue in the absence of cell division. This article describes the cloning of the cdc14 gene and the identification of its product, a protein of 240 amino acids, p28cdc14. A null allele of the cdc14 gene shows that the gene is essential for septum formation and completion of the cell-division cycle. Overexpression of the gene product, p28cdc14, causes cell-cycle arrest in late G2 before mitosis. Cells leaking past the block activate p34cdc2 kinase and show condensed chromosomes, but the normal rearrangements of the microtubules and microfilaments that are associated with the transition from interphase to mitosis do not occur. Overexpression of p28cdc14 in mutants, in which the timing of mitosis is altered, suggests that these effects may be mediated upstream of the mitotic inhibitor wee1. These data are consistent with the idea that p28cdc14 may play a role in both the initiation of mitosis and septum formation and, by doing so, be part of the mechanism that coordinates these two cell-cycle events.

Phosphatidylinositol phospholipase C mediates carbon sensing and vegetative nuclear duplication rates in Aspergillus nidulans

In this work, we disrupted one of three putative phosphatidylinositol phospholipase C genes of Aspergillus nidulans and studied its effect on carbon source sensing linked to vegetative mitotic nuclear division. We showed that glucose does not affect nuclear division rates during early vegetative conidial germination (6-7 h) in either the wild type or the plcA-deficient mutant. Only after 8 h of cultivation on glucose did the mutant strain present some decrease in nuclear duplication. However, decreased nuclear division rates were observed in the wild type when cultivated in media amended with polypectate, whereas our plcA-deficient mutant did not show slow nuclear duplication rates when grown on this carbon source, even though it requires induction and secretion of multiple pectinolytic enzymes to be metabolized. Thus, plcA appears to be directly linked to high-molecular-weight carbon source sensing.

Molecular cloning and characterization of a conserved nuclear serine(threonine) protein kinase.

Human, Drosophila melanogaster, and Caenorhabditis elegans cDNA clones encoding homologues of a serine(threonine) protein kinase (EC 2.7.1.37) (designated Ndr protein kinase) have been isolated and sequenced. The human and Drosophila cDNAs predict polypeptides of 54 kDa and 52 kDa, respectively, which share approximately 80% amino acid similarity. Northern analysis of human tissues revealed a ubiquitously expressed 3.9-kb transcript. Recombinant GST-Ndr underwent intramolecular autophosphorylation on serine and threonine residues in vitro but failed to transphosphorylate several standard protein kinase substrates. Transfection of the human cDNA into COS-1 cells resulted in the appearance of an intense nuclear staining in cells analyzed by indirect immunofluorescence; deletion mutagenesis identified a short basic peptide, KRKAETWKRNRR, responsible for the nuclear accumulation of Ndr. Thus, Ndr is a conserved and widely expressed nuclear protein kinase. The closest known relative of this previously uncharacterized kinase is Dbf2, a budding yeast protein kinase required for the completion of nuclear division.

Index to conferences relating to nuclear science.

Includes index.

Index to conferences relating to nuclear science /

"This supplement updates TID-4043(rev.3) Index of conferences relating to nuclear science, July 1976, by indexing conferences included in Nuclear science abstracts, volume 20 (1966) and 21 (1967).".

Role of phospholipase C and protein kinase C in Aspergillus nidulans during growth on pectin or glucose: Effects on germination and duplication cycle

The effects of PLC and Pkc inhibitors on Aspergillus nidulans depend on the carbon source. PLC inhibitors Spm and C48/80 delayed the first nuclear division in cultures growing on glucose, but stimulated it in media supplemented with pectin. Less intense were these effects on the mutant transformed with PLC-A gene rupture (AP27). Neomycin also delayed the germination in cultures growing on glucose or pectin; however, on glucose, the nuclear division was inhibited whereas in pectin it was stimulated. These effects were minor in AP27. The effects of Ro-31-8425 and BIM (both Pkc inhibitors) were also opposite for cultures growing on glucose or pectin. On glucose cultures of both strains BIM delayed germination and the first nuclear division, whereas on pectin both parameters were stimulated. Opposite effects were also detected when the cultures were growing on glucose or pectin in the presence of Ro-31-8425.

The S. pombe cdc15 gene is a key element in the reorganization of F-actin at mitosis.

The S. pombe cdc15 gene is essential for cell division. cdc15ts mutants do not form a septum, but growth and nuclear division continue, leading to formation of multinucleate cells. The earliest step in septum formation and cytokinesis, rearrangement of actin to the center of the cell, is associated with appearance of hypophosphorylated cdc15p and formation of a cdc15p ring, which colocalizes with actin. Loss of cdc15p function impairs formation of the actin ring. The abundance of cdc15 mRNA varies through the cell division cycle, peaking in early mitosis before septation. Expression of cdc15 in G2-arrested cells induces actin rearrangement to the center of the cell. These data implicate cdc15p as a key element in mediating the cytoskeletal rearrangements required for cytokinesis.

An essential role for the Leishmania major metacaspase in cell cycle progression.

Metacaspases (MCAs) are distant orthologues of caspases and have been proposed to play a role in programmed cell death in yeast and plants, but little is known about their function in parasitic protozoa. The MCA gene of Leishmania major (LmjMCA) is expressed in actively replicating amastigotes and procyclic promastigotes, but at a lower level in metacyclic promastigotes. LmjMCA has a punctate distribution throughout the cell in interphase cells, but becomes concentrated in the kinetoplast (mitochondrial DNA) at the time of the organelle's segregation. LmjMCA also translocates to the nucleus during mitosis, where it associates with the mitotic spindle. Overexpression of LmjMCA in promastigotes leads to a severe growth retardation and changes in ploidy, due to defects in kinetoplast segregation and nuclear division and an impairment of cytokinesis. LmjMCA null mutants could not be generated and following genetic manipulation to express LmjMCA from an episome, the only mutants that were viable were those expressing LmjMCA at physiological levels. Together these data suggest that in L. major active LmjMCA is essential for the correct segregation of the nucleus and kinetoplast, functions that could be independent of programmed cell death, and that the amount of LmjMCA is crucial. The absence of MCAs from mammals makes the enzyme a potential drug target against protozoan parasites.

A 2D/3D image analysis system to track fluorescently labeled structures in rod-shaped cells: application to measure spindle pole asymmetry during mitosis.

BACKGROUND: The yeast Schizosaccharomyces pombe is frequently used as a model for studying the cell cycle. The cells are rod-shaped and divide by medial fission. The process of cell division, or cytokinesis, is controlled by a network of signaling proteins called the Septation Initiation Network (SIN); SIN proteins associate with the SPBs during nuclear division (mitosis). Some SIN proteins associate with both SPBs early in mitosis, and then display strongly asymmetric signal intensity at the SPBs in late mitosis, just before cytokinesis. This asymmetry is thought to be important for correct regulation of SIN signaling, and coordination of cytokinesis and mitosis. In order to study the dynamics of organelles or large protein complexes such as the spindle pole body (SPB), which have been labeled with a fluorescent protein tag in living cells, a number of the image analysis problems must be solved; the cell outline must be detected automatically, and the position and signal intensity associated with the structures of interest within the cell must be determined. RESULTS: We present a new 2D and 3D image analysis system that permits versatile and robust analysis of motile, fluorescently labeled structures in rod-shaped cells. We have designed an image analysis system that we have implemented as a user-friendly software package allowing the fast and robust image-analysis of large numbers of rod-shaped cells. We have developed new robust algorithms, which we combined with existing methodologies to facilitate fast and accurate analysis. Our software permits the detection and segmentation of rod-shaped cells in either static or dynamic (i.e. time lapse) multi-channel images. It enables tracking of two structures (for example SPBs) in two different image channels. For 2D or 3D static images, the locations of the structures are identified, and then intensity values are extracted together with several quantitative parameters, such as length, width, cell orientation, background fluorescence and the distance between the structures of interest. Furthermore, two kinds of kymographs of the tracked structures can be established, one representing the migration with respect to their relative position, the other representing their individual trajectories inside the cell. This software package, called "RodCellJ", allowed us to analyze a large number of S. pombe cells to understand the rules that govern SIN protein asymmetry. CONCLUSIONS: "RodCell" is freely available to the community as a package of several ImageJ plugins to simultaneously analyze the behavior of a large number of rod-shaped cells in an extensive manner. The integration of different image-processing techniques in a single package, as well as the development of novel algorithms does not only allow to speed up the analysis with respect to the usage of existing tools, but also accounts for higher accuracy. Its utility was demonstrated on both 2D and 3D static and dynamic images to study the septation initiation network of the yeast Schizosaccharomyces pombe. More generally, it can be used in any kind of biological context where fluorescent-protein labeled structures need to be analyzed in rod-shaped cells. AVAILABILITY: RodCellJ is freely available under http://bigwww.epfl.ch/algorithms.html, (after acceptance of the publication).

Compounds used to produce cloned animals are genotoxic and mutagenic in mammalian assays in vitro and in vivo

The compounds 6-dimethylaminopurine and cycloheximide promote the successful production of cloned mammals and have been used in the development of embryos produced by somatic cell nuclear transfer. This study investigated the effects of 6-dimethylaminopurine and cycloheximide in vitro, using the thiazolyl blue tetrazolium bromide colorimetric assay to assess cytotoxicity, the trypan blue exclusion assay to assess cell viability, the comet assay to assess genotoxicity, and the micronucleus test with cytokinesis block to test mutagenicity. In addition, the comet assay and the micronucleus test were also performed on peripheral blood cells of 54 male Swiss mice, 35 g each, to assess the effects of the compounds in vivo. The results indicated that both 6-dimethylaminopurine and cycloheximide, at the concentrations and doses tested, were cytotoxic in vitro and genotoxic and mutagenic in vitro and in vivo, altered the nuclear division index in vitro, but did not diminish cell viability in vitro. Considering that alterations in DNA play important roles in mutagenesis, carcinogenesis, and morphofunctional teratogenesis and reduce embryonic viability, this study indicated that 6-dimethylaminopurine and cycloheximide utilized in the process of mammalian cloning may be responsible for the low embryo viability commonly seen in nuclear transfer after implantation in utero.

Toxicogenomic activity of gemcitabine in two TP53-mutated bladder cancer cell lines: special focus on cell cycle-related genes

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

In vitro assessment of the cytotoxic, apoptotic, and mutagenic potentials of Isatin

Isatin (1H-indole-2,3-dione) is a chemical found in various medicinal plant species and responsible for a broad spectrum of pharmacological and biological properties that may be beneficial to human health, as an anticonvulsant, antibacterial, antifungal, antiviral, and anticancer agent. The aim of the present study was to determine in vitro the cytotoxic, mutagenic, and apoptotic effects of isatin on CHO-K1 and HeLa cells using the MTT viability assay (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide), micronucleus (MN) test, apoptosis index, and nuclear division index (NDI). The 5 isatin concentrations evaluated in the mutagenicity and apoptosis tests were 0.5, 1, 5, 10, and 50 μM, selected through a preliminary MTT assay. Positive (doxorubicin, DXR) and negative (phosphate buffered saline, PBS) control groups were also included in the analysis. Isatin did not exert a mutagenic effect on CHO-K1 after 3 and 24 h of treatment or on HeLa cells after 24 h. However, 10 and 50 μM concentrations inhibited cell proliferation and promoted apoptosis in both CHO-K1 and HeLa cells. Data indicate that the cytotoxic, apoptotic, and antiproliferative effects of isatin were concentration independent and cell line independent. The authors thank Profa Dra Eiko Nakagawa Itano for the use the spectrophotometer and the Conselho Nacional para o Desenvolvimento Científico e Tecnológico for master's scholarships to P. M. Cândido-Bacani and grants to T. R. Calvo, W. Vilegas, E. A. Varanda and I. M. S. Cólus. The Conselho Nacional para o Desenvolvimento Científico e Tecnológico provided funding for this study. © 2013 Taylor & Francis Group, LLC.

Genotoxicidade humana e fármacos antidepressivos: avaliação da duloxetina em culturas de linfócitos

Fármacos antidepressivos são largamente utilizados no tratamento sintomático do transtorno depressivo. Inúmeras pesquisas atuais sobre a depressão vêm contribuindo para o avanço da terapia farmacológica e para o surgimento de novos fármacos antidepressivos. Diretrizes atuais para testes de genotoxicidade de novos medicamentos sugerem a importante utilidade de ensaios que detectem danos ao DNA, ou seja, testes para avaliar a indução de quebras no DNA. Entretanto, o número escasso de dados sobre a genotoxicidade de fármacos faz com que seja reduzido o número de fármacos que realmente podem ser usados em segurança. Portanto, é de extrema importância estudos sobre a avaliação genotoxicológica de fármacos, principalmente, drogas utilizadas por um longo período de tempo como é o caso dos antidepressivos. A duloxetina é um antidepressivo novo, pertencente à classe dos inibidores seletivos da recaptação de serotonina e noradrenalina (ISRSN), utilizada no tratamento sintomático da depressão. Apesar da existência de trabalhos demonstrando que alguns fármacos antidepressivos são genotóxicos, não existe até hoje nenhum estudo sobre a possível genotoxicidade da duloxetina em células de origem humana. Assim, o presente estudo tem como objetivo explorar o possível potencial genotóxico in vitro da duloxetina em culturas primárias de linfócitos humanos através das técnicas de detecção de aberrações cromossômicas e micronúcleos. Culturas primárias de linfócitos sanguíneos de voluntários sadios foram expostas a diferentes concentrações de duloxetina (10-150 ng/ml) e ciclofosfamida (6 μg/ml) como controle positivo. Aberrações cromossômicas estruturais, índice mitótico, índice de divisão nuclear, índice de binucleação, número de células com um, dois, três e quatro micronúcleos e o número de células com pontes nucleoplasmáticas foram avaliadas. Todos os índices das culturas incubadas com duloxetina foram significativamente menores que aqueles dos grupos controles, indicando um certo grau de citotoxicidade da droga. Entretanto, só as concentrações de 100 e 150 ng/ml provocaram o aumento significativo da presença de aberrações cromossômicas e micronúcleos. Considerando que essas concentrações ficam perto do limite superior da faixa terapêutica da droga usada em humanos, nossos resultados alertam já sobre a necessidade de aprofundar no conhecimento da genotoxicidade humana da duloxetina.

Compounds used to produce cloned animals are genotoxic and mutagenic in mammalian assays in vitro and in vivo

The compounds 6-dimethylaminopurine and cycloheximide promote the successful production of cloned mammals and have been used in the development of embryos produced by somatic cell nuclear transfer. This study investigated the effects of 6-dimethylaminopurine and cycloheximide in vitro, using the thiazolyl blue tetrazolium bromide colorimetric assay to assess cytotoxicity, the trypan blue exclusion assay to assess cell viability, the comet assay to assess genotoxicity, and the micronucleus test with cytokinesis block to test mutagenicity. In addition, the comet assay and the micronucleus test were also performed on peripheral blood cells of 54 male Swiss mice, 35 g each, to assess the effects of the compounds in vivo. The results indicated that both 6-dimethylaminopurine and cycloheximide, at the concentrations and doses tested, were cytotoxic in vitro and genotoxic and mutagenic in vitro and in vivo, altered the nuclear division index in vitro, but did not diminish cell viability in vitro. Considering that alterations in DNA play important roles in mutagenesis, carcinogenesis, and morphofunctional teratogenesis and reduce embryonic viability, this study indicated that 6-dimethylaminopurine and cycloheximide utilized in the process of mammalian cloning may be responsible for the low embryo viability commonly seen in nuclear transfer after implantation in utero.

Cytotoxic and genotoxic effects of high concentrations of the immunosuppressive drugs cyclosporine and tacrolimus in MRC-5 cells

Immunosuppressive drugs are used to suppress immune system activity in transplant patients and reduce the risk of organ rejection. The present study evaluated the potential cytotoxic, genotoxic and mutagenic of the immunosuppressive drugs cyclosporine (CsA) and tacrolimus (FK-506) on normal human fibroblasts (MRC-5 cells). Based on plasma concentrations of the immunosuppressive drugs, which were obtained from the records of kidney transplant patients at the Kidney Institute of Londrina, Brazil, 11 concentrations of each immunosuppressive were chosen to evaluate cell viability using the MIT assay. From these results, CsA and FK-506 concentrations of 135, 300, 675, and 1520 ng/ml and 8, 16, 24, and 32 ng/ml, respectively, were evaluated using (i) the comet assay, (ii) the nuclear division index (NDI), (iii) the micronucleus test (CBMN) and (iv) cell proliferation curves generated by quantifying cell numbers and protein levels. In this study, 1520 to 3420 ng/ml CsA decreased cell viability after 48 h of exposure. Genotoxic effects were observed only with a concentration of 1520 ng/ml after 3 h of exposure and with concentrations of 675 and 1520 ng/ml after 24 h of exposure. Mutagenic effects were observed only for the concentration of 1520 ng/ml. FK-506 decreased cell viability after 72 h of exposure for concentrations up to 20 ng/ml; genotoxic effects were observed with concentrations up to 8 ng/ml for both treatment times (3 and 24 h) and mutagenic effects were observed with concentrations of 24 and 32 ng/ml after 24 h of treatment. The cell proliferation curves demonstrated the absence of cytostatic effects of these drugs, and these data were confirmed by the NDI analysis. Our results suggest that concentrations lower than 300 ng/ml of CsA and 16 ng/ml of FK-506 are safe for use, as they did not induce genotoxic and mutagenic damage or affect MRC-5 cell viability and proliferation. (C) 2014 Elsevier GmbH. All rights reserved.